| The cellular slime mould Dictyostelium discoideum (D.discoideum) belongs to Acra-siomzcetes. It shows promising characteristics as an expression system for the production of recombinant glycoproteins owing to its ability to perform a whole spectrum of post-translation modifications. Limited maximal cell densities (1-2×l07mL-1) and slow growth rates, however, disfavor its application. For the production of high-value products like heterogenous proteins, efforts should be made to improve its cultivation.The influence of certain components and culture conditions on the growth of D.discoideum AX3-pMB74-hFasL-H was investigated based on the complex medium HL-5C. It was found that the optimal pH for cell growth was 6.5. Glucose was the best carbon source, and optimal concentration should be 15g L-1. Adding certain amino acid to standard HL-5C medium would decrease the growth rates of AX3-pMB74-hFasL-H, whereas the maximal cell density would be raised when moderate tryptophan or cysteine was used.Since the way of immobilization was often applied in order to achieve high cell concentrations, the immobilization of D. discoideum with cotton cloth was investigated in the present work. The results showed that cotton cloth was a suitable fibrous matrix for efficient immobilization of D. discoideum. On shake flask scale, the effects of cotton cloth types, volume and other culture conditions on cell immobilization were examined systematically. Under the optimized conditions, the highest cell density of D. discoideum in the immobilization volume of cotton cloth was 7.87×107 mL-1. When the fibrous matrix (cotton cloth) was added to the shake flask at the very begining, the generation time of D. discoideum was doubled. So we divided the whole immobilization culture process into two stages, suspension culture and immobilization. Firstly, D. discoideum was cultivated in suspension till the cell density reached 1×107 mL-1. Then the cotton cloth was added, in which cells were immobilized and cultivated. After 120h of cultivation, the immobilized cell density reached 1×108 mL-1, which was 4 times higher than that observed in suspension culture under similar conditions.Fibrous-bed bioreactors were evaluated in D. discoideum immobilization. The results showed that rotating fibrous-bed bioreactor (RFB) with cotton cloth and PUF as carriers was suitable for the immobilization culture of D. discoideum, in which D. discoideum reached a immobilized cell density of 1×108 mL-1 . Then a external loop fibrous-bed bioreactor was used for comparison. The fibrous matrix used for cell immobilization was made of a piece of cotton cloth (14 cm×70 cm), mounted on a stainless steel mesh. The matrix was spirally wound around the vertical axis and packed in a glass column.D. discoideum was first allowed to grow in the 5-L recirculation reactor until a cell density of 0.5-2×107mL-1 was attained. The broth was then circulated through the FBB system for immobilization culture. At the end of the cultivation the immobilized cell density reached 1.37×108 mL-1, which was 4 times higher than that observed in conventional suspension culture. By immobilized cells cultivatied in external loop fibrous-bed bioreactor, the shFasL could rise to 173.7μg 1-1. Furthermore, with repeated batch culture, the fermentation was prolonged to over 360 hours without any loss of cell density or shFasL productivity.
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