A novel fibrous-bed airlift bioreactor for recombinant yeast fermentation

A 20 L novel bioreactor was introduced in which an internal loop airlift device incorporated a fibrous-bed to support the immobilization of recombinant yeast cells. The production of glucoamylase by immobilized cells of recombinant Saccharomyces cerevisiae strain (C468/pGAC9) was studied. Plasmid stability of recombinant Saccharomyces cerevisiae was measured and compared for both freely-suspended and fiber-immobilized cells using three modes of operation, namely batch, repeated-batch and continuous fermentation.;In yeast extract-peptone-glucose (YEPG) non-selective medium, the batch cultures showed that cell growth was typically diauxic and glucoamylase production was cell growth-associated. Results of continuous free cell cultures confirmed plasmid instability of recombinant yeast when grown in this medium. The plasmid stability was growth rate (dilution rate) dependent, as was the product yield, productivity, and specific glucoamylase enzyme productivity. As the dilution rate was increased from 0.05 to 0.37 h-1, plasmid stability, productivity, and specific productivity also increased. However, product yield decreased as the dilution rate increased. A mathematical model was proposed to evaluate the plasmid instability parameters. The results indicated that the growth rate ratio of the specific growth rate of the plasmid-carrying to that of the plasmid-free cells was almost constant. It was found that the probability of plasmid loss coefficient, decreased with increasing dilution rate, indicating an increase in the segregational stability.;A 9 L scale down version of the 20 L airlift bioreactor with and without a fibrous packed downcomer, was used to test the recombinant yeast growth kinetics. Continuous culture experiments were carried out at different dilution rates in the YEPG nonselective growth liquid medium. The immobilized cell fibrous-bed bioreactor gave a higher glucoamylase activity and maintained recombinant protein production for a longer period of time compared to the corresponding free cell system Immobilizing the recombinant yeast cells in a hydrophilic cotton fiber gave the best performance. Immobilizing ∼101 gdw/L cell concentration increased the activity of glucoamylase from 86 U/L for the free cell system to 213 U/L for the immobilized system at 0.05 h -1 dilution rate, and productivity and specific productivity from 2.3 to 3.3 U/L.h and 0.47 to 0.86 U/g cell.h, for free and immobilized systems, respectively. The more stable glucoamylase production was due to a reduced plasmid loss in the immobilized cell fibrous-bed bioreactor system. By operating the immobilized cell bioreactor system in a repeated batch mode, further reduction in plasmid instability of the recombinant S. cerevisiae strain was achieved. A mathematical model was also developed to describe the kinetics of plasmid loss. The model successfully described the experimental results and provided important information for better understanding of the stabilizing mechanisms of the immobilized recombinant yeast cells. It is concluded that a reduced cell specific growth rate accompanied by an increased plasmid copy number are the main reasons for the effective enhanced plasmid stability in the immobilized cell system compared to free cell system. In the present research study, the adhesion of recombinant yeast cells could be a dynamic reserve of highly concentrated plasmid-carrying cells having a high plasmid copy number and less segregational instability.;More research is needed to further optimize the cell immobilization in the fibrous bed bioreactor to achieve controlled growth and immobilization of productive plasmid-carrying cells and removal of the non-productive plasmid-free cells for the stable long-term operation of immobilized cell fermentation process.;Batch culture studies showed that the medium composition affected both the growth characteristics of S. cerevisiae and stability of the plasmid. Superior plasmid stability was obtained in yeast nitrogen base selective medium and in complex medium with 0.5 to 2% D-glucose. Plasmid stability of 92% was obtained in complex medium with 2% D-glucose yielding 48 units of glucoamylase/g of cells compared to 54% plasmid stability achieved with 2% soluble starch, which yielded 23 units of glucoamylase/g of cells. The plasmid stability was also studied when soluble starch was used as carbon source. It was found that plasmid stability decreases with increasing starch concentration in the complex media, as compared to glucose medium.;Keywords: fibrous-bed, recombinant yeast, cell adhesion, immobilization, plasmid stability, glucoamylase, airlift, modeling, kinetics, bioreactor.