An optimal medium (300 g·L

-1

initial glucose) comprising 6.3 mmol·L

-1

Mg

2+

5.0 mmol·L

-1

Ca

2+

15.0 g·L

-1

peptone and 21.5 g·L

-1

yeast extract was determined by uniform design to improve very high gravity (VHG) ethanol fermentation

showing over 30% increase in final ethanol (from 13.1% to 17.1%

by volume)

29% decrease in fermentation time (from 84 to 60 h)

80% increase in biomass formation and 26% increase in glucose utilization. Experiments also revealed physiological aspects linked to the fermentation enhancements. Compared to the control

trehalose in the cells grown in optimal fermentation medium increased 17.9-

2.8-

1.9-

1.8- and 1.9-fold at the fermentation time of 12

24

36

48 and 60 h

respectively. Its sharp rise at the early stage of fermentation when there was a considerable osmotic stress suggested that trehalose played an important role in promoting fermentation. Meanwhile

at the identical five fermentation time

the plasma membrane ATPase activity of the cells grown in optimal medium was 2.3

1.8

1.6

1.5 and 1.3 times that of the control

respectively. Their disparities in enzymatic activity became wider when the glucose levels were dramatically changed for ethanol production

suggesting this enzyme also contributed to the fermentation improvements. Thus

medium optimization for VHG ethanol fermentation was found to trigger the increased yeast trehalose accumulation and plasma membrane ATPase act

ivity.