As a means of integrating cell growth and immobilization into one-step, recombinant Saccharomyces cerevisiae cells with invertase activity were immobilized in liquid-core alginate capsules and cultured to a high density.
The capsules should be made without tails to prevent immobilized cells from leaking. The optimum conditions making the capsules without tails were 1.3% (w/v) calcium chloride and 0.6% (w/v) sodium alginate, respectively, and the amount of the xanthan gum was 20% of the total calcium chloride by weight when the cavity depth was 1.0 cm in the reactor of 7.5 cm in diameter and 3.0 cm in height. When Saccharomyces cerevisiae cells immobilized in the capsules were cultured in the growth medium not containing $CaCl_2$, the capsules swelled to twice its initial size and the wall became thicker from 0.2 mm to 1.5 mm. But adding 5.0 g $CaCl_2$ to a litre of the growth medium kept the capsule from swelling. To prevent capsules from rupturing because of the $CO_2$ formation, 10 g surfactant (Tween 20) was added to a litre of the $CaCl_2$ solution during the step of capsule formation. The capsules with the surfactant did not burst open when the culture was carried out. The dry cell concentration of the cells immobilized into capsules was 83 g/L based on the inner volume.
In batch culture, the specific invertase activity of immobilized cells was similar to that of free cells, although much higher cell concentration was obtained in immobilized cells. Thus, increasing cell density using this microencapsulatioon technique could cause a proportionate increase in total invertase activity. Both free cells and immobilized cells showed a maximum specific invertase activity at the same temperature of 65℃, but the thermal stability of invertase of immobilized cells was better than that of free cells. It was concluded that the optimum reaction temperature was between 30℃ and 50℃ considering both maximum activity and thermal stability. When the immobilized cells were stored in a Na-acetate buffer (pH4.9, 0.2M) for a month, the activities of immobilized cells decreased by about 7%, 8%, 20% and 20% at 4℃, 10℃, 30℃ and 45℃, respectively. When stored at 65℃, however, the immobilized cells almost deactivated in less than 5 hr.
Stable reaction of sucrose hydrolysis was possible during 25 repeated batches for about 7 days. Using the packed bed reactor, continuous hydrolysis of 0.3M sucrose was carried out with a conversion more than 90% at the dilution rate of 1.08 $h^{-1}$, but the crush of capsules was observed after 7 days of operation. In the stirred tank reactor, we obtained a maximum productivity of 80.8 g/L/hr at 0.8 $h^{-1}$ dilution rate using 1.0M sucrose solution and a 0.5M sucrose solution was hydrolyzed during a week with 95% conversion at the dilution rate of 0.6 $h^{-1}$.
