2.4. Conducting experiments
This study was conducted in three phases: (1) a control fermentation
of soybean molasses diluted with S. cerevisiae inoculum with
no added enzyme, (2) enzymatic hydrolysis of SM followed by fermentation
with S. cerevisiae and (3) simultaneous SM enzymatic
hydrolysis and fermentation. For all of the experiments, the following
parameters were measured: the total sugars at the beginning
of fermentation (TS0), the residual sugar at the end of fermentation
(RS) and the amount of ethanol at the end of alcoholic fermentation.
The Y1 and Y2 yields were also determined for each experiment.
Preliminary experiments to determine the concentration of SM
that would provide the best yield (Y2), higher ethanol productivity
(P) and lowest concentration of residual sugar (RS) were performed
under conditions of natural soybean molasses fermentation with no
enzymatic hydrolysis. The kinetic profiles of substrate consumption
and ethanol production, the amount of cells and a central composite
design (CCD) that evaluated the influence of the concentration of
SM and inoculum in the responses mentioned above were all determined.
The CCD in the concentration of SM ranged from 520.0 g/L to
180.0 g/L and the concentration of inoculum ranged from 18.0 g/L
to 52.0 g/L. An alpha orthogonality of 1.147 was used in planning.
After running the control experiments, we examined the fermentation
of SM that had been hydrolyzed by -galactosidase.
Preliminary experiments to determine the concentration of soybean
molasses to be enzymatically hydrolyzed were performed to
provide better ethanol yields and residual sugar concentrations.
The kinetic profiles of alcoholic fermentation in relation to substrate
consumption, the ethanol production and a number of cell
and experimental design parameters were also determined. The
CCD was used to evaluate the influence of the enzyme concentration
and the temperature of fermentation on the parameters of
interest (Y2, P and RS). The enzymatic hydrolysis was performed at
pH 4.5 and 55 ◦C with 250 g/L of soybean molasses, as determined
by the preliminary experiments. After 10 h of enzymatic hydrolysis,
the samples were inoculated in the same fermentation reactor with
a 35 g/L initial concentration of inoculum at pH 4.5 and a temperature
of 35
±
1 ◦C. The amount of enzyme used (X1) in the CCD ranged
from 0.021 to 0.079% based on the weight of the substrate, and temperature
(X2) ranged from 49.3 to 60.7 ◦C. An alpha orthogonality
of 1.147 was used in planning. The ranges of enzyme concentration
and temperature adopted in the CCD were selected according to
information from the manufacturer of the enzyme.
Next, we examined the simultaneous enzymatic hydrolysis and
fermentation of soybean molasses (SHF of SM). Preliminary experiments
were performed to determine the optimal concentration of
SM, the kinetic profiles of fermentation and a CCD to assess the
influence of the enzyme concentration, amount of inoculum and
temperature in Y1 and RS. These experiments were also conducted
in a batch reactor with the initial pH adjusted to 4.5 and the concentration
of soybean molasses determined by the test concentration
of hydrolyzed soybean molasses. The amount of enzyme used (X1)
ranged from 0.009 to 0.091% based on the weight of the substrate,
the inoculum concentration (X2) ranged from 14.7 to 55.3 g/L, and
the temperature (X3) ranged from 25.5 to 44.5 ◦C. The alpha orthogonality
used in CCD was 1.353.
The actual factor levels corresponding to coded factor levels are
shown in Table 2.
2.4. Conducting experiments
This study was conducted in three phases: (1) a control fermentation
of soybean molasses diluted with S. cerevisiae inoculum with
no added enzyme, (2) enzymatic hydrolysis of SM followed by fermentation
with S. cerevisiae and (3) simultaneous SM enzymatic
hydrolysis and fermentation. For all of the experiments, the following
parameters were measured: the total sugars at the beginning
of fermentation (TS0), the residual sugar at the end of fermentation
(RS) and the amount of ethanol at the end of alcoholic fermentation.
The Y1 and Y2 yields were also determined for each experiment.
Preliminary experiments to determine the concentration of SM
that would provide the best yield (Y2), higher ethanol productivity
(P) and lowest concentration of residual sugar (RS) were performed
under conditions of natural soybean molasses fermentation with no
enzymatic hydrolysis. The kinetic profiles of substrate consumption
and ethanol production, the amount of cells and a central composite
design (CCD) that evaluated the influence of the concentration of
SM and inoculum in the responses mentioned above were all determined.
The CCD in the concentration of SM ranged from 520.0 g/L to
180.0 g/L and the concentration of inoculum ranged from 18.0 g/L
to 52.0 g/L. An alpha orthogonality of 1.147 was used in planning.
After running the control experiments, we examined the fermentation
of SM that had been hydrolyzed by -galactosidase.
Preliminary experiments to determine the concentration of soybean
molasses to be enzymatically hydrolyzed were performed to
provide better ethanol yields and residual sugar concentrations.
The kinetic profiles of alcoholic fermentation in relation to substrate
consumption, the ethanol production and a number of cell
and experimental design parameters were also determined. The
CCD was used to evaluate the influence of the enzyme concentration
and the temperature of fermentation on the parameters of
interest (Y2, P and RS). The enzymatic hydrolysis was performed at
pH 4.5 and 55 ◦C with 250 g/L of soybean molasses, as determined
by the preliminary experiments. After 10 h of enzymatic hydrolysis,
the samples were inoculated in the same fermentation reactor with
a 35 g/L initial concentration of inoculum at pH 4.5 and a temperature
of 35
±
1 ◦C. The amount of enzyme used (X1) in the CCD ranged
from 0.021 to 0.079% based on the weight of the substrate, and temperature
(X2) ranged from 49.3 to 60.7 ◦C. An alpha orthogonality
of 1.147 was used in planning. The ranges of enzyme concentration
and temperature adopted in the CCD were selected according to
information from the manufacturer of the enzyme.
Next, we examined the simultaneous enzymatic hydrolysis and
fermentation of soybean molasses (SHF of SM). Preliminary experiments
were performed to determine the optimal concentration of
SM, the kinetic profiles of fermentation and a CCD to assess the
influence of the enzyme concentration, amount of inoculum and
temperature in Y1 and RS. These experiments were also conducted
in a batch reactor with the initial pH adjusted to 4.5 and the concentration
of soybean molasses determined by the test concentration
of hydrolyzed soybean molasses. The amount of enzyme used (X1)
ranged from 0.009 to 0.091% based on the weight of the substrate,
the inoculum concentration (X2) ranged from 14.7 to 55.3 g/L, and
the temperature (X3) ranged from 25.5 to 44.5 ◦C. The alpha orthogonality
used in CCD was 1.353.
The actual factor levels corresponding to coded factor levels are
shown in Table 2.
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