The present invention relates to a process for immobilizing microorganisms, and more particularly to an immobilization process for preparing microbial beads by using calcium alginate, polyethylene glycol (PEG) and polyethylene imide (PEI).
Studies on immobilization of microorganisms confirmed the gaining of highest biomass in smallest unit volume, and the buffering and protective effects against external environmental impact. The immobilization techniques of microorganisms afford a great deal of potential of improving the wastewater treatment.
The immobilization methods of microorganisms in which various natural or synthetic polymeric substances are used to bring about the immobilization of the activated microorganisms, have received a great deal of attention in recent decades, and achieved some success in the industrial application, as exemplified by their applications in the production of biochemical products such as high fructose syrup, 6-APA, L-amino acid, etc. The immobilization methods of microorganisms include generally two types, namely, the adhesion type natural immobilization methods, and the entrapment type artificial immobilization methods. In the entrapment type artificial immobilization methods, calcium alginate is most commonly used in preparing the immobilized microbial beads. Other commonly used substances for preparing the immobilized microbial beads include polyethylene glycol (PEG), polyvinyl alcohol (PVA), K-carrageenan, aga, gelatin, etc. However, polymeric substances are toxic and expensive, and their low mechanical strength make them difficult to form beads of spherical shape. It is, therefore, important that we are in need of developing a new and inexpensive immobilization material which is not toxic to microorganisms and has a strong gel strength so as to ensure that the bead formation is successful.
PEI (polyethylene imide), PEG (polyethylene glycol and calcium alginate are non-toxic to microorganisms. When mixed well, the mixture of PEI, PEG and calcium alginate has advantages that it is non-toxic to both human being and microorganisms, and that it is provided with a mechanical strength sufficiently strong enough to ensure the success of bead formations, and further it can be used in industry and produced in quantity economically. Therefore, the mixture of PEI, PEG and calcium alginate is an ideal material for use in immobilizing microorganisms.
Various patented methods of immobilizing microorganisms by PVA have been disclosed in recent years, as exemplified by the exemplified by the Japanese patent applications Kokai 57-14129 (1982) and 61-139385 (1986) in which the entrapment methods are characterized that the gelations of the mixture containing the PVA aqueous solution and the microorganisms are carried out by the methods associated with the freezing and thawing technique. Another Japanese patent application Kokai 1-454372 (1989) discloses a method, in which the mixture of the PVA aqueous solution and the microorganisms is exposed to the ultraviolet radiation so as to form a photocrosslinking gelation. Another gelation technique is shown in U.S. Pat. No. 5,290,693, in which PVA, boric acid and phosphate are used for immobilizing microorganisms.
The prior art described above have the following shortcomings. In the first place, they are time-consuming and complicated, thereby resulting in a substantial increase in the expenditure for large-scale production facilities without increasing the productivity. Secondly, an environment in which cryogenic temperature, vacuum and boric acid are present is antagonistic to the living microorganisms intended to be immobilized. In the third place, their high density structure affect the delivery of gas and substances, thereby causing the gelations not suitable for a long term use.