Bacillus Alkalophilus Immobilized On Monofilament Curtain For CGTase Production

The strain growth for enzyme production was the key step in the process of cyclodextrinindustrial production. However, due to the influence of the environment and various operatingconditions, they were extremely unstable. Immobilized cell technology can increase thegrowth stability of bacteria, shortening the seed culture period, and can be used repeatedly.There are lots of nice features on monofilament curtain, such as environmental friendly,corrosion resistance, recyclable, etc. Monofilament curtain is commonly used as matrices ofseed-adhering in aquaculture. In this paper, by first use of monofilament curtain as the carrierfor immobilizing Bacillus circulans ATCC21783, the immobilization conditions, thefermentation conditions of shaking flask and fermentation tank, and the benefits in theproduction of cyclodextrins were studied. Then a preliminary feasibility report was preparedidentifying Bacillus circulans ATCC21783immobilized on the monofilament curtain that canbe used in the industrial production of cyclodextrins.The immobilization methods and matrices were firstly selected. Monofilament curtain,loofa sponge, and monofilament curtain–alginate, loofa sponge–alginate were used asmatrices for the immobilization of Bacillus circulans ATCC21783cells. With free cells as acomparison, the results displayed that the immobilized cells showed better behavior ofrecycling enzyme producing than free cells. The cells immobilized on brown blademaintained83.5%cyclodextrin glycosyltransferases (CGTase) production abilities in theseventh cycle; but in monofilament curtain–alginate and loofa sponge–alginate, maintained80%of CGTase production ability. Brown blade was finally chosen for further studiesbecause of its renewable character and simple method of immobilization.The influence for immobilized method to CGTase activity was analyzed. The resultdemonstrated that enzyme activity was related to the biomass. By the determination ofbiomass for each cycle and the analysis of scanning electron microscopy image ofmonofilament curtain immobilized cells, the preliminary reasons that immobilized cells cancontinue to produce high CGTase activity was described as the following: immobilized cellswere physicaly adsorbed on monofilament curtain which had special concave surfacestructure, then the immobilized cells can shorten the lag phase of the cell growth and maintainhigh cell density in the fermentation medium, which providing proper enzyme producingconditions.The immobilized conditions and immobilized cell fermentation conditions wereoptimized in shake flask. Firstly, the best immobilized condition was found as theimmobilization period of1day, and the loading volume of40mL and inoculum density of3%and three brown blade discs. Secondly, the temperature, initial pH, shaking speed, loadingvolume were found using single factor experiment explores as follows: temperature40℃,initial pH9.5, shaking speed220r/min, the loading volume40ml. Enzyme activity wasdetected as5469.25U/ml. Finally, the sequence of factors that affecting the activity wasanalyzed through orthogonal experiment. It was temperature>rotate speed>loading volume>pH. An extended scale cultivation in5L fermentation tank was further studied. Throughanalyzing the relations between biomass, enzyme activity, and pH, dissolved oxygen, thesolubility of oxygen was found essential to enzyme activity. The fermentation conditions wereoptimized as speed400r/min, ventilation4.5L/min, and the enzyme activity was detected as5539.36U/mLunder this condition. Further, the fermentation period was shortend to24h, witha16h decrease than initial fermentation.The economic feasibility of immobilized cell being applied in the industrializedproduction of β-cyclodextrin was analyzed.The results showed that the difference of theeconomic feasibility between new process and original process followed the followingfunction(x—the reduced time that new process compared to the original process, the unit:y=3699799/(8-x)-28262.2-491180=3699799/(8-x)-519442.2When y equal to0, x equal to0.88day. Which means the income would reduce when theproduction time is more than0.9day, and the income would increase when the productiontime is less than0.9days, and will be increased9100RMB/month with each reduced day.