CHAPTER I

BACKGROUND OF THE STUDY

INTRODUCTION
With the decline of oil reserves around the world, finding an alternative and a renewable
resource is quite a challenge. In the Philippines, the Republic Act 9367 or the “Biofuels Act of
2006” was enacted on January 12, 2007 to prevent the country to depend on imported fuels and
ensure the availability of alternative and renewable clean energy without damaging the natural
ecosystem, biodiversity and food reserves of the country. On the Section 5 of the Republic Act
explains the mandatory use of biofuels which stated “a minimum of ten percent (10%) blend of
bioethanol by volume and a minimum of two percent (2%) blend of biodiesel by volume into all
gasoline fuel distributed and sold by each oil company in the country.” According to the National
Biofuels Board (NBB) of the country, supplying for the required blend for ethanol has been
problematic in ten years of the Biofuel Act’s implementation due to the inadequate production
from the local sources while the plan to increase the 2% blend of biodiesel was postponed due to
the increasing prices of biofuels (Global Agricultural Information Network [GAIN], 2016).

According to the news about the oil prices last May 2018, the Philippines is one of the
country who is at risk from rising oil prices which will continue to hurt the economy. According
to PhilStar, the country’s trade deficit “may increase by 0.5 percent of GDP for every $10 per
barrel rise in oil prices” With this the need in alternative and new resources for fuels remains.

One biofuel emerging in the market because of its advantages is butanol, also known as
biobutanol, which is produced from fermentable sugars, synthesis gas and glycerol. As stated by
Gable (2017) Biobutanol is less miscible with water and has higher energy content than ethanol,
so there is a much lower loss of fuel economy. With an energy content of about 105,000
BTUs/gallon (versus ethanol’s approximate 84,000 BTUs/gallon), biobutanol is much closer to
the energy content of gasoline (114,000 BTUs/gallon). Butanol meets the renewable fuel 20%
greenhouse gas-emission reduction threshold. It is less susceptible to separation in the presence
of water (than ethanol), it can be distributed via conventional infrastructure (pipelines, blending
facilities and storage tanks). There’s no need for a separate distribution network. (Gable, 2017)

Breakthroughs within the production of butanol have been made available. One of which
is the fermentation of biomass using “butanol-producing microorganism”. Butanol is naturally
produced by some Clostridia species, but due to inherent problems with clostridial
fermentations, industrially more relevant organisms have been genetically engineered for n-
butanol production. Although the yeast Saccharomyces cerevisiae holds significant advantages
in terms of scalable industrial fermentation, n-butanol yields and titers obtained so far are only
low (Schadeweg and Boles, 2016).
Saccharomyces cerevisiae is the most common species of the genus Saccharomyces, commonly
known as baker’s or brewer’s yeast. Butanol tolerance is a critical factor affecting the ability of
microorganisms to generate economically viable quantities of butanol. There are many different
microorganisms that is used for butanol extraction but not all produce great amount of it.
Generally, a barrier to growth exists between 1% and 2% butanol and a few microorganisms can
tolerate 2% butanol. Saccharomyces cerevisiae exhibits limited growth in 2% butanol.

In the Philippine sugar industry, bagasse is considered a major biomass waste produced
after the extraction of juice from the sugarcane stalk. Sugar mills are estimated to produce more
than 6 million metric tons of bagasse per year or 350,000 tons of dry bagasse per hectare (Sugar
Regulatory Administration [SRA], 2016). This abundance of sugarcane bagasse in the country
has been taken advantage of by millers in power generation for their boilers and by distilleries in
the production of ethanol, butanol and other biofuels.

The purpose of this study is to increase the production of butanol using fermentation
organism. It aims to initiate a new way to produce and increase a clean, renewable and locally
available source of energy without causing harm to health, natural ecosystem and environment.

STATEMENT OF THE PROBLEM

This study aims to determine the butanol fermentation performance of Sacchromyces
cerevisiae with sugarcane bagasse as a substrate. Specifically, this study aims to:

a. To determine the effect of Sacchromyces cerevisiae in the fermentation of sugarcane

bagasse in terms of butanol yield, total sugar consumed, cell growth and fermentation
efficiency;
b. To evaluate the effect of Sacchromyces cerevisiae using different parameters including
specific growth rate and other parameters with respect to productivity per cell growth
phase;
c. To determine the optimum condition for the Sacchromyces cerevisiae to produce the highest
butanol yield.

SIGNIFICANCE OF THE STUDY

The significance of this study is to increase the production of butanol using fermentation
organism. It aims to initiate a new way to produce and increase a clean, renewable and locally
available source of energy without causing harm to health, natural ecosystem and environment.

If achieved the desired results, it may open a new opportunity to a new source of
renewable resource which can contribute to a big change in the country and the world as a whole.
HYPOTHESIS
Null Hypothesis

1. The effect of Sacchromyces cerevisiae in the fermentation of sugarcane bagasse is

undetermined in terms of butanol yield, total sugar consumed, cell growth and
fermentation efficiency;
2. Mistakes in evaluation to the effect of Sacchromyces cerevisiae using the different
parameters.
3. The optimum condition for the Sacchromyces cerevisiae to produce the highest butanol
yield is undetermined

Alternative Hypothesis

1. The effect of Sacchromyces cerevisiae in the fermentation of sugarcane bagasse is

determined in terms of butanol yield, total sugar consumed, cell growth and fermentation
efficiency;
2. The effect of Sacchromyces cerevisiae using the different parameters is evaluated.
3. The optimum condition for the Sacchromyces cerevisiae to produce the highest butanol
yield is determined.

SCOPE AND LIMITATION

Because of the financial restraints, time constraints, personal obligations and duties to
family and school, and the general unavailability of equipment and resources required, the
researchers can only guarantee that the experiment can only be performed at least once. After
this experimental trial, the researchers cannot guarantee that they have the available resources,
funding, or time to perform a second trial of this experiment.

DEFINITION OF KEY TERMS

The following terms are used in the research paper and defined conceptually and
operationally for better understanding.
Sacchromyces cerevisiae - commonly known as baker’s or brewer’s yeast. commonly
used in the baking and brewing industries.

Biobutanol - is produced from fermentable sugars, synthesis gas and glycerol, and
suitable for alternative fuel.

Sugarcane bagasse - is the dry pulpy fibrous residue that remains after sugarcane or
sorghum stalks are crushed to extract their juice.
CONCEPTUAL FRAMEWORK

Gathering and
Preparation of Pretreatment of
Preparation of
Stock Culture bagasse
materials

Fermentation Substrate
Additional trials Process and preparation and
analytical methods Inoculum Build Up
 CHAPTER II
REVIEW OF RELATED LITERATURE

Butanol, like ethanol, can be produced from fermentable sugars, synthesis gas, and
glycerol. Butanol has a number of notable qualities that make it a suitable alternative fuel. Its
energy content is 30% more than ethanol (Qureshi and Ezeji, 2008). It can be mixed with
gasoline in any proportion or be used as the sole fuel component (100% butanol) in unmodified
car engines (Ramey, 2007). It carries less water and, therefore, it can be transported through
existing gasoline pipelines (Dürre, 2007). Reports of biological butanol formation date back to
Louis Pasteur. He reported an alcohol product from a clostridial culture (Dürre, 2007). The ABE
fermentation was essential during World War I. Acetone was needed to prepare munitions, and it
was in great shortage at the time. Production of acetone by fermentation meant a constant supply
of acetone to Britain and its allies (Dürre, 2007).

Biofuels produced by microbial fermentations of renewable resources represent an

important replacement for fossil fuels. Today, bioethanol is the most important renewable
gasoline substitute. However, higher alcohols like butanol are regarded as more suitable fuel
substitutes, due to their higher energy density, lower hygroscopicity, and therefore lower
corrosiveness (Generoso, Schadeweg , Oreb & Boles, 2015).

Biobutanol is derived mainly from the fermentation of the sugars in organic feedstocks
(biomass). Historically, up until about the mid-50s, biobutanol was fermented from simple
sugars in a process that produced acetone and ethanol, in addition to the butanol component. The
process is known as ABE (Acetone Butanol Ethanol) and has used unsophisticated (and not
particularly hearty) microbes such as Clostridium acetobutylicum (Gable,2017). Butanol
tolerance is a critical factor affecting the ability of microorganisms to generate economically
viable quantities of butanol. Current Clostridium strains are unable to tolerate greater than 2% 1-
butanol thus membrane or gas stripping technologies to actively remove butanol during
fermentation are advantageous. To evaluate the potential of alternative hosts for butanol
production, we screened 24 different microorganisms for their tolerance to butanol. We found
that in general, a barrier to growth exists between 1% and 2% butanol and few microorganisms
can tolerate 2% butanol. Strains of Escherichia coli, Zymomonas mobilis, and non-
Saccharomyces yeasts were unable to surmount the 2% butanol growth barrier. Several strains of
Saccharomyces cerevisiae exhibit limited growth in 2% butanol, while two strains of
Lactobacillus were able to tolerate and grow in up to 3% butanol (Knoshaug & Zhang, 2008).
 CHAPTER III
RESEARCH METHODOLOGY

A. Materials

a. culture of Saccharomyces cerevisiae m. ice

b. wire loop n. 5μm filter paper
c. agar slants o. anaerobic jars (six large, three medium,
d. oxygen absorber packs and three small) with airlock
e. sugarcane bagasse p. pipettor
f. thermometer q. 50 mL centrifuge tubes
g. grinder r. weighing scale
h. 40 mesh sieve s. Eppendorf tubes
i. 1% and 2 M sodium hydroxide (NaOH) t. Scanning Electron Microscope
j. 250 mL beaker u. pH meter
k. microwave oven v. Gas chromatography
l. 0.005 M sulfuric acid (H2SO4) solution
B. Preparation of Stock Culture
Suspend 65g of the Agar in 1L of distilled water. Heat to boiling while stirring to dissolve all
ingredients completely. Autoclave for 15 minutes at 121°C. Pour around 25-30mL of the agar
medium on to sterile plates and let it set in a laminar flow chamber. Streak (yeast obtained from
plate)/spread (yeast sourced from broth cultures) cells on the agar plates and incubate at 30°C.
C. Pretreatment of Bagasse
The bagasse will be dried at 50°C overnight. It will then be grinded into 40 mesh particle
size. Using a solid to liquid ratio of 1:12, the grinded bagasse will be soaked in 1% sodium
hydroxide (NaOH) at 121°C for 2 hours to delignify the bagasse. The pH of the bagasse will be
neutralized by washing with water then dry.
In a 250 mL beaker, 0.005 M sulfuric acid (H2SO4) solution will be added to the
pretreated bagasse in the ratio of 10 mL H2SO4/g bagasse. The mixture will be heated in a
microwave oven at 180°C for 30 minutes. After heating, it will be cooled in an ice bath until the
room temperature is reached. The mixture will be filtered using a 5μm filter paper. The solid
particles from the filtration will be washed with water to remove the acid adhering and to stop
further reaction from taking place. The solid particles will be dried at 105° for 24 hours. On the
other hand, the liquid solution will be neutralized using 2 M NaOH.
D. Preparation of Anaerobic Jars
To ensure anaerobic working condition, anaerobic jars of three different sizes – large,
medium, and small, will be used as vessels. The airlocks of the jars will be filled with H2SO4 to
absorb moistures and prevent air form entering the jars. For every setup where samples will be
exposed in the atmosphere, continuous flushing with O2-free gas will be done to remove oxygen
from the system. Oxygen-free gas will also be flushed for 10 minutes before covering the vessels
and tubes.
E. Substrate Preparation
Six large anaerobic jars with 300 mL of the dilute hydrolysate, three small anaerobic jars
with 50 mL of the dilute hydrolysate, and three medium anaerobic jars with 90 mL of the dilute
hydrolysate will be prepared. Among the six large anaerobic jars, two will be supplemented with
0.2% chitin and another two with 0.4% chitin. The remaining two large anaerobic jars will be
used as controls. The jars will be flushed with O2-free gas for 10 minutes before covering and
will be sterilized for 15 psi and 121°C for 15 minutes. The jars will be stored in a cool room.
F. Inoculum Build-up
The prepared stock cultures of Saccharomyces cerevisiae will be inoculated into each
prepared 50 mL dilute hydrolysates. For the aseptic transfer, 5 mL of the dilute hydrolysate from
the jar will be obtained using a pipettor with sterilized tips and will be transferred into the slant.
Fresh microorganisms will be scraped-off from the agar slant using a wire loop. After which, the
medium containing the cells will be poured back into the jar. The samples will be incubated at
37°C for 60 hours.
Actively growing strains will be made by streaking an amount from refrigerated slants to
new agar slants. These slants will be incubated for 24 hours in an anaerobic jar with oxygen
absorber packs and will be inoculated into each of the small anaerobic jars with 50 mL substrate
media.
After 60 hours, 10 mL will be obtained from each of the small anaerobic jars with 50 mL
dilute substrate and will be transferred to each medium anaerobic jar with 90 mL substrate media
using a pipettor with sterilized tips. The samples will be incubated again at 37°C for 60 hours.
G. Butanol Fermentation
 One mL sample will be obtained from each of the inoculum for optical density test to
ensure uniform cell concentration in all the fermentation setups. The required volume of
inoculum will be transferred into 50 mL centrifuge tubes for centrifugations. After
centrifugation, the cells will be separated from the supernatant liquid and will be resuspended
using 30 mL of dilute substrate.
A sample from the total fermentation volume will be obtained for the calculation of the
initial sugar concentration, initial pH, initial cell concentration, and initial butanol concentration.
The airlock of the anaerobic jar will be filled with concentrated H2SO4 to absorb
moisture that can be produced during the fermentation process and to ensure that only CO2 gas
will be released from the setup. The jars will be weighed every 12 hours to calculate the butanol
content based on CO2 released.
Ten milliliters of sample will be obtained every 12 hours using a pipettor with sterilized
tips and will be transferred in 15 mL centrifuge tubes. In the 60th hour, an additional 5 mL
sample will be obtained for biomass analysis. One mL from each 10 mL samples will be
transferred into Eppendorf tubes for cell count using haemacytometry. To ensure correct cell
count, the samples will be analyzed immediately.
H. Analytical Method
Fiber and surface analyses of bagasse will be done using a scanning electron microscope.The pH
changes in this study will be done using a pH meter.Viable cell counts at different sampling time
will be determined by haemacytometry. The phenol-sulfuric acid assay method will be used to
determine the total sugar concentration. Gas chromatography will be used to measure the butanol
concentration every 12 hours.
REFERENCES

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Cafugauan, J. (2013). Kinetic study on the ethanol fermentation of molasses using

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Christine, Gable, S., Christine, & Gable, S. (2017, March 15). The Process, Pros, and Cons of
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85645

Knoshaug, E. P., & Zhang, M. (2008, December 17). Butanol Tolerance in a Selection of
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Limbo, K. P. (April 2018). BUTANOL PRODUCTION FROM SUGARCANE (Saccharum

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the Philippines, Los Baños, College, Laguna.

Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers. (2014,

October 04). Retrieved January 20, 2019, from
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Virginia Schadeweg, & Eckhard Boles. (2016, February 24). N-Butanol production in
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Retrieved January 20, 2019, from
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 Rizal High School
Dr. Sixto Antonio Avenue, Caniogan, Pasig City

Butanol Production from Sugarcane Bagasse using

Saccharomyces cerevisiae

A partial fulfillment with one of the requirements

in the subject General Chemistry II

Tuico, Jenina C.
Benitez, Kaian V.
Elican, Joana Loraine G.
Morota, Arabella Nicole S.
Martinez, Kaya May O.
Roluna, Mariel D.

XI – STEM C
January 2019