71NISR Research GRANT
2005 Young Investigator Research Grant

Development of Target-adhesive Bacteria and Its Application
to Fermentation Process
Kazuaki NINOMIYA
Department of Biotechnology, Graduate School of Engineering, Osaka University

Research aims
Recently, the use of bioethanol as alternative transportation fuel has rise considerable interest due to the public
conscious of environmental problem and petroleum price.
One of the most common methods for bioethanol production is through fermentation of renewable biomass such as
starch using yeast, Saccharomyces cerevisiae. Since theyeast lacks a -amylase and glucoamylase, we have developed some recombinant yeast that displays these amylolytic
enzymes on the surface of the yeast (arming yeasts) for direct ethanol fermentation from starch. Moreover, we havealso constructed arming yeasts system using flocculent
yeast as a host (flocculent arming yeast), since the flocculent yeast can be recovered without centrifugation, contributing to saving of equipment cost especially for onsite
bioethanol production1,2).
In general enzyme reactions, an enzyme and its substratediffuse and associate together to initiate the reaction. On theother hand, in the direct fermentation from starch using thearming yeast, a -amylase is immobilized on the yeast andthe starch granule as substrate is huge molecule with a very
low diffusion rate. In the case of flocculent arming yeast, tomake things worse, it is considered that the starch granules
cannot approach to a -amylase molecules existed inside of
the floc, where the apparent specific activity of a -amylase
might decrease drastically. These decrease in an apparent
activity of a -amylase would be critical for a specificethanol production rate, since the rate limiting step in thedirect ethanol fermentation using the arming yeasts was theconversion step from starch to oligosaccharide catalyzed bya -amylase, as described previously1).
Therefore in the present study, to verify the effect ofyeast flocculency on the performance in the direct ethanol
fermentation as predicted above, we firstly conducted thefermentation using flocculent and non-flocculent arming
yeast. Moreover, the microscopic observation was carriedout to examine the surface contact between yeast cells andstarch granules during the fermentation using the arming
yeasts. Finally, to promote the surface contact between thearming yeast and starch granules, we demonstrated the direct fermentation under the packed culture condition, where
the yeast cells and starch granule precipitated together.
Methods
We used flocculent arming yeast (YF207/pGA11/pUFLA) and non-flocculent (YF237/pGA11/pUFSA) con-structed previously1,2). One gram of corn starch (Wako Pure
Chemical Industries, Ltd, Tokyo) and 0.5 ml of the yeast
suspension (400 g wet cell · l1) were taken in a test tube
and filled up to 10 ml with YP medium (yeast extract
10 g · l1, polypeptone 20 g · l1, pH 5.0). The tube wascapped with a backflow valve and shaked vigorously at30°C in the suspended condition. On the other hand, in thepacked condition, the test tube was centrifuged at 1600gfor 5 minutes and 8 ml of the supernatant was kept in another bottle, then the remainder containing the cells andstarch granules was mixed by a vortex and finally the kept
supernatant was overlaid carefully on the mixture. The tube
was capped with the backflow valve and incubated statically at 30°C. With a time interval, the sedimented cells andstarch granules were resuspended and centrifuged to prepare the sample for the ethanol quantification. The ethanol
concentration of the supernatant was analyzed by a gas
chromatography.
Results
To investigate the effect of yeast flocculency on the performance in ethanol fermentation from starch, we conducted the fermentation using flocculent and non-flocculent
arming yeast. It was found that the specific ethanol production rate (r ) for the flocculent arming yeast was 0.04 g · g
dry cell1 · h1, which was almost comparable to the r value
for non-flocculent arming yeast (0.06 g · g dry cell1 · h1).
Moreover, the microscopic observation proved that many
starch granules were captured inside the yeast flocs during
the fermentation using the flocculent arming yeast. Therefore, it could be said that the captured starch granules contributed to increase in the apparent a -amylase activity, because of an increase in the ratio of a -amylase moleculecontacting with starch granule. In other words, the surface
contact between yeast cells and starch granules seems to be
an important factor that influences the performance of direct ethanol fermentation of raw starch by the arming
yeasts.
Therefore, to promote the surface contact between thearming yeast and starch granules, we conducted the direct
fermentation under the packed culture condition. It wasfound that the larger r value was obtained from the packed
culture condition, compared with the usual suspended culture condition. The r values were 0.09 g · g dry cell1 · h1
and 0.07 g · g dry cell1 · h1 under the packed and suspended culture condition, respectively. It was consideredthat these results were due to more surface contact between

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starch granule and yeast under packed condition, whichleads to the increase in apparent a -amylase activity toward
starch. On the other hand, it could be said that the yeast
cells did not always contact with a starch granule in the suspended condition, even though the amylolytic enzymes displayed on the cell surface have starch binding domains.

concentration of sugars at the initial stage of fermentation,
cost saving for equipment (as the solid state fermentation
requires smaller volume of fermenter tank than the conventional liquid fermentation) and less power consumption for
distillation of ethanol. Our researches for development ofsolid state fermentation process are in progress.

Conclusion

References

In conclusion, we have demonstrated that the specificethanol production rate in the direct ethanol fermentation
could be enhanced by increasing the surface contact between the starch granules and the flocculent arming yeasts.
These results provides important information to develop a
more feasible process for direct ethanol fermentation using
the arming yeasts, such as solid state fermentation wherethe arming yeasts and starch existed closely. The advantages of solid state fermentation over conventional liquid
fermentation are; less osmotic pressure caused by the high1) Khaw T. S., Katakura Y., Koh J., Kondo A., Ueda M., and Shioya S.
Evaluation of performance of different surface-engineered yeast
strains for direct ethanol production from raw starch. Appl Microbiol
Biotechnol. 70, 573–579 (2005).
2) Khaw T. S., Katakura Y., Ninomiya K., Bito Y., Katahira S., KondoA., Ueda M., and Shioya S. Effect of flocculation on performance ofarming yeast in direct ethanol fermentation. Appl Microbiol Biotechnol. 73, 60–66 (2006).
3) Khaw T. S., Katakura Y., Ninomiya K., Kondo A., Ueda M., and Shioya S. Enhancement of ethanol production by promoting surface
contact between starch granules and arming yeast in direct ethanol
fermentation. J. Biosci. Bioeng. (2007) in press.