sample_proposal_2012x - SMTP - Hwa Chong Institution

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Feb 20, 2013 (4 years and 4 months ago)

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1

Investigating the Production
of Alcohol by Rice Wine
Yeast (
Saccharomycopsis
fibuligera
)

Project Proposal

XXX

XXX

XXX


Hwa Chong Institution (High School)

Mentor:
YYY



2

Introduction


Rationale

Biofuels

like ethanol play a major role in reducing reliance on fossil fuels.
However, ethanol derived from food crops has dr
iven
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 fib
uligera
, 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
Saccharomyc
opsis fibuligera


Hypotheses

1.

Saccharomycopsis fibuligera

is able to produce ethanol from starchy wastes.


3

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 m
any other
differe
nt 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, a
cid 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,
Le
mmel
,
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

4

hydrolysed by α
-
amylase.
On top of that, se
veral studies (Lemmel
et al
., 1980; Chi
et al
.,
2009) have
also shown that glucoamylase is an

industriall
y important enzymes that
catalys
e the release of glucose from the non
-
reducing ends of starch molecules.

An

important application of
S.
f
ibuligera

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 v
ery
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 Method
s


Materials



Potato dextrose broth



Yeast extract



α
-
amylase



Peptone



Phosphate
-
buffered saline



Potassium Dichromate (K
2
Cr
2
O
7
)



Starchy w
astes (Rice, barley and
noodle wastewater)



Ethanol




5

Apparatus



Centrifuge



Spectrophotometer


Variables

Independent Variables



Starchy w
astes (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)


Preparation
o
f
Medium
w
ith Starchy Wastewaters

Y
east 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.





6

Growth

of Yeast
s

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 et
hanol 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 h
emocytometer.



7

Determination of Ethanol Concentration

The yeast cultures
are

centrifuged at 7000rpm for 10 minutes and the supernatant
collected. Acidified potassium dichromate,

K
2
Cr
2
O
7
,
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 e
ach 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
Saccha
romycopsis fibuligera
). The ethanol content
are
then
found by the following method:


D
iff
erent concentrations of ethanol
are then

prepared and
acidified potassium
dichromate
will be added
into the ethanol in a 3:1 ratio.

The solutions
are

h
eat
ed

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 a
mount 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.



8

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,
20
10 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/rep
rint/54/384/901


Lemmel
,

S.A., Heimsch
,

R.C. &

Korus
,

R.A. (1980). Kinetics of
g
rowth and Amylase
p
roduction of
Saccharomycopsis fibuligera

on
p
otato
p
roc
e
ssing 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
s
equences of
Saccharomycopsis fibuligera

g
enes for
e
xtracellular r
-
g
lucosidases as
e
xpressed in
Saccharomyces cerevisiae
.
Applied and Environmental
M
icrobiology
, 54
,

1
-
9.
Retrieved
on February 20, 2010 from

http://www.n
cbi.nlm.nih.gov/pmc/articles/PMC204441/pdf/aem00117
-
0273.pdf/?tool=pmcentrez


9

Reiser
,

V.
&

Gasperik
,

J. (1995). Purification and characterization of the cell
-
wall
-
associated and extracellular x
-
glucosidases from
Saccharomycopsis fibuligera
.
Biochemistry

J
ou
rnal
, 753
, 1
-
8. Retrieved
O
n February 20, 2010 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1136789/pdf/biochemj00061
-
0061.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

March 2010



Confirmation of Methods



Begin Experiments

July 2010



Complete Experiments



Analyse Results



Write Up
Proposal