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In this study, we investigated whether galactooligosaccharide (GOS) can be stably and steadily synthesized using immobilized beta-galactosidase (β-gal) inclusion body (IB)- containing E. coli cells during long-term repeated-batch operation. To improve the operational stability of this enzyme reactor system, immobilized E. coli cells were crosslinked with glutaraldehyde (GA) after immobilization of the E. coli. When we treated with 2% GA for E. coli crosslinking, GOS production continued to an elapsed time of 576 h, in which seven batch runs were operated consecutively. GOS production ranged from 51.6 to 78.5 g/l (71.2 ± 10.5 g/l, n = 7) during those batch operations. In contrast, when we crosslinked E. coli with 4% GA, GOS production ranged from 31.5 to 64.0 g/l (52.3 ± 10.8, n = 4), and only four consecutive batch runs were operated. Although we did not use an industrial β-gal for GOS production, in which a thermophile is used routinely, this represents the longest operation time for GOS production using E. coli β-gal. Improved stability and durability of the cell immobilization system were achieved using the crosslinking protocol. This strategy could be directly applied to other microbial enzyme reactor systems using cell immobilization to extend the operation time and/or improve the reactor system stability.

In this study, we investigated the performance of an immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-β-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the beta-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that β-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.

In this study, we investigated the feasibility of sustainable long-term bioethanol production from the hydrolysate of a brown seaweed, Sargassum sagamianum. Because the hydrolysate was prepared as a liquid solution using a hightemperature liquefying system, a repeated-batch operation was utilized as the operational strategy for bioethanol production. Additionally, we used surface aeration to improve bioethanol production from the hydrolysate containing C5 monosaccharides such as xylose. In this study, the C5 monosaccharide-utilizable yeast strain Pichia stipitis was used for bioethanol production. Therefore, based on this repeated-batch flask culture, we designed a surface-aerated repeated-batch fermentor culture, in which the aeration was finely controlled at 100 ml/min and delivered into the headspace of a 2.5-l fermentor. When the medium was replaced every 48 h, bioethanol was continuously produced for 200 h under repeated-batch fermentor culture, where the level of bioethanol production was about 9~10 (g/l). Additionally, the bioethanol yield based on the reducing sugar was about 0.386, which was the average value throughout four consecutive cultures and was about 74.5% of the theoretical value. In addition, the bioethanol yield based on quantitative TLC analyses of glucose and xylose was about 0.431, which was the average value throughout four consecutive cultures and was about 84.3% of theoretical value. Consequently, throughout this repeated-batch operation, we demonstrated that it was actually feasible to produce bioethanol from the hydrolysate of seaweed S. sagamianum. In addition, the approach described here is a practical strategy for commercial bioethanol production from seaweed, particularly for finely controlling aeration through surface aeration.