Year 11 Biology Project:

Investigating Biofuels

By: Shental Rohan

Contents:

Abstract 3
Plan of Action 4
Introduction 4
Experiment 6
Results 8
Conclusion 9
Self Evaluation 10
Abstract

This project was about investigating the sources for glucose; starch and
cellulose, which can be used in the production of biofuels. Biofuels are
extremely important because they are sustainable and renewable sources of
energy whereas fossil fuels are not. Ethanol, a type of biofuel, is produced by
yeast when they anaerobically respire on glucose. My first step in this project
was to create a Plan of Action and I decided to do this by creating a Gantt Chart
which allowed me to break down the project into smaller parts and divide my
time so that deadlines for particular tasks would be met. After conducting
thorough research using secondary sources, I designed an experiment to
investigate the optimum pH at which amylase works so that starch can be
broken down into glucose in the most efficient manner, to be used by the yeast
in respiration. I hypothesised that this would be at pH 7 since amylase in the
saliva works at a neutral pH. After I had done the experiment and recorded my
results in a table, I found the rate of reaction at each pH and used the average to
plot a graph on Sheets. I also added error bars to study the accuracy of my data.
My graph showed that the rate of reaction was highest at a pH of 6.8, proving
that my hypothesis was correct to a fairly large extent. Following the evaluation
of my data and the graphs, my final step in the project was self-reflection and
considering what I had learned, science-wise and skill-wise, in this project.
Plan of Action

Introduction

To allow them to be used by yeast, starch and cellulose must be treated through
hydrolysis because the yeast can only use glucose during fermentation. This is
difficult because cellulose has a crystalline structure and starch has a
semi-crystalline structure, which means they are stable under most chemical
conditions. Starch and cellulose can be hydrolysed either chemically or
enzymatically. Chemical hydrolysis involves using concentrated strong acids
under high temperature and pressure, however, this is not preferred because
toxic byproducts that remain will affect the yeast during fermentation. In
enzymatic hydrolysis, bacteria secrete enzymes, mainly cellulase, which break
down the cellulose and starch chains into glucose units. (Reference 5)

Once the starch has been hydrolysed, the glucose is put into the solution in the
fermenter which contains the yeast at a suitable temperature and pH. The yeast
then anaerobically respires using the glucose, and the absence of oxygen turns
glucose into ethanol and carbon dioxide. The ethanol is then removed from the
mixture and then distilled so it becomes more concentrated and can be used.
(Reference 3)

Starch is considerably simpler to be treated and be used by yeast as the refined

corn kernels only need to be mixed with amylase to be broken down to glucose
to be used by yeast. However, ethanol production is slower when dealing with
cellulose because it first needs to be mixed with refineries acids, mainly to
break down the lignin which is not fermentable, and then enzymes must be
added to break down the cellulose in a complicated process. Additionally, when
using corn it is easy to predict how much starch it contains but cellulose
contents in a plant continuously differ. However, although it is a long process,
the advantage of using cellulose is that it uses abundant plant waste whereas to
use starch, a human food source of corn must be used, which could be used to
feed people. This is why it is more ethical to use cellulose. (Reference 2)

Biofuels are necessary because they are sustainable and renewable sources of
energy and hence reduce greenhouse gas emissions as they are used in the place
of fossil fuels which are unrenewable and release an abundance of toxic gases
such as carbon dioxide which contributes to enhanced global warming. In
addition to being biodegradable and recyclable, it is also easy to use in engines,
infrastructure, and vehicles as it is almost identical to petroleum fuel in terms of
storage and pumping. Thirdly, biofuels promote economic development as it
will improve the country’s economy and provide new sources of income and
jobs for farmers in the industry. Therefore, biofuels are the necessary long-term
energy solution. (Reference 1)

There are many differences between biofuels and fossil fuels. Biofuel is a
renewable energy source made from vegetable oils and animal fats and is hence
considered a carbon-neutral source since the plants that produce biofuel will be
regrown to absorb the carbon dioxide emitted when the biofuel is burned.
However; fossil fuels, coal, oil, and natural gas, are formed from the
fossilisation of dead animals, a process that takes millions of years which makes
them nonrenewable. When they are burned, they also release high quantities of
carbon dioxide whereas biofuel does not produce toxic byproducts. (Reference
5)
Experiment
The aim of this experiment was to investigate the optimum pH that amylase
works at to break down starch into glucose. Finding this out will enable a more
efficient production of glucose to be used by yeast in industry, which will lead
to more efficient production of ethanol (biofuel).

Hypothesis

The enzymes involved in hydrolysing starch into glucose are most effective at a
neutral pH 7, which I predict is their optimum pH.

My reason behind this hypothesis is because the major enzyme involved in

breaking down starch into glucose units is amylase. This enzyme is found in the
saliva of humans which is pH 7, which is why I predict a neutral pH will be
optimum for enzyme activity

Variables

Independent Variable: pH of the solution

Dependent Variable: Time taken for the iodine to turn from black to brown

Constant Variables:
1. The temperature of the solution
2. The surroundings,
3. the volume of pH buffer used
4. The volume of the enzyme used
5. The volume of starch used
6. The concentration of the starch solution used

Equipment

1. 10 test tubes
2. Test Tube rack
3. 10 dimple trays
4. 5% starch solution
 5. 0.1% amylase solution
6. pH buffer of pH 4.5, 5, 5.5, 6, 6.5, 7, 7,5, 8, 8.5, 9
7. Two pipettes
8. Stopwatch

Method

1. Place the test tubes into the test tube rack

2. Place 1 drop of iodine in all rows of the 10 dimple trays
3. Place 2cm3 of starch solution into each test tube using a pipette
4. Label each test tube in the following order: pH 4, 4.5, 5, 5.5, 6, 6.5, 7,
7,5, 8, 8.5, 9
5. Place 1cm3 of pH buffer into each test tube according to the label using a
pipette
6. Place 2cm3 of the enzyme into the pH 4 test tube using the other pipette,
shake it, and start the stopwatch
7. After 15 seconds, use the pipette to place 1 drop of the solution onto 1
drop of iodine
8. Repeat this after every 15 seconds until the solution remains
orange-brown
9. Count the number of iodine drops used and multiply by 15 to find the
total amount of time in seconds. Record this in the table of results
10.Repeat steps 5-8 for the remaining test tubes
11.Repeat the whole experiment 2 more times, so you have 3 sets of data,
and find the average time for each pH

Risk Assessment

1. Wear protective gloves especially when working with pH buffer solution

2. Wear safety goggles

Experimental Issues

1. Preparing buffer solutions covering a range of pH

2. Observing colour change without a colorimeter
4. Obtaining amylase to use in the experiment
Results

Raw data from experiment, including average and standard deviation:

Rate of Reaction / Arbitrary units

Standard
pH Run 1 Run 2 Run 3 Average Deviation
4.5 0.917 0.909 0.833 0.887 0.046
5 3.333 4.167 5.556 4.352 1.123
5.5 12.987 11.111 14.286 12.795 1.596
6 27.778 25.000 31.250 28.009 3.131
6.5 38.462 40.000 35.714 38.059 2.171
7 37.037 40.000 34.483 37.173 2.761
7.5 24.390 25.641 20.408 23.480 2.733
8 10.638 11.111 10.000 10.583 0.558
8.5 3.030 3.571 3.226 3.276 0.274
9 0.641 0.667 0.625 0.644 0.021
Graph Analysis:

Initially, as the pH increases, the enzyme activity increases, gradually at first

and then at a faster rate. The enzyme activity peaks at pH 6.8 and then begins to
decrease as the solution becomes more alkaline.

I had expected that the highest enzyme activity would occur at pH 7 because
amylase is found in saliva in the mouth which is neutral,but the optimum pH
was 6.8, which was slightly acidic. However, there were no anomalous readings,
with all the recordings easily fitting on the graph.

From the error bars plotted on the graph, we can see that the data from pH 9 was
the most accurate, closely followed by the data from pH 8.5 and pH 4.5.
However, the data collected at pH 6 was the least accurate because of the large
error bar, so it would be advisable to repeat the experiment at this pH more
times or collect data at points around pH 6, such as pH 5.8, pH 6.1 etc.

The similarity was that both the experimental results and information from
secondary sources showed the amylase works best around a neutral pH.
However, in several secondary sources which experimented on gluco-amylase,
the optimum pH was much more acidic, around pH 4.5. The differences
between the secondary sources and the experimental results could be because of
the different type of amylase used. Both could have used amylase from different
sources (ie different animals) or different types such as gluco-amylase. The
similarities in pH are probably because amylase works at quite a stable pH, and
the different types of amylase still have similar components and properties.

The method used was straightforward and easy to understand, however instead
of judging the colour change by eye, a more accurate instrument to use would
be a colorimeter. The experiment was also repeated three times to reduce the
chance of an anomalous result. Risks were managed very well by wearing
standard lab coat safety uniform including a lab coat, safety goggles and gloves,
especially when working with the pH buffer. The results show that amylase is
most effective between pH 6.5-7, when it is slightly acidic.
Conclusion:
Overall, the results support the hypothesis to a fairly large extent. In the
hypothesis, I predicted that the enzyme’s optimum pH would be neutral (pH 7),
however from the graph, we can see that the peak activity happens at a pH of
around 6.5, which is slightly acidic.

To sum up, in this project I have found that both starch and cellulose can be
used as suitable sources for biofuels. However, although it is easier and quicker
to obtain glucose from starch, I believe that using cellulose as a source s more
sustainable and ethical because starch from corn can be used to feed our
population whereas cellulose is found in many plants, wood and grass in
abundance, and is hence worth the extra cost and time.

Self Evaluation
This project has allowed me to gain a greater understanding of biofuels, and the
importance of them for a sustainable future. Science skills I was able to practice
in this project included hypothesis testing, designing an experiment and creating
a methodology. I also considered ethical guidelines and health and safety, which
I found quite interesting to research about.

However, I have also learned many general skills that will help me in all my
subjects as I enter A Levels such as formatting features on Google Sheets and
creating graphs with error bars, which was a new skill I learned. I also was able
to understand the importance of referencing and the consequences of plagiarism,
with a bibliography being a must-have in all research reports I produce in the
future.

Self-reflection is the final skill I practiced through this project. Evaluating and
reflecting on what I have learned along with considering what could be
improved allows me to keep striving to reach greater heights in all the projects I
do in the future.

I am proud of myself for producing a good-quality piece of work, but an

improvement would be to conduct more research on ethical guidelines, which is
something I am interested in, and explore more experiments conducted which
investigate pH change on amylase.

Bibliography

Reference list

REF 1: Dale, Bruce & Holtzapple, M.. (2015). The need for biofuels. 111. 36-40.

REF 2: HowStuffWorks. (2009). How Cellulosic Ethanol Works. [online] Available at:
https://science.howstuffworks.com/environmental/green-tech/energy-production/cellulosic-et
hanol1.htm.

REF 3: Mohd Azhar, S.H., Abdulla, R., Jambo, S.A., Marbawi, H., Gansau, J.A., Mohd Faik,
A.A. and Rodrigues, K.F. (2017). Yeasts in sustainable bioethanol production: A review.
Biochemistry and Biophysics Reports, [online] 10, pp.52–61. Available at:
https://www.sciencedirect.com/science/article/pii/S2405580816302424.

REF 4: M.Sc, C.S.C.S., Diego, B.S.S. has a B.S. in earth science from the U. of C.S.,
Oceanography, is currently a graduate student in geological sciences at C.S.P.U.P.H. has done
scientific research in planetary science at the S.I. of, Astrophysics, astrophysics at the C. for,
Diego, S.S. at U.S.D.C.S. has a B.S. in E.S. from the U. of C.S., University, H. is currently a
graduate student working on an M.S. in geological sciences from C.S.P., science, P.H.
master’s research is in planetary, surveying, he has participated in published research related
to cosmochemistry while working at the S.I.G.L.H. also has experience with archaeological,
excavation, sensing, collections H. skills include satellite remote, Mapping, G., Excavation,
A., surveying, geology, mass spectroscopy C. has also published popular articles for
magazines related to, astronomy, archaeology, anthropology and history. (n.d.). Difference
Between Biofuel and Fossil Fuel | Difference Between. [online] Available at:
http://www.differencebetween.net/science/difference-between-biofuel-and-fossil-fuel/.

REF 5: Stanford.edu. (2010). Breaking Down Cellulose. [online] Available at:

http://large.stanford.edu/courses/2010/ph240/jin2/.