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Production of Alcohol From Starch by Direct Fermentation: Mita Banerjee, Sipra Debnath, and S. K. Majumdar

1) Saccharomyces diastaticus has the ability to produce the enzyme glucoamylase which allows it to break down and ferment starch. 2) Experiments were conducted to determine optimal conditions for growth of S. diastaticus and production of glucoamylase including starch concentration, pH, incubation time. 3) Under these optimized conditions, direct alcohol production from starch by S. diastaticus was examined in experiments using a fermentor.

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0% found this document useful (0 votes)
76 views4 pages

Production of Alcohol From Starch by Direct Fermentation: Mita Banerjee, Sipra Debnath, and S. K. Majumdar

1) Saccharomyces diastaticus has the ability to produce the enzyme glucoamylase which allows it to break down and ferment starch. 2) Experiments were conducted to determine optimal conditions for growth of S. diastaticus and production of glucoamylase including starch concentration, pH, incubation time. 3) Under these optimized conditions, direct alcohol production from starch by S. diastaticus was examined in experiments using a fermentor.

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Ephrem Kasse
Copyright
© © All Rights Reserved
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Production of Alcohol from Starch by Direct

Fermentation

Mita Banerjee, Sipra Debnath, and S. K. Majumdar


Department of Food Technology and Biochemical Engineering,
Jadavpur University, Calcutta 700 032, India
Accepted for publication September 18, 1987

Recent developments in both price and availability of (0.5%),yeast extract (0.3%), malt extract (0.3%), and agar
crude oil have motivated a worldwide search for cheap alter- *
(2%) at a pH of 4.5. After growth at 28 2°C for 48 h on
natives. Starch is an alternate source of energy because it is agar slants the culture was stored at 4°C.
both renewable and available throughout the globe in large A liquid medium composed of soluble starch (2%; ob-
quantities. There are a variety of products that can be ob- tained from Merck Co., Darmstadt, Germany), (NH,),SO,
tained from starch biomass via hydrolysis. Alcohol is one of (0.2%), KH,PO, (0.1%), MgSO, * 7H,O (0.05%), and
the largest volume of products that can be produced from yeast extract (0.1%) at pH 5.0 was used for growth of
biomass. Recently, there has been active research aimed at S . diastaticus and synthesis of glucoamylase. The yeast
attaining an increase in ethanol productivity by fermenta- was grown using 2% (v/v) inoculum in 50 mL of the sterile
tion or immobilized cell techniques. medium contained in a 250-mL Erlenmeyer flask, aerobi-
Starchy raw materials for production of alcohol need pre- cally, in a rotary shaker (120 rpm) at 28°C for different time
treatment, viz., liquefaction and saccharification. Common periods. The cells were separated by centrifugation at 5000
alcohol-producing yeasts Saccharomyces cerevisiae, S. rpm for 10 min, and washed with 20 mL of saline solution
carlsbergensis, S . sake, or bacterium Zymomonas mobilis two times, and then used for enzyme assay and determina-
lack the starch-splitting enzymes or amylases. So an organ- tion of cell growth. The cells were dried at 65 t 5°C for
ism possessing a fermentative power comparable to that of 24 h and the dry weight of cells were taken as a measure of
S. cerevisiae traditionally used and a strong amylolytic ac- cell concentration. The effect of pH and time of incubation
tivity would simplify the process by producing alcohol in a on cell growth and glucoamylase production, and the in-
single step. Many yeasts are known to grow on starch and hibitory effect of ethanol on cell growth of S . diastaticus,
ferment it. ’ As a result of extensive screening of yeasts, we were studied next.
have isolated a yeast from natural sources and identified it Experiments on alcohol fermentation were performed us-
as Saccharomyces diastaticus. The culture differs from the ing 10% (w/v) soluble starch. The starch slurry was pre-
other Saccharomyces species in regard to its pattern of pared as follows: 10 g starch was solubilized in 100 cc
starch assimilation and fermentation. S. diustiaticus has glu- boiled 0.02 N H,SO, (pH 2.5) and then sterilized by auto-
coamylase activity and has the ability to assimilate and fer- claving for 15 min at 121°C. It was then cooled to room
ment starch. This yeast has a short generation time and temperature and the pH adjusted to 6.0 by using 4 N NaOH
grows well in synthetic minimal media with a wide variety aseptically. Other components of the medium were the same
of substrates including starch. The alcohol-producing capa- used for cell growth and glucoamylase production. The fer-
bilities of S. diastaticus has not been studied extensively, al- mentation was carried out in a 250 mL Bellco Fermentor
though there are a few r e p ~ r t s ”on
~ the production of alcohol containing 150 mL of the medium set anaerobically at 37°C
from glucose and dextrin by S. diastuticus. for 72 h.
The work reported here is on the investigation of certain The amount of alcohol produced in cell-free fermentation
parameters related to the growth of S. diastaticus, synthe- broth was determined by alcohol dehydrogena~e~ (obtained
sis of glucoamylase, and direct production of alcohol from from Sigma Chemical Co., St. Louis, MO) according to the
starch. following equation:

Alcohol +N Alcohol
A D ’ w e Acetaldehyde + NADH + H’
MATERIALS AND METHODS
The concentration of NADH as measured spectrophotomet-
rically at 340 nm is proportional to the amount of alcohol.
Cultures and Substrate Preparation
Total reducing sugars were determined by the dinitrosalicylic
The culture of S.diastaticus was maintained in the labo- acid (DNS) m e t h ~ dThe
. ~ residual starch was hydrolyzed to
ratory on agar slants containing glucose (1%), peptone glucose and the glucose then determined by the DNS method.

Biotechnology and Bioengineering, Vol. 32,Pp. 831-834 (1988)


0 1988 John Wiley 81 Sons, Inc. CCC 0006-35921881060831 -04$04.00
Glucoamylase activity was assayed after incubating the
reaction mixture at 37°C for 5 min. The reaction mixture
consisted of 1.0 mL of 2% soluble starch solution in 0.2M
sodium acetate buffer, pH 4.8, and 1.O mL of the cell suspen-
sion, which acted as an enzyme source. The reaction was
stopped by cooling on ice and then boiling for 10 min. The
reaction mixture was then centrifuged at 5000 rpm for 10 min
to remove the insoluble materials. The released glucose was
then estimated by the glucose oxidase and peroxidase (ob-
CO.5
tained from Sigma Chemical Co., St. Louis, MO) method.6
The activity of enzyme is expressed as micromoles of glu-
cose released per minute per milligram dry cell.

RESULTS AND DISCUSSION

Growth of S. diastaticus and Glucoamylase


Production
The yeast cells were grown in different concentrations of
starch (1.0-4.0%) at 28°C for 48 h. The cell growth was
maximum in the medium containing 2% starch (Figure 1)
and thereafter the yield of cell biomass decreased with in-
creasing concentration of starch. The effect of time of in-
cubation on cell growth and glucoamylase production by
S . diustuticus is presented in Figure 2. Cell concentration 1 1 1 1 1 1 I
24 4s 72 96 I20
is maximum at 72 h but maximum enzyme activity is ob-
served at 48 h of incubation. The influence of initial pH (af- TlYL P E R I O D (HK)

ter sterilization) in the starch medium on the growth of the Figure 2. Optimum time of incubation for growth and glucoamylase ac-
yeast and glucoamylase production is shown in Figure 3. tivity of S. diasfaticus.
The fermentation was carried out for 48 h at 28°C. A maxi-

a-60
2
-
90 50
5JI
i5u

--
- 1-0 2-0
STARCW(*/*)
3-0 4.0

Figure 1. Optimum cell growth in different concentration of starch.


Figure 3. Influence of pH on growth and glucoamylase activity of
S.diastaticus.

832 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32, SEPTEMBER 1988


mum growth is observed at pH 5.0 but glucoamylase activity alcohol fermentation in the medium containing 10% starch,
of the cell is maximum at pH 5.5. About 60% of an initial a higher amount of inoculum (2.0 g wet wt) was added to
2% starch concentration has been utilized during growth at 150 mL of the medium. For the development of inoculum
pH 5.5 of the medium. Figure 4 shows the effect of ethanol the cells were grown in the medium containing 2% starch
on cell growth when added initially. It is evident that ethanol under fermentation conditions favorable for cell growth and
inhibits yeast g r ~ w t hGrowth
.~ of S. diastaticus is inhibited glucoamylase synthesis.
here up to 14.8%when ethanol concentration in the medium The effect of initial pH on final ethanol concentrations and
is 5%. In the presence of 7.5 and 10% ethanol, cell growth final cell weight is shown in Figure 5 . The maximum alco-
is hindered by 48 and 72%, respectively. hol level was produced in the range of pH 6.0-6.5. Next,
fermentation was carried out at various temperatures for
different time periods (Figure 6). The maximum alcohol
Effect of pH Time and Temperature on
Production of Alcohol by S. diastaticus fermentation rate is observed at 37°C. At lower or higher
temperatures, the fermentation rate is much lower. The op-
The previous work has shown that S. diastaticus produces timum time period is 72 h, about 86% of an initial 10%.
the glucoamylase necessary to partially convert starch to fer- Starch is utilized in 72 h fermentation at 37°C.
mentable sugars. As shown in Table I, S. diastaticus grows Table I1 shows the effect of the starch concentration on
sufficiently in starch-salt-yeast extract medium containing the production of alcohol. Different initial concentrations
2% starch and this growth in starch medium is necessary for of starch in the range of 5.0-17.5% were used. The maxi-
the induction of the enzyme glucoamylase. So for studies on mum alcohol yield was found with 12.5% starch.

CONCLUSIONS
Saccharomyces diastaticus, possessing both amylolytic
and fermentative power, has been studied for cell growth,

?
t glucoamylase synthesis, and production of alcohol. The
yeast can grow well in a starch-salt-yeast extract medium
containing 2% starch. Glucoamylase activity is maximum
at 48 h of incubation when incubated aerobically. The opti-

YT
2
L
4-0

\
o ! \A\

I.0 LL
-
0 Id 2-5

%
5-0

LTHANOL
7.3 10
(V/V)
I8

Figure 4. Effect of ethanol on growth of S. diastaticus.

Table I. Effect of starch and glucose on growth and glucoamylase ac-


tivity of S. diastaticus.

Carbohydrates Enzyme activity


glucose Starch Cell growth ( p moles of glucose,
(S) (%) (g dry cell/L) min-' mg-' dry cell)
~ _ _ _ _ ~

2.0 - 3.930 0.108


1 .o 1.0 4.208 0.168
2.0 4.112 0.33
Figure 5. Influence of pH on the production of alcohol by S. diastaticus.

COMMUNICATIONS TO THE EDITOR a33


Table 11. Effect of the starch concentration on the production of alcohol
by S. diastaticus.

Starch % Alcohol
(%) (w/v) % Conversion

5.0 2.3 81.27


10.0 4.48 79.15
12.5 5.68 80.3
15.0 4.7 55.35
17.5 4.56 46.00

optimum production of alcohol takes place in 72 h at 37°C.


With 12.5% starch, alcohol yield is maximum. Alcohol is
found to have an inhibitory effect on cell growth.
The financial support of the University Grants Commission of India
is gratefully acknowledged.

References
1. J. Lodder, Ed., The Yeasts. A Taxonomic Study, (N. H. Publishing
Company, 1970).
2. Z . Duvnjak, N. Kosaric, and S . Kliza, Biotechnol. Eioeng., 24, 2297

44 48
TIME
72
( HR) - 96 (PO

Figure 6 . Optimum time and temperature for the production of alcohol


(1982).
3. Z . Duvnjak and N . Kosaric, in Advances in Eiotechnology, vol. 2,
Murray Moo-Young, Ed. (Pergamon Press, New York, 1981), pp. 175.
4. E. Bernt and I. Gutman, in Methods of Enzymatic Andysis, vol. 3,
H. U. Bergmeyer, Ed. (Academic Press, New York, 1974), pp. 1499.
by S. diastaticus.
5. A. Nason, Ed., Analytical Biochemistry, vol. 1, 2nd ed. (Academic
Press, New York, 1960), pp. 127.
6. H. U. Bergmeyer and E. Bernt, in Methods of Enzymatic Analysis,
vol. 3, H.U. Bergmeyer, Ed. (Academic Press, New York, 1974).
mum initial pH of the medium for growth of S. diustuticus pp. 1205.
is 5.0, that for glucoamylase synthesis is 5.5, while alcohol 7. B. M. Nakmanovich and V. L . Yarovenko, Mikrobiologiya, 1, 70
production is maximum in the pH range of 6.0-6.5. The (1974).

834 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32, SEPTEMBER 1988

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