Investigating the Production
of Alcohol by Rice Wine
Yeast (Saccharomycopsis
fibuligera)
Project Proposal
XXX
XXX
XXX
Hwa Chong Institution (High School)
Mentor: YYY
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Introduction
Rationale
Biofuels like ethanol play a major role in reducing reliance on fossil fuels.
However, ethanol derived from food crops has driven up the prices of food crops. The
development of biofuels should not compete with crops used for food.
Thus, this project will focus on producing ethanol from starchy wastes, which will
reduce reliance on food crops. Hence, instead of using crops for fuel, starch wastes is
actually being recycled. However, fermentation from starchy wastes is not economically
feasible as pure amylase has to be added to break down the starchy wastes into simple
sugars before fermentation can occur. Therefore, the yeast, Saccharomycopsis fibuligera,
is used instead of the yeast commonly used in fermentation, Saccharomyces cerevisiae¸
as Saccharomycopsis fibuligera, produces extracellular amylase, thus saving costs.
Objectives
1. Investigate the production of ethanol from starchy wastes (e.g. rice, noodle and
barley wastewaters)
2. Compare the amount of ethanol produced by Saccharomyces cerevisiae and
Saccharomycopsis fibuligera
Hypotheses
1. Saccharomycopsis fibuligera is able to produce ethanol from starchy wastes.
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2. Saccharomyces cerevisiae is able to produce ethanol from amylase-treated starchy
wastes.
3. Saccharomycopsis fibuligera is able to produce a higher amount of ethanol than
Saccharomyces cerevisiae.
Literature Review
Recently, there has been growing interests and investments in the research on
Saccharomycopsis fibuligera. According to Chi, Liu, Wang, Ju, Zhang (2009), S.
fibuligera has the ability to assimilate glucose, sucrose, cellobiose, trehalose and soluble
starch. S. fibuligera has been considered in the realm of ascomycetous yeast as it is a
good producer of extracellular amylolytic enzymes (Reiser & Gasperik, 1995). These
enzymes, which are associated with the degradation of starch, are excellent and widely
used raw materials in many fermentation processes (Reiser & Gasperik, 1995). On top of
that, S. fibuligera has very high amylolytic activity as compared to the many other
different types of amylolytic yeasts (Saelim, Dissara and H-Kittikun 2007).
The saccharifying properties of S. fibuligera are of interest because of their
various applications: accumulate trehalose from starch, secrete amylases, acid protease
and β-glucosidase used in industrial applications, and the production of ethanol from
starch (Chi et al., 2009). Initially, it was assumed that only one amylase was produced
and that glucose is the predominant hydrolytic end product of the enzyme. However,
Lemmel, Heimsch, Korus (1980) reported that S. fibuligera is actually able to produce an
amylolytic complex that consists of an α-amylase, a glucoamylase, and a maltase. Studies
by Waghorn, TalõÂa, Elba, Liliana (2003) have shown that most starch is being
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hydrolysed by α-amylase. On top of that, several studies (Lemmel et al., 1980; Chi et al.,
2009) have also shown that glucoamylase is an industrially important enzymes that
catalyse the release of glucose from the non-reducing ends of starch molecules.
An important application of S. fibuligera is the production of ethanol. Chi et al.
(2009) reported that ethanol is a popular liquid biofuel and also an excellent raw material
used for synthetic chemicals. Starch, existing in abundance and low in cost, is a very
good substrate for ethanol production (Chi et al., 2009).
Based on several studies (Chi et al., 2009; Machida et al., 1988), Saccharomyces
cerevisiae was initially used in the production of ethanol from starch. Unfortunately, the
disadvantage of using S. cerevisiae is that it would require a high-energy input. This is
because S. cerevisiae cannot produce amylase. Artificial amylases are hence required to
be added manually in the fermentation process, which results in high overall costs (Chi et
al., 2009).
Outline of Methods
Materials

Potato dextrose broth

Yeast extract

α-amylase

Peptone

Phosphate-buffered saline

Potassium Dichromate (K2Cr2O7)

Starchy wastes (Rice, barley and

Ethanol
noodle wastewater)
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Apparatus

Centrifuge

Spectrophotometer
Variables

Starchy wastes (noodle, rice and barley waster)

Yeast (S.cerevisiae and S.fibuligera)
Dependent Variables

Amount of ethanol produced
Controlled Variables

Amount of yeast cells transferred to setups

Amount of starchy waste in each of the setups

Growth medium (potato dextrose broth)
Independent Variables
Preparation of Medium with Starchy Wastewaters
Yeast extract peptone broth (YEP) containing 1% yeast extract and 0.5% peptone
will first be prepared. 20ml of each of the wastewaters are then prepared and 1ml of 1%
amylase is then added to 10ml of each of the wastewaters and left in room temperature
for 15 minutes. YEP and the amylase-treated and untreated wastewaters will be
autoclaved at 10psi for 10 minutes to sterilize. 10ml of each of the wastewaters are added
to 20ml of YEP and a control, consisting of 20ml YEP and 10ml sterile water is set up.
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Growth of Yeasts
Saccharomycopsis fibuligera and Saccharomyces cerevisiae are inoculated into
10ml of potato dextrose broth. The cultures are then incubated at 30°C overnight. 1ml of
the cultures will then be transferred into each of the setups as below.

20ml YEP+10ml amylase-treated noodle wastewater

20ml YEP+10ml amylase-treated rice wastewater

20ml YEP+10ml amylase-treated barley wastewater

20ml YEP+10ml untreated noodle wastewater

20ml YEP+10ml untreated rice wastewater

20ml YEP+10ml untreated barley wastewater

20ml YEP+10ml sterile water
The rationale is that the starchy wastes act as a carbon source for the yeasts to
grow.
The setups are then incubated at 30°C for 7 days with shaking.
The data will then be analysed as below.
The following data will be collected:
1. The density of yeast cells in the samples
2. The concentration of ethanol in the samples
The density of yeast cells are compared between each wastewater (barley, rice and
noodle wastewaters) and between the two yeasts (Saccharomyces cerevisiae and
Saccharomycopsis fibuligera). Absorbance readings for each sample are taken at 660nm.
The density of yeast cells are found out by counting the cells using a hemocytometer.
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Determination of Ethanol Concentration
The yeast cultures are centrifuged at 7000rpm for 10 minutes and the supernatant
collected. Acidified potassium dichromate, K2Cr2O7, is added to the supernatants in a 3:1
ratio and the solutions are then heated over a boiling water bath for 15 minutes.
Absorbance readings for each of the solutions are taken at 590nm with a
spectrophotometer.
The concentration of ethanol in the samples are compared between each of the
wastewaters (barley, rice and noodle wastewaters) and between the two yeasts
(Saccharomyces cerevisiae and Saccharomycopsis fibuligera). The ethanol content are
then found by the following method:
Different concentrations of ethanol are then prepared and acidified potassium
dichromate will be added into the ethanol in a 3:1 ratio. The solutions are heated over a
boiling water bath for 15 minutes and the absorbance readings for each concentration of
ethanol will be taken at 590nm with a spectrophotometer.
A calibration curve is then plotted with the absorbance readings from each
concentrations of ethanol and the amount of ethanol in each of the products are then
found.
Safety Precautions
During experiments, latex gloves and lab-coats will be worn. When working with
the yeasts, laminar flow hoods will be used. All microbial cultures and apparatus used to
contain the yeasts will be sterilized with an autoclave before disposal.
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References
Chi, Z., Liu, G., Wang, F., Ju, L., Zhang, T. (2009). Saccharomycopsis fibuligera and its
applications in biotechnology. Biotechnology Advances, 1, 1-9. Retrieved February 20,
2010 from http://www.elsevier.com/locate/biotechadv
Waghorn, J.J, TalõÂa del Pozo, Elba A. Acevedo, Liliana A. Cardemil (2003). The role
of two isoenzymes of a-amylase of Araucaria araucana (Araucariaceae) on the digestion
of starch granules during germination. Journal of Experimental Botany, 54, 1-11.
Retrieved May 1, 2010 from http://jxb.oxfordjournals.org/cgi/reprint/54/384/901
Lemmel, S.A., Heimsch, R.C. & Korus, R.A. (1980). Kinetics of growth and Amylase
production of Saccharomycopsis fibuligera on potato processing Wastewater. Applied
and Environmental Microbiology, 39, 1-7. Retrieved February 20, 2010 from
http://aem.asm.org/cgi/reprint/39/2/387
Machida, M., Ohtsuki, I., Fukui, S. & Yamashita, I. (1988). Nucleotide sequences of
Saccharomycopsis fibuligera genes for extracellular r-glucosidases as expressed in
Saccharomyces cerevisiae. Applied and Environmental Microbiology, 54, 1-9. Retrieved
on February 20, 2010 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC204441/pdf/aem001170273.pdf/?tool=pmcentrez
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Reiser, V. & Gasperik, J. (1995). Purification and characterization of the cell-wallassociated and extracellular x-glucosidases from Saccharomycopsis fibuligera.
Biochemistry Journal, 753, 1-8. Retrieved On February 20, 2010 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1136789/pdf/biochemj000610061.pdf/?tool=pmcentrez
Saelim, K., Dissara, Y. & H-Kittikun, A. (2007). Saccharification of cassava starch by
Saccharomycopsis fibuligera YCY1 isolated from Loog-Pang (rice cake starter).
Songklanakarin Journal of Science and Technology, 1, 1-7. Retrieved February 20, 2010
from http://rdo.psu.ac.th/sjst/ejournal/journal/30-Suppl-1/0125-3395-30-S1-65-71.pdf
Timeline
January 2010

Research on Project Topics
February 2010

Literature Review

Write up Proposal

Confirmation of Methods

Begin Experiments

Complete Experiments

Analyse Results

Write Up Proposal
March 2010
July 2010
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