CHAPTER 3

METHODOLOGY

This chapter focused on research methodology related to the study. It represents the research

design, the subject of the study, data gathering procedure, date analysis procedure, and ethical

consideration. The following information is provided.

Research Design

The study used statistical experimental research. Rice Hull (O. rubribarbis) was employed as

a control in the experimental design of the study to assess if it contained ethanol, while cellulase

enzyme served as the independent variable. The samples will undergo testing at a lab. To achieve this

by getting fermented Rice Hull (O. rubribarbis) it if ethanol was present.

The Subject of the Study

This research study utilized Rice Hull (O. rubribarbis) as the main lignocellulosic materials

for the study because of its accessibility, as well as its abundance in the area where this research

study was conducted. It also has the potential to be used as an alternative to current fuel production

methods. For preparation, the researchers will use grounded rice hulls. The grounded rice hulls will

undergo dilute acid pretreatment using 10% sulfuric acid solution and will kept at 102°C for under

six hours to breakdown lignin and make cellulose easier to access. The pretreated rice hulls will now

undergo enzymatic hydrolysis by using cellulase enzyme to convert cellulose into simple reducing

sugars. After the enzymatic hydrolysis, the solution will now move on to fermentation process

through the help of saccharomyces cerevisiae to convert the produced sugar into ethanol.
Data Gathering Procedure

Figure 2 below shows the process of bio ethanol production

Lignocellulosic waste
(Rice Hull)

Pretreatment
Fragmentation of solid
Dilute acid (Sulfuric acid)
Buffer ( Sodium Hydroxide)
Alteration of lignocellulosic

Enzymatic hydrolosis
(Cellulase Enzyme)

Fermentation

(Saccharomyces cerevisiae)

Bioethanol fuel

Pretreatment

Pretreatment of lignocellulosic biomass prior to hydrolysis is required for bioethanol

production because it determines the yield of bioethanol obtained after fermentation. The primary

objective of pretreatment is to reduce the compactness, strength, and crystalline nature of cellulose,

which will aid in the hydrolysis of lignocellulosic biomass to convert into simple sugar units. The

study used dilute acid pretreatment; 80 g of rice hull was mixed with 400 mL of 10% H2SO4 with 2
molars, and stored at 100 °C for two hours. The filtrate was separated from the solid residues by

filtering the mixture. 40 grams of Sodium Hydroxide (NaOH) was then diluted to 950 mL of distilled

water. After the NaOH was mixed to distilled water, it was then poured to the filtrate to neutralize pH

level of the solution to 5.0.

Hydrolysis

The aim of hydrolysis is to further degrade the polysaccharides present in the pretreated

lignocellulosic biomass of rice hull into monosaccharide subunits. The monosaccharides produced by

hydrolysis will improve the fermentation process by S. cerevisiae. Sulfuric acid was used in this

study because it was readily available and easy to handle. 10% sulfuric acid was prepared and mixed

with the lignocellulosic biomass of rice hull produced by the various pretreatment processes. The

setup was heated to 102 °C for two hours before cooling. Prior to fermentation, a glucose assay was

performed using Benedict's test to confirm the presence of reducing and non-reducing sugars. There

was a color change observed after hydrolysis, and the intensity was pretreatment dependent.

Glucose Assay

Prior to fermentation, the total reducing sugar produced from rice hull hydrolysis was

estimated. Confirming the presence or absence of sugar prior to fermentation saves a significant

amount of time and effort. Benedict's test for reducing sugars was used to determine the amount of

sugar generated by pretreatment techniques. Color changes were observed in the samples after adding

a few drops of Benedict's solution and heating in a water bath for five minutes. The color change

caused by Benedict's reaction provides a semi-quantitative or rough estimate of the reducing sugars

present in a sample. The amount of reducing sugar in a sample can be determined by measuring the
color change as follows: blue (no sugar), green (0.5% sugar), yellow (1% sugar), orange (1.5%

sugar), red (2% sugar), and brown (highest level of sugar).

Fermentation Process

Fermentation is the final stage in the production of bioethanol. The study used Saccharomyces

cerevisiae to convert the monosaccharides and some disaccharides produced during hydrolysis into

ethanol using invertase and zymase enzymes found in S. cerevisiae. The S. cerevisiae cells were

suspended in deionized water and the pretreated rice hull was used as the only carbon source for the

yeast cells.

Data Analysis Procedure

The research used a dependent-samples t-test to analyze the interval of days of fermentation

and to determine whether the fermented rice hull produced more bioethanol with regards to the days

of fermentation process using S. cerevisiae.

Ethical Consideration

In this study, the researchers made certain that all information was acknowledged and

accurately represented. The following principles will be used in this study:

Data collection. If the experiment requires gathering information from sources besides

subjects, it is important to make sure that the data is gathered ethically. This may include getting

consent to access the data, making sure the data is anonymized and cannot be linked to specific

sources, and making sure the use of the data is appropriate and required for the research.
 Integrity and Transparency. It's essential to maintain the research's integrity and to make

sure that the results are published honestly and rightfully. This means making sure that the study is

carried out with precision and objectivity and that the findings are assessed and presented objectively.

Competency. The researcher supports and develops their professional skills and competence.

Through conducting this research, the researcher continuously improves, maintains competency, and

expands their expertise.

Confidentiality. The privacy of the research study is protected. The data protection and

confidentiality protocols are in this study, and only the teachers, principal, and research members are

authorized to manage and access any data on this information.

Carefulness. The researchers avoid making careless mistakes and show no cautiousness in

the experiment. The researchers prevent thoughtless mistakes and negligence, and each of us

thoroughly reviews each member's work.

By taking these ethical considerations into account, researchers can ensure that their scientific

experiments are conducted ethically and responsibly and that the honesty of the research is

maintained.