{Title of project} - AOS-HCI-2011-Research-Program

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20 Φεβ 2013 (πριν από 4 χρόνια και 10 μήνες)

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1


Sembawang Shipyard’s

“Greenwave” Project 201
1




Biofuel from microorganisms


Group Members:

Glenn Chua (Leader)

Rick Wong


Sponsor Teacher:

Mrs Goh
-
Yip Cheng Wai


School:

Hwa Chong
Institution (High School)



















2


Abstract


Most energy used in the world today comes from burning fossil fuels such as coal and

petroleum
. The combustion of these fossil fuels generates a high concentration of
carbon dioxide in the Earth’s
atmosphere, leading to global warming. Moreover, the
supply of fossil fuels is dwindling and they are non
-
renewable sources. Hence, t
his
project,
b
iofuel from microorganisms,

aims to
find ways to address this problem by
ferment
ing

sugar to ethanol by using

wastes such as fruit peels
and sugarcane bagasse
as a source of sugar for the growth of the bacteri
um

Zymomonas mobilis.
Two methods
of ethanol fermen
t
ation were
explored,
namely, ethanol fermen
t
ation by free and
immob
i
lized cells. Reducing sugars were fo
und to be present in different
concentrations

in

the wastes used, and the
re

was a correlation between the concentration of
reducing
sugar
s and

the ethanol yield

for both

methods.
Orange peel gave the highest yield of
ethanol, followed by sugarcane bagasse
and watermelon peel. T
o
further
increase

the

ethanol yield,
cellul
a
se was added to hydrolyse the cellulose in sugarcane bagasse.
There was evidently a higher reducing sugar concentration
after cellulase treatment
. In
summary, the findings of this study can potentially be applied
in

the
scale
-
up of
production of biofuel from wastes which are renewable sources that are highly abundant,
thus saving costs by recycling these wastes
. This
also
helps to alleviate

environ
mental
problems such as the excessive release of greenhouse gases from combus
tion of non
-
renewable fossil fuels.




3


Introduction

Fossil fuels such as petroleum and coal are widely used in today

s society. It is
the main source of energy, that is needed for

various things, from running a car engine,
to the
producti
on of electricity. However, there is a disadvantage of using fossil fuels.
First of all, fossil fuels are a non
-
renewable source of energy, and according to latest
trends, the amount of fossil fuel
s has been declining at an alarming rate
. Thus,
scientists have been looking
for alternatives to the fossil fuel supplies
, which has been
projected to become exhausted by

the next few decades. S
e
condly,
the combustion of
fossil fuels have a detrimental
effect on the environment as well
, with there being large
volumes of greenhouse gases, such as
carbon dioxide
, being released during
combustion.

Due to the reason above, m
icrobial production of biofuel from
food crops has
gained much
significance in recent

years. Ethanol
is a viable

alternat
ive

fuel for the
future (Gunasekaran and Chandra Raj, 1999)
, especially with it being a renewable
source of energy
. However, according to Banschbach and Letovsky (2010), there is a
controversy in using food crops such as

corn, sugarcane and soybean in producing
biofuels. For example, when Brazilian rainforests
we
re cleared for growing sugarcane,
the carbon stored in the forests
was
released through cutting and burning the trees,

which emitted approximately 50% more greenh
ouse gases than producing and burning
gasoline (Tilman and Hill, 2007).

Given the food crop controversy, the production of ethanol from food wastes
instead is worth exploring.
According to the 2010 statistics of wastes generated and
recycling from the Na
tional Environmental Agency, Singapore, food wastes are
4


generated at a rate of 0.64 million tones / year. They are recycled at a rate of 0.10
million tonnes / year, thus representing only 16% of the total waste output. Wastes are
recycled, incinerated or l
andfilled. By increasing the rate of recycling, there will be a
lower need for incineration which releases
toxic
gases into the atmosphere
, and landfills
which are gradually reaching their maximum capacity.

Thus, in order to address the problems of using
fossil fuels, the controversy of
using food crops in the production of ethanol, and the increasing amount of food wastes
generated over recent years, this project aims to investigate the feasibility and efficiency
of producing ethanol from food wastes

such

as fruit peels and sugarcane bagasse
.


Objectives

The objectives of the project are to:

1.

Determine the
concentration

of reducing sugars present in fruit peels and
sugarcane waste

2.

Quantify the amount of ethanol produced by free and immobilized
Zymomonas
mob
ilis

cells using the

sugars in the
se wastes as substrates


Hypothes
e
s

We hypothesize the following:

1.

Fruit peels and sugarcane
wastes
contain
varying

concentrations of reducing
sugars.

2.

Free and
immobilized

Zymomonas mobilis

cells produce different
concentrations

of ethanol when grown in different wastes.

5


Literature Review

The yeast
Saccharomyces cerevisiae

is commonly used in ethanolic
fermentation from sugars. However, t
his project utilizes
Zymomonas mobilis
, which

is a
rod shaped gram
-
negative
bacterium
, in the production of ethanol. This bacterium

can
be found in sugar rich plant saps. Its ability to
utilize

sucrose, glucose and fructose
m
akes it
a versatile organism in ethanol fermentation
.
Other benefits of
Z. mobilis
include
a higher sugar
uptake

and
a higher
tolerance to ethanol
as

compared
to yeast

(Gunasekaran

and Chandra Raj,

1999).

A

few
studies
have been carried out

on ethanol fermentation
by

Z
.

m
obilis
.

For
example,
Gunasekaran
et. al
. (1986)

studied
the fermentation pattern of
Z
.

mobilis

strains on different substrates

such as cane juice and molasses
.
Ethanol yield was
highest at pH 7 and using an initial sugar concentration of 15%.
Doelle and Greenfield
(1985) carried out single
-
batch
ethanol
fermentation
from refined sucrose, su
garcane
juice and syrup and obtained a high yield of ethanol within 30 hours.
Amin
et al
. (1987)
immobilized
Z. mobilis

cells in polyurethane foam and investigated the production of
ethanol from sucrose. A final ethanol concentration of 6.3% was obtained.
More recently,
Zhang and Feng (2010) attempt
ed

to produce ethanol from low
-
cost
, non
-
grain
feedstock such as

raw
sweet potato,

and this offered an advantage over the use of food
crops such as corn and wheat in
producing

ethanol, especially in developing co
untries.

Though
quite some
work has been done in this field,
much less
work has been
done in attempting to produce ethanol from waste materials.
Usage

of waste
s

as
substrates for e
thanol
fermentation is

more
economically
feasible.
This is what this
project aims to achieve.

6


Methods


Experimental variables

The independent variables were the types of wastes used (sugarcane bagasse, orange
and watermelon peels). The dependent variables that were measured were the mass of
Z. mobilis

cells obtained after growth in sugars from wastes and the concentration of
ethanol obtained. The controlled variables that were kept constant were the volume of
Z.
mobilis

preculture added to fermentation medium, the number of
Z. mobilis

beads
added to w
aste extracts, and the temperature of growth of bacteria.


Outline of methods

The overview of the methods
is shown in Figure 1.





















Fig
ure

1
:

Overview of methods


Preparation of extracts from wastes

DNS test for reducing sugars in wastes

Growth of
Zymomonas mobilis

in GY medium

Ethanol fermentation by free
cells


Transfer cells to fermentation
medium with wastes

Ethanol fermentation by
immobilized cells

Prepare beads and add them
to extracts of wastes

Distillation and
d
etermination of
e
thanol yield

by the
dichromate test

7


Preparation of extracts from wastes

30

g of fruit peels

or
sugarcane waste
were blended
in 300

ml of deionised water using
a blender

(Figure 2)
.
T
he liquid
was passed
through a sieve to remove the
residue.







Figure
2: The preparation of wastes


DNS test for reducing sugars in waste

To 0.5

ml of the extract,
0
.5

ml of DNS (
d
initrosalicylic acid)

was added
.
The mixture
was left

in a boiling water bath for 5 minutes. 4

ml of water

was then added. The
samples were placed in spectrophotometer cuvettes and the
absorbance
was taken
at
530

nm using a spectrophotometer

(Figure 3)
.
The
concentration of reducing sugar
s

in
µmol/ml was read
from a
maltose
standard curve.






Figure 3:
DNS test for reducing sugars


8


Growth of
Zymomonas mobilis

Z
.

mo
b
ilis

cells were inoculated
in 20

ml GY medium (2% glucose, 0.5% yeast extract)

and i
ncubate
d

at 3
0
º
C for 2 days

(Figure 4)
.






Figure 4: Inoculation of cells


Ethanol fermentation by free

cells

3

ml of
Z
.
mobilis

preculture
was transferred
to fermentation medium

(
1% yeast extract,
0.1%
a
mmonium
s
ulfate, 0.1%
d
ipotassium phosphate, 0.05%
m
agnesium sulfate

in 50
ml waste extract
)

and i
ncubate
d

at 30
º
C for 2 days

for ethanol fermentation to t
ake
place
.
The cultures were centrifuged
at 7000

rpm for 10 minutes to pellet the cells.
The

wet weight of cells

was recorded
.
The
supernatant
was collected
and sen
t

for distillation
to obtain the ethanol.


Ethanol
fermentation

by Immobilized cells

T
he
Z.
mobilis

preculture

was centrifuged

at 7000 rpm for 10 minutes
and
the cell pellet
was
resuspend
ed

in 7.5

ml of GY

medium
.
The
absorbance
of the culture was taken
at
600

nm
.

7.5

ml
of 2%
sodium alginate
was added
to
the
cell suspension and mix
ed

well.
T
he mixture
was dropped
into 0.1

mol dm
-
3

c
alcium
c
hloride solution to form
Z. mobilis

9


alginate
beads.
Th
e beads
were rinsed
with 0.85%
s
odium
c
hloride solution.

200 beads
were added
to 50

ml
waste
extract

(Figure 5). A control was prepared in which 200
emp
ty alginate beads were added to the same volume of waste extract instead. All the

set
-
ups were incubated with shaking
at 30
º
C for 2 days

for ethanol fermentation to
occur
.
T
he beads

were
then
remove
d

and

the extract
s

were
distill
ed
to obtain ethanol.






Figure 5: Preparation of
alginate
beads

containing
Z. mobilis

cells and addition of beads
to waste extracts


Determination of ethanol yield
with the d
ichromate test

2.5 ml of a
cidified
potassium dichromate solution was added

into
0.5 ml of

distillate

in
a

ratio of 5:1.
The samples were p
lace
d

in a boiling water bath for 15

minutes.
The
absorbance was m
easure
d

at 590

nm using a spectrophotometer

(Figure 6)
, and the
concentration of ethanol
was read
from a
n ethanol

standard curve.










Figure 6:
Dichromate test for ethanol

10


Results

The maltose

standard curve used for the determination of concentration of reducing
sugars present in the wastes is shown in Figure 7.











Figure
7: Maltose standard curve for the determination of reducing sugar concentration



The
ethanol standard curve for the determination of ethanol concentration is shown in
Figure 8. It
is used to determine the
percentage of ethanol after fermentation
by

Z
.
mo
bilis

using
sugars in
waste extracts as substrates.





11












Figure 8: Ethanol standard curve for the determination of ethanol concentration


Ethanol f
ermentation using free
Z. mobilis

cells

The concentration of reducing sugars i
n extracts of
wastes

is shown in Figure 9. Orange
peel was found to have the highest concentration of reducing sugars among the three
samples tested. The ANOVA test had a p value of 0.0003, indicating a significant
difference in the concentration of reducing sugars in t
he sugarcane waste, orange and
watermelon peels.
The yield of ethanol corresponded with the concentration of reducing
sugars in the wastes. As such, orange peel resulted in the highest ethanol yield,
followed by sugarcane waste and watermelon peel (Figure
10). The ANOVA test yielded

a

p value of
4.17 x 10
-
7
, showing

significant differences in the concentration of ethanol
obtained from the different wastes used.


12











Figure 9: Concentration of reducing sugars in wastes used in ethanol fermentation by
free
Z. mobilis

cells











Figure 10: Yield of ethanol from free
Z. mobilis

cells


13


Ethanol f
ermentation using free
Z. mobilis

cells

A different batch of wastes was used in ethanol fermentation using
Z. mobilis

cells
immobilized in calcium alginate. The concentration of reducing sugars showed the same
trend as before, with orange peels having the highest, followed by sugarcane waste and

watermelon peel (Figure 11).
The ANOVA test revealed a significant difference in the
concentration of reducing sugars (p

value of
6.76 x 10
-
7
).
The yield of ethanol
corresponded with the concentration of reducing sugars in the wastes. As such, orange
peel

resulted in the highest ethanol yield, followed by sugarcane waste and watermelon
peel (Figure 11). The yield of ethanol corresponded again with the concentration of
reducing sugars in the wastes. As such, orange peel resulted in the highest ethanol
yield
, followed by sugarcane waste and watermelon peel (Figure 12). The ANOVA test
showed ……………….












14












Figure

11:
Concentration of reducing sugars in wastes used in ethanol fermentation by
immobilized
Z. mobilis

cells











Figure
12:
Yield of ethanol from immobilized
Z. mobilis

cells

15


Conclusion and
Discussion


Orange peels contain
ed

the
highest

concentration of reducing sugars, and g
ave

the greatest yield of ethanol when used as a substrate for fermentation to ethanol by
Z
.

m
obilis

using both free cells and immobili
z
ed cells.

There was a correlation between
reducing sugar content and yield of ethanol, due a higher rate of utilization of these
sugars by
Z. mobilis

in ethanol fermentation. Also, a higher reducing sugar content
support
ed a higher growth rate of free
Z. mobilis

cells, thus resulting in a higher
biomass and yield of ethanol.


The use of immobilised cells has more advantages compared to the use of free
cells.

According to Amin
et al
. (1987), cell immobilization reduces the

problems of
washout of cells in continuous cultures, which limit productivity. Moreover, product
inhibition of cells in batch cultures can be reduced by using immobilized cells. The
findings from this study have shown that significant yields of ethanol we
re obtained
using immobilized cells, comparable to free cells.


Application
s

of project


There are a few benefits of using food wastes as substrates for producing
ethanol. The wastes are recycled, thus saving costs in the production of ethanol.
The
production of ethanol from agricultural feedstocks is based on renewable resources.
There have been debates over the use of large areas of agricultural lands for planting
food crops such as corn and rice for ethanol production, instead of being consume
d as
food, especially in developing countries. Moreover, the large
-
scale planting and
16


harvesting machinery of corn are powered by fossil fuels, resulting in the emission of
considerable amounts of carbon dioxide,
to the extent that driving a car fueled by
corn
-
based ethanol only reduces the

emission of greenhouse gases by a small margin,
compared to a similar car powered by gasoline (Tilman and Hill, 2007).

However, with
the use of agricultural wastes instead of crops to produce ethanol, these problems can
be alleviated. The cost feasibility and its impact on truly reducing the reliance on fossil
fuels
and waste management
will definitely make further development into this area
worthwhile.


Further Work



Since initial findings indeed show a high potential
of using wastes in ethanol
fermentation, the next step would be to scale up the production of ethanol by increasing
the wastes used as the starting substrate, so as to increase the concentration of sugars.
The volume of fermentation medium can be increased

to support a much higher growth
rate of
Z. mobilis

cells and thus a greater yield of ethanol.


References



Amin, G., Doelle, H.W. and Greenfield, P.F. (1987). Ethanol production from sucrose
by immobilized
Zymomonas mobilis

cells in polyurethane foam.
Biotechnology
Letters
, 9(3), 225
-
228.

17




Banschbach
, V.S.

and Letovsky
, R. (2010). The use of corn versus sugarcane to
produce ethanol fuel: a fermentation experiment for environmental studies.
The
American Biology Teacher
, 72(1), 31
-
36.



Doelle, H.W. and G
reenfield
, P.F. (1985). The production of ethanol from sucrose
using
Zymomonas mobilis
.
Applied Microbiology and Biotechnology
, 22, 405
-
410.



Gunasekaran, P.
and

Chandra Raj, K.

(1999).
Ethanol fermentation technology


Zymomonas mobilis
.
Current Science,
77(1),

56
-
68.



Gunasekaran, P., Karunakaran, T., & Kasthuribai M. (1986).
Fermentation pattern of
Zymomonas mobilis
strains on different substrates

a comparative study.
Journal of
Bio
s
cience
,
10
(2)
, 181
-
186.



Tilman, D. and Hill, J. (2007). The ethanol conundrum: grown improperly, biofuels
might make our environmental problems worse.
Washington Post National Weekly
Edition
, April 2
-
8, p.27.



Zhang, K.
and

Feng, H. (2010). Fermentation potentials of
Zymomonas mobilis
and
its application in ethanol production from low
-
cost raw sweet potato.
African Journal
of Biotechnology,
9
(37)
, 6122
-
6128.


Safety

Zymomonas mobilis

ATCC 29191 is

a
biosafety level
-
1

bacterium
. Work involving
microorganisms
were

perfo
rmed in the biological safety cabinet. Gloves and labcoats
were

worn. Bacterial cultures
we
re discarded in biohazard bags and autoclaved at
121°C for 20 min before disposal.


18


Acknow
ledgements

We would like to thank our
m
entor
Mrs Goh
-
Yip Cheng Wai

for
guiding

us throughout
the entire project,
and
Mdm Lim

Cheng Fui

for assisting us during experimentation
. We
would also like to
thank
Hwa Chong Institution
, for providing us with the facilities to
conduct our experimentation.