Biochemical path ways and products of ethanol fermentation

1. Alcohol Fermentation of Corn Starch
Digested by Chalara paradoxa Amylase
without Cooking*
Katsuhiko Mikuni, Mitsuru Monma and Keiji Kainumat
National Food Research Institute,
Ministry of Agriculture, Forestry and Fisheries 2-1-2,
Kannondai, Yatabe, Tsukuba, lbaraki 305 Japan
Accepted for publication April 3, 1986
Alcohol fermentation of corn starch without cooking was
performed by using Chalara paradoxa glucoamylase
preparation, which had stronger raw starch digesting ac-
tivity than those of the conventionally known glucoam-
ylases. A raw corn starch-enzyme-yeast mixture was
fermented optimally at pH 5.0 and 30°C for five days and
produced ethanol. The yields of ethanol were between
63.5 and 86.8% of the theoretical value by baker's yeast
(Saccharornycescerevisiae),and between 81.I and 92.1%
of the theoretical value by sake yeast (Saccharornyces
sake).
INTRODUCTION
Recently, alcohol fermentation without cooking has
received much attention as a way of saving the cost of
energy consumption by cooking. In conventional al-
cohol fermentation from starch materials, precooking
is necessary for liquefaction and saccharification of the
raw materials. This precooking requires a large amount
of heat energy, ca. 3040% of all energy spent for
alcohol production.
Ueda and co-worker~'-~reported that ethanol was
produced from raw starch in a single-step process by
using Aspergillus niger and Aspergillus awamori glu-
coamylase preparation and Rhizopus koji. Matsumoto
and co-workers4 reported that ethanol was produced
from maize heated at low temperature in an industrial
scale by using Rhizopus koji.
Kainuma et al. reported the
discovery of a new raw starch digesting amylase from
Chalara paradoxa, the studies of the optimum culture
conditions, and purification and characterizationof the
amylase. In the present article, we report the effec-
In previous
* Studies on the novel raw starch-digesting amylase were obtained
from Chalara paradoxa (part 6).
i To whom all correspondences should be addressed, at the Na-
tional Food Research Institute 2-1-2, Kannondai, Yatabe, Tsukuba
Ibaraki, 305 Japan.
tiveness of alcohol fermentationof corn starch without
cooking by using a Chalara paradoxa amylase prep-
aration.
MATERIALS AND METHODS
Materials
Corn starch was donated by Nihon Shokuhin Kako
Co. (Tokyo, Japan). Amylase preparation of Chalara
paradoxa was prepared as by Ishigami and co-workers6
by a large-scale tank at the Meiji Seika Co. (Tokyo,
Japan). The yeast strains used were compressed bak-
er's yeast (Saccharomyces cerevisiae) produced by
the Oriental Yeast Industry Co. (Tokyo, Japan). Sake
yeast (Saccharomyces sake IF0 2164) was cultured in
this laboratory.
EnzymeAssay
Raw starch digesting activity was assayed by a method
previously reported with slight modification. A reac-
tion mixture consisting of 10 mg corn starch, 0.4 mL
0.1M acetate buffer solution (pH 5.0),0.5 mL distilled
water, 0.1 mL suitably diluted enzyme solution was
incubated for 30 min at 40°C with shaking. The amount
of glucose liberated was determined by the Somo-
gyi-Nelson method8 with glucose as the standard. As
we know, the reaction product of the enzyme on raw
starch is only glucose.s
Gelatinized starch-saccharifying activity was as-
sayed using substrate solution consisting of 0.25 mL
2% soluble starch (Merck Co., Darmstadt, Germany)
solution and 0.25 mL 0.1M acetate buffer solution (pH
5.0) and 0.5 mL suitably diluted enzyme solution. The
amount of reducing sugars released by enzyme action
for 30 min at 40°C was determined by the Somogyi-
Biotechnology and Bioengineering, Vol. XXIX, Pp. 729-732 (1987)
0 1987 John Wiley & Sons, Inc. CCC 0006-3592/87/060729-04$04.00

2. Nelson methods with glucose as the standard. One unit
of the enzyme is defined as the amount of the enzyme
which produces 1 pmol glucose/min under the condi-
tions mentioned above.
The a-amylase activity was determined by the blue
value m e t h ~ d . ~The unit of activity for a-amylase is
defined as a 1% decrease of blue value per minute
based on the initial blue value at 660 nm and 40°C of
1% soluble starch.
Analytical Methods
For the determination of the total sugar, the phenol-
sulfuric acid methodlo was employed with glucose as
the standard.
The protein concentration was measured by modi-
fied Hartree method," with bovine serum albumin as
the standard.
Alcohol Fermentation and Assay
The basal composition of fermentation broth was
composed of 10 g raw corn starch, 625 units (as gel-
atinized starch-saccharifying activity) of Chalara par-
adoxa amylase preparation, and 1.O g compressed bak-
er's yeast, mixed in a 100-mL-Meissel's fermentation
vessel. The broth was made up to 50mL with tap water
adjusted to pH 5.0 with 2M phosphoric acid and 0.2M
NaOH.
The Meissel's fermentation vessel was incubated at
30°C with occasional shaking and weighed every 24 h.
At the end of the fermentation, the alcohol content of
broth was determined by gas liquid chromatography
after centrifugation and filtration.
Preparation of Yeast Cells for Fermentation
Sake yeast (Saccharomycessake I F 0 2164) was cul-
tured in 500-mL Sakaguchi flasks containing 100 mL
YPG medium (5.0 g/L yeast extract, 10.0g/L peptone,
40.0 g/L glucose, 5.0g/L KH2P04,and 2.0 g/L MgS04)
on a rotary shaker (110rpm) at 30°Cfor 24 h. The cells
were collected by centrifugation and inoculated into
the fermentation medium to give a cell population of
4.2 x 10VmL.
Table I. Composition of Chalara paradoxu amylase preparation.
Total sugar content 564.7 mg/g
Reducing sugar content 62.4 mp/g
Protein content 250.4 mg/g
Glucoamylase
Raw starch-digesting activity 918.5 IU/g
a-Amylase activity 1420.5 U/g
Gelatinized starch-saccharifying activity 2244.1 IUlg
In.-
260
21 40
20
d
f0
I
'0 20 40 60 80 100
Incubation Time (hr)
Figure 1. Effects of concentration of substrate and condition of
shaking on starch hydrolysis. Amylase (1795 units), 10 mL 0.1M
acetate buffer (pH 5.0), 90 mL distilled water, and a few drops of
toluene were mixed in a 500-mL Erlenmeyer flask and incubated at
40°C: (0)10 g with shaking 140 rpm, (0)10 g with shaking occa-
sionally, (H) 30 g with shaking 140 rpm, (0)30 g with shaking
occasionally, (A)50 g with shaking 140 rpm, and (A)50 g with
shaking occasionally.
RESULTS AND DISCUSSION
Table I shows the composition of crude Chalara
paradoxa amylase powder. The raw starch digestion
activity of the enzyme is considerably high compared
with the conventional glucoamylase. The ratio of the
activity for raw starch to the activity for gelatinized
starch was 40.9%. This value is much higher than the
value (26.0%) previously reported.6
Saccharification Conditions
Figure 1 shows the effects of concentration of sub-
strate and condition of shaking on starch hydrolysis.
The initial velocity of hydrolysis with 140 rpm shaking
was faster than that achieved with only occasional
shaking. However, the final degreesof hydrolysis were
close with both methods. In the case of substrate con-
taining 30 and 50 g, the reaction progressed to levels
of 89.5 and 63.0%,respectively, by removing formed
glucose by dialysis. It was assumed that the reaction
was inhibited by the end-product inhibition of glucose.
60
a
f 40._c
-'0 10 20 30 40
Initid Conc. of GI(%)
Figure 2. Effects of glucose concentration on raw starch digesti-
bility of Chalaraparadoxu amylase preparation. Symbol G ,indicates
glucose.
730 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 29, APRIL 1987

3. - l o o p = q80 5.0I
0-0
EtOH Conc. (VOl X)
Figure 3. Effects of ethanol concentration on raw starch digesti-
bility of Chalara paradoxa amylase preparation: (0)0.25 units and
(0)1.25 units.
To verify this assumption, we tested the effect of initial
concentrationof glucoseon raw starch saccharification
by the enzyme shown in Figure 2. We found that the
relative activities were linearly decreased by the in-
creased concentration of glucose, up to 20%.
Effects of Ethanol on Raw Starch Digestion
We considered that alcohol fermentation in a single-
step process was prevented by end-product inhibition.
We tested the effect of ethanol concentration on raw
starch saccharification, and the results are shown in
Figure 3. When 1.25 units of enzyme were used, the
relative activities were more than 90% at &lo% ethanol
concentration. When 0.25 units of the enzyme were
used, the relative activity of the enzyme was nearly
80% at 10% ethanol concentration. Figure 3 suggests
that the desirable concentration of starch is ca. 20%,
since the enzyme was inhibited by higher concentra-
tion than 10% ethanol. Ethanol concentration was af-
fected not on starch but on enzyme, as reported by
Kunisaki and Matsumoto.'2
Incubation Time (day)
Figure 5. Relationship between enzyme concentration and alcohol
yield (raw corn starch equals 8.6 9): (0)250 units, (0)625 units,
and (0)1250 units.
at three different temperatures: 30, 35, and 40°C. As
shown in Figure 4, the incubation at 30°Cgave the best
yield of alcohol in five days.
Effects of Amylase Quantity on Ethanol
Formation
The effects of various quantities of the ChaIaru par-
adoxa amylase preparation on alcohol fermentation at
30°C were tested by varying only the quantity of the
enzyme. The results are shown in Figure 5. When 250
units of the enzyme were used, the yield of ethanol
was only 63.2% of the theoretical yield of ethanol.
When 625 or 1250 units were added, yields were ca.
85%.
Effects of the Amount and Strains of Yeasts on
Alcohol Fermentation.
The effects of yeast concentration on the yield of
alcohol were also examined and the results are shown
in Figure 6. We did not observe significant effects of
the concentration of the yeast among the conditions
Alcohol Fermentationat Various Temperatures
Alcohol fermentation was carried out by the pro-
ceduredescribed in the Materials and Methods section,
Incubation Time (day)
Figure 4.
different temperatures: (0)30"C, (0)35"C, and (0)40°C.
Alcohol fermentation of raw corn starch (8.6 g) at three
0
0 1.0 2.0 3.0
Yeast (g)
0 0.6 1.2 l.8
Number of cells (xlO*/rnl)
I 4
Figure 6. Effects of yeast concentration on the alcohol fermenta-
tion (raw corn starch equals 6.5 g):(0)alcohol yield and (0)alcohol
concentration. "Yield" shows the yield of ethanol to the theoretical
value of ethanol fermentation.
MIKUNI, MONMA, AND KAINUMA: ALCOHOL FERMENTATION FROM RAW STARCH 731

4. Table 11. Alcohol fermentation and yield from raw corn starch without cooking by using Chalara paradoxa
amylase preparation.
Total starch
(d50 mL)
Before After COzformed" EtOH formed EtOH yieldb
Strain fermentation fermentation (€9 (g) (%)
Baker's yeast 6.7 0.1 3.4 3.3 86.8
Baker's yeast 8.8 0.1 4.1 4.2 84.0
Baker's yeast 13.1 3.1 4.8 4.7 63.5
Sake yeast 6.7 0.1 3.2 3.5 92.1
Sake yeast 8.8 0.1 4.1 4.4 88.0
Sake yeast 13.1 0.5 6.0 6.0 81.1
Fermentation mixtures were incubated for five days at 30°C with shaking occasionally.
a The CO, is the weight decrease by CO, gas formation.
The yield is the percentage of observed EtOH to theoretical EtOH.
employed in this study. When 1 g compressed baker's
yeast was added, the yield of alcohol was slightlybetter
than the other batches. The purpose of the alcohol
fermentation in this study was fermentation without
sterilization of the substrate. It required that the initial
concentration of yeast was high enough to prevent the
infection of other microorganisms. The initial concen-
tration of yeast was desirable at 0.6 x lo9cells/mL.
Table I1 shows the results of the comparison of yeast
strains. Sake yeast was better than baker's yeast in
regard to both alcohol concentrations and yield. When
sake yeast was added, the best yield of alcohol was
92.1%; the highest alcohol concentration was 6.0 g in
50 mL.
Ueda et al.l 3 reported alcohol yield from raw cassava
starch was 90% using Aspergillus niger. Park and
Rivera14reported alcohol yield from raw corn starch
of 90% using Aspergillus awamori. The data which we
obtained is on the same level of theirs, but the quantity
of amylase we used is different from theirs. We cannot
strictly compare our results with these other research-
ers because of the differences in the method of the
measurement of amylase activity.
In a laboratory scale, it is obvious that alcohol fer-
mentation of raw starch was carried out efficiently.The
results of large-scale alcohol fermentation will be de-
scribed in later reports.
CONCLUSIONS
Direct ethanol fermentation from corn starch gran-
ules without sterilization was examined using the com-
bination of novel raw starch-digesting amylase ob-
tained from Chalara paradoxa and Saccharomyces sp.
We found it feasible to use the new enzyme for raw
starch digestion process for one-step ethanol fermen-
tation from starch granules. Although there are several
problems to be solved, we obtained an ethanol yield
close to 90% of the theoretical yield of fermentation.
The authors thank Dr. Shoichi Kobayashi, Dr. Toru Hayashi,
and Mr. Akihiro Hino of National Food Research Institute
for their valuable discussions in this work. We also thank Dr.
Hidemasa Hidaka and his research group at Meiji Seika Co.
for large-scale cultivation of the microorganisms. This work
was supported by agrantfrom the Biomass Conversion Project,
Ministry of Agriculture, Forestry and Fisheries (BCP-86-V-
1-8).Japan.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
S. Ueda and Y.Koba, J. Ferment. Techno!., 58, 237 (1980).
H. Matsuoka, Y. Koba, and S. Ueda, J. Ferment. Techno!.,60,
599 (1982).
Y. Fujio, P. Suyanadona, P. Attasampunna, and S. Ueda, Bio-
technol. Bioeng., 26, 315 (1984).
U. Matsumoto, 0. Fukushi, and 0. Fukuda, Nippon NBgei-
kagaku Kaishi,59, 271 (1985).
K. Kainuma, H. Ishigami, and S. Kobayashi,J. Jpn. Soc.Starch
Sci., 32, 136 (1985).
H. Ishigami, H. Hashimoto, and K. Kainuma, J.Jpn. Soc. Srarch
Sci., 32, 189 (1985).
H. Ishigami, H. Hashimoto, and K. Kainuma, J.Jpn. Soc. Srarch
Sci., 32, 197 (1985).
N. Nelson, J . Bio!. Chem., 153, 375 (1944).
H. Fuwa, J. Biochem., 41, 583 (1954).
M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.
Smith, Anal. Chem., 28, 350 (1956).
E. F. Hartree, Anal. Biochem., 48, 422 (1972).
S. Kunisaki and N. Matsumoto, J. Jpn. SOC.Starch Sci.,32,
152 (1985).
S. Ueda, C. T. Zenin, D. A. Monterio, and Y. K. Park, Bio-
rechnol. Bioeng., 23, 291 (1981).
Y. K. Park and B. C. Rivera, Biotechnol.Bioeng., 24,495 (1982).
732 BIOTECHNOLOGYAND BIOENGINEERING, VOL. 29, APRIL 1987