Optimization of clear liquid fermentation condition for ethanol production from Canna edulis Kerl

doi:10.4236/ns.2010.22019

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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; [email protected]

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()



＝

％

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-

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

18.2

18.4

18.6

18.8

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.

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.

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

17.5

18.5

19.5

20.5

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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