Production of Alcohol From Starch by Direct Fermentation: Mita Banerjee, Sipra Debnath, and S. K. Majumdar
Production of Alcohol From Starch by Direct Fermentation: Mita Banerjee, Sipra Debnath, and S. K. Majumdar
Fermentation
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.
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
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
Starch % Alcohol
(%) (w/v) % Conversion
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