Vol.2, No.2, 115-119 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.22019
Optimization of clear liquid fermentation condition for
ethanol production from Canna edulis Kerl
Ying Shen, Qing-li Tang, Tian-Xiang Wu*
College of Life Sciences,Guizhou University, Guiyang, China;
Received 27 October 2009; revised 24 November 2009, accepted 15 December 2009.
ABSTRACT
In this paper, clear liquid fermentation condition
for ethanol production from Canna edulis Kerl
with orthogonal design was studied. We have
studied five factors that influenced clear liquid
fermentation condition for ethanol production
from Canna edulis Kerl by the single-factor test.
The five factors were α-amylase amount, glu-
coamylase amount, pH, nitrogen and auxiliary
materials amount. Selected the best amounts of
α-amylase, glucoamylase, pH, nitrogen source
and auxiliary materials by means of the singe-
factors test. And then, we used the Orthogonal
test to optimize the fermentation process. The
study showed that the best fermentation condi-
tions were α-amylase amount 7 u/g C.eduli, glu-
coamylase amount 145u/g C. edulis, pH 4.4 and
the amount of urea 0.08%, 1% of rice bran as
auxiliary materials. The rate of alcohol which was
produced by the best fermentation conditions is
21.5% that increased by 1.3% than the rate of
alcohol by the single-factor test.
Keywords: Alcohol; Canna edulis Kerl ;
Clear Liquid Fermentation; Orthogonal Design
1. INTRODUCTION
Canna edulis Ker1 belongs to Canna ceae and Canna L,
in chinese herbs it was called jiang-yu or jiao-ou. At the
first of 20th century, it was planted widely at slope and
hills in Sichuan, Guizhou and Yunan because of its ex-
cellent feature. The content of starch in rhizomes of
Canna edulis Ker1 is very abundant. The content of
starch is about 40%–60% [8-13]. So Canna edulis Ker1
has great development potential in supply of starch. The
planting of Canna edulis Ker1 can increase earning for
montanic people. At the same time it has con-
tinuable-development significance in regulating agricul
tural configuration and developing rural economics. But
the starch of Canna edulis Ker1 is made into noodle
largely only now, so its value is low. The molecular
structure and physical and chemical properties of the
starch of Canna edulis Ker1 were studied largely. Now,
many scholars are investigating other purposes of the
starch of Canna edulis Ker1 . For exemple, the starch of
Canna edulis Kerl has achieved a breakthrough in alco-
holic fermentation. The solid-state fermentation, clear
liquid fermentation of Canna edulis Kerl and the com-
prehensive utilization of fermentation residue have been
researched largely in laboratory [1,7-11]. On this basis, we
have got the best clear liquid fermentation process from
Canna edulis Kerl with the single-factor test. Then we
improve the utilization rate of Canna edulis Kerl raw
after optimizing the technics of clear liquid fermentation
condition for ethanol production from Canna edulis Kerl
[8,12,15,16].
2. MATERIALS AND METHODS
2.1. Experimental Material
Canna edulis Ker1: Collected from Xingyi Guizhou, the
content of Starch is 60.1%, the content of moisture is
14.8%;
Thermal Resistance α-amylase: wuxi xing-da Bio-
Engineering Co., Ltd., enzyme activity: 20000u/mL;
New liquid glucoamylase: wuxi xing-da Bio-
Engineering Co., Ltd., enzyme activity 100000u/Ml.
2.2. Methods
2.2.1. The Preparation of Clear Liquid Culture
Medium
Firstly, mixed the powder of Canna edulis Kerl with
water in a certain proportion, joined in α-amylase, then
heated them to 85~90℃ rapidly, keep them together
30~60min. When the time was over, cooled them to
60℃, adjusted pH 4.2. A certain proportion moderate
glucoamylase was joined into the mixture solution, a few
minites later, the Solid-state glycation Saccharification
dregs and clear liquid can be gained by centrifugal tech-
nology, then put the clear liquid into the autoclave whose

Y. SHEN et al. / Natural Science 2 (2010) 115-119
116
tempreture was 121℃, the clear liquid was taken out
after twenty minutes. When the clear liquid tempreture
fell to 30℃, the yeast which was activated was inocu-
lated into the clear liquid, the fermentation started in the
incubator [2,3].
2.2.2. Determination of Alcohol Volume Fraction
The clear liquid, which was the outcome of Canna
edulis Kerl fermented, was measured 100mL by meas-
uring graduates (100mL), inoculated into a rockered
flask (500ml) which contained 100mL distilled water.
The distillation was collected by volumetric flask
(100mL), determined alcohol level by alcohol meter
[4-6].
2.2.3. Calculation of the Producing Ethanol Rate
of Raw Materials and Starch Utilization [17]
The producing ethanol rate of raw materials
95ethanol yield()100%
amount of raw materials()
kg
kg

＝
％
The starch utilization
the actual liquor rate of starch100
the theoretical liquor rate of starch

＝
％
2.2.4. Determination of Reducing Sugar
Concentration
The distilled water was inoculated into the volumetric
flask which contained separated clear liquid 0.1mL till
25mL. The mixed solution was determinated for reduc-
ing sugar concentration by DNS analytical method [5].
2.2.5. Determination of Mass Concentration of
Total Sugar
The distilled water was inoculated into the volumetric
flask which contained separated clear liquid 0.1mL till
25mL. The mixed solution was determinated for total
sugar concentration by H2SO4-Pheuol analytical method
[5].
3. RESULTS AND ANALYSIS
3.1. Single-Factor Test and Medium
Optimization
Based on former experiments, we have found the best
solid-state fermentation conditions of the ethanol pro-
duction from Canna edulis Kerl, the best conditions
were ratio of material to water 1:2.2, α-amylase amount
5u/g C.edulis, liquefaction time 45 min, glucoamylase
amount 150u/g C.edulis, saccharification time 23.2 min,
pH 5.5, urea 0.1%, fermentation temperature 27.9 ºC,
ADY 0.15%, fermentation time 48h and inert carrier 1%.
On this basis, the paper studied the clear liquid fer-
mentation conditions of the ethanol production from
Canna edulis Kerl. By a large number of experiments,
we found that α-amylase amount, glucoamylase amount,
pH and urea amount have an important influence on the
whole fermentation technology.
So we were proceeding to the in-depth study for these
four factors, these studies as follows:
3.1.1. The Effect of α-amylase Amount on the
Alcohol Fermentation
In order to confirm the appropriate amount of α-amylase
on clear liquid fermentation from Canna edulis Kerl,
different amount of α-amylase were tested on the ethanol
rate of raw materials (Canna edulis Kerl) [7-11,14]. The
results were showed in Figure 1.
The Figure 1 showed that when the α-amylase
amount was 6u/g C.edulis, the ethanol rate of raw mate-
rials (Canna edulis Kerl) was highest. So the conclusion
was α-amylase amount 6u/g was the best condition on
clear liquid fermentation for ethanol production from
Canna edulis Kerl.
3.1.2. The Effect of Glucoamylase Amount on
the Alcohol Fermentation
Glucoamylase played an important role in the clear liq-
uid fermentation for ethanol production from Canna
edulis Kerl, it can influence the cost of production. Dif-
ferent glucoamylase amount were tested on the ethanol
rate of raw materials [7-11].
The different results were revealed in Figure 2.
The Figure 2 described that when the glucoamylase
amount exceeded 150u/g, the ethanol rate of raw materi-
als (Canna edulis Kerl) was steady. In order to cut down
the consumption of glucoamylase, the glucoamylase
amount 150u/g was appropriate on clear liquid fermenta-
tion for ethanol production from Canna edulis Kerl.
3.1.3. The Effect of Different pH Conditions on
Alcohol Fermentation
The pH of Clear Liquid fermented were adjusted to 3.6, 4.2,
4.5, 5.0 and 6.0 (the pH of Canna edulis Kerl Glycosy-
lated liquid was original) [7-11]. The outcomes were dr-
0
5
10
15
20
0510 15
α-amylase amount (u/g)
95% ethnoal yield (%)
Figure 1. The effect of α-amylase amount on
the alcohol fermentation.

Y. SHEN et al. / Natural Science 2 (2010) 115-119
117
18
18.2
18.4
18.6
18.8
19
19.2
19.4
19.6
19.8
100 120 140160 180 200
glucoamylase amount（u/g）
95% ethnoal yield (%)
Figure 2. The effect of glucoamylase amount on the alcohol
fermentation.
13
14
15
16
17
18
19
20
21
3.5 4 4.5 55.5 6 6.5
fermentation pH
95% ethanol yield（%）
Figure 3. Effect of pH on ethanol fermentation.
ew in Figure 3.
The conclusion can be ascertained: the yield of alco-
hol was gradual increasing when pH was 3.5 to 4.2.
When pH was 4.2 to 6.0, the yield of alcohol had a
basically stable situation and the best effect was pH 4.2.
3.1.4. The Effect of Nitrogen on the Alcohol
Fermentation
Under suitable environment and nutritional conditions,
yeast can bear alcohol in high concentration, but the
vigour of yeast was lower when nitrogen was scarce in
culture medium. In this article, the yeast was restrained
because the amount of nitrogen was deficient in glyco-
sylated liquid. For accelerating growth of yeast and en-
hancing the ability of alcohol-resistant yeast, a certain
amount of nitrogen was necessary to be added in the
culture medium. With an eye to the results were various
because of different nitrogen, therefore, urea, ammonium
sulfate, ammonium bicarbonate, peptone and yeast ex-
tract were choiced as the element inspected in this ex-
periment. The amount of nitrogen must be based on
0.1% nitrogen content of urea, the nitrogen content of
urea, ammonium sulfate, ammonium bicarbonate, pep-
tone and yeast extract were 46.7%, 20%, 17.7%, 13.5%,
7% respectively. The outcomes were shown in Figure 4.
It was found that five kinds of nitrogen had great in-
fluence on alcohol fermentation from Canna edulis Kerl
although urea showed the best consequence, maybe it
can protect some enzymes, for example the alcohol de-
hydrogenase, promoted the activity of enzyme.
In order to confirmed the best amount of urea, at the
next experiment, different dosage of urea which were
0.05%, 0.1%, 0.15%, 0.2% and 0.3% were studied on
clear liquid fermentation condition for ethanol produc-
tion from Canna edulis Kerl at the same condition .The
results were revealed in Figure 5.
It was showed that the rate of alcohol reduced gradu-
ally from 0.15% to 0.3% dosage of urea, the rate of al-
cohol rised steadily from 0 to 0.1% dosage of urea. The
best data appeared at 0.1% dosage of urea.
3.1.5. The Effect of Inert Carrier on Alcohol
Fermentation
Fine-Grained material maybe reduce the content of CO2
which could suppress yeast colony, different content of
bran and corn cob were tested respectively on ethanol
Figure 4. The effect of nitrogen on alcohol fermentation.
0
2
4
6
8
10
12
00.05 0.1 0.15 0.20.3
Urea(g/100g CE meal)
Ethanol concentration
(%,v/v)
Figure 5. Effect of urea concentration on ethanol fermentation.

Y. SHEN et al. / Natural Science 2 (2010) 115-119
118
Figure 6. Effect of inert carrier on ethanol fermentation.
fermentation. The content of them were 0, 0.5%, 1%, 2%
respectively. The results were showed in Figure 6.
The diagram indicated that bran and corn cob can
promote alcohol fermentation of Canna edulis Kerl. 1%
bran had the best effect. The same information had been
reported in other documents.
3.2. Orthogonal Test
According to the results of the single-factor test, α-amyl-
ase amount, glucoamylase amount, pH and the amount
of urea were optimizated by orthogonal experiment
[8,12,15].
The α-amylase amount 6u/g C.edulis, glucoamylase
amount 150u/g C.edulis, pH 4.2 and the amount of urea
0.1% were used as intermediate level in the orthogonal
tests. The experimental design and results of analysis
were showed in Table 1, Table 2 and Figure 7 (In the
Table 1 and Table 2, A, B, C and D stand for the
16.5
17
17.5
18
18.5
19
19.5
20
20.5
21
21.5
A1 A2 A3B1 B2 B3C1 C2 C3D1 D2 D3
factor level
Alcohol yield/(g·L
-1
)
Alcohol yield
Figure 7. The trend of factors and indexs in orthogonal ex-
periment.
α-amylase amount, glucoamylase amount, pH and urea
amount respectively. In the Tables 1 and 2 stand for
three different amount levels of the four factors respec-
tively.)
The Table 2 showed that α-amylase amount had a
great influence on clear liquid alcohol fermentation of
Canna edulis Kerl. Based on the results of experiment,
the influence of the other factors on the clear liquid
alcohol fermentation of Canna edulis Kerl were ordered
from larger to little as the amount of urea>glucoα-
mylase amount>pH.
As can be seen from Figure 7, the best combination
was A3B1C3D1, that is to say, the α-amylase amount 7u/g
C.edulis, the glucoamylase amount 145u/g C.edulis, pH
4.4, and the amount of urea 0.08%.
At last, after three parallel experiments which have
been carried out on ethanol fermentation by the optimal
Table 1. Factor and level of orthogonal experiment.
level α-amylase amount (u/g) glucoamylase amount
(u/g) pH the amount of urea(%)
1 5 145 4.0 0.08
2 6 150 4.2 0.1
3 7 155 4.4 0.12
Table 2. Results and analysis of orthogonal experiment.
factor
Test No. A B C D Alcohol yield(%)
1 1 1 1 1 19.8
2 1 2 2 2 17.1
3 1 3 3 3 18.0
4 2 1 2 3 20.1
5 2 2 3 1 2.0
6 2 3 1 2 18.8
7 3 1 3 2 21.3
8 3 2 1 3 20.5
9 3 3 2 1 21.4
mean 1 18.3 20.4 19.7 20.7
mean 2 20.0 19.5 19.5 19.1
mean 3 21.1 19.4 20.1 19.5
pole difference 2.8 1.0 0.6 1.7

Y. SHEN et al. / Natural Science 2 (2010) 115-119
119
technological conditions, we found the production rate
of alcohol were 21.7%, 21.4%, 21.5% respectively, and
the average was 21.5%.
4. CONCLUSIONS
1) Canna edulis Kerl is a non-alimentarn crop. The ad-
vantages of fuel ethanol produced by Canna edulis Kerl
were raising the utilization rate of resources, reducing
the pollution of the environment, lower manufacturing
Cost and higher productivity. The fuel ethanol produced
from Canna edulis Kerl can not only solve the problem
of food security, but also promote the development of
fuel alcohol industry in china.
2) We have known that α-amylase amount, glucoa-
mylase amount, pH, nitrogen and auxiliary materials can
influence clear liquid fermentation for ethanol produc-
tion from Canna edulis Kerl. Then optimizing four fac-
tors by the orthogonal test .The best process conditions
areα-amylase amount 7u/g C.edulis, glucoamylase
amount 145u/g C.edulis, pH 4.4, the amount of urea
0.08% and 1% of rice bran. The production rate of alco-
hol was 21.5% which increased by 1.3% than the pro-
duction rate of alcohol by the single-factor test.
3) We are researching the best conditions of clear liq-
uid fermentation for ethanol production from Canna
edulis Kerl. These investigations laid a good foundation
for reducing the cost of alcohol production, comprehen-
sive utilization of Resources and efficient, saving, unde-
filed technics of alcohol production. So clear liquid fer-
mentation for ethanol Production from Canna edulis
Kerl was an epoch-making event in the realm of indus-
trial alcohol.
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