Project: Second Generation Biofuels

Goal: Determine the effectiveness of cellulosic sources of biomass to generate ethanol for biofuel.

How is the follow-up project similar/different to the experiment you did in Lab 3?

● In Lab 3 you measured fermentation in first generation biofuels that contain a significant amount
of glucose. The lab manual discusses the ethical concerns around corn and sugarcane as
biomass for biofuel. In this project you will explore second generation biomass: cellulosic
sources. All plant cells contain a cell wall, which is made up of cellulose. Cellulose is a long chain
of glucose molecules.
● Yeast cells do not have the ability to cleave cellulose into the glucose it needs for fermentation,
therefore in this project, you will need to treat the cellulosic sources of biomass with an enzyme
called cellulase prior to the fermentation reaction.
● You will choose three sources of second generation biomass that are more ethical and
sustainable than corn and sugarcane and prepare the extracts at home to bring to the lab for
weeks 2 and 3 of the project. (For example, you can consider plant materials like food waste,
grass, paper, sawdust, etc) and have three replicates for each biomass.
● You will measure the glucose content of each extract before you treat with cellulase and after
you treat with cellulase. The cellulase reaction should break down the cellulose into glucose,
and then you will use these second generation extracts to measure fermentation in the same
way you did in Lab 3.
● You will measure the OD of the yeast culture each week

Checklist of data needed for Portfolio Figures:

Individual work to collect the data and make the figure:

o Microscopy image of yeast with scale bar (each student must create their own
microscopy image)

The rest of the data collection is a team effort, depending on how your team distributes the
workload. Although the data is shared, each person uses the shared data to make their own
figures for the Portfolio Assignment.

o Graph or Table that shows the OD for the culture on both days
o Results from the experiment:
 o Table that shows the pH of each extract, and glucose content before and after
cellulase (minimum of three biomasses tested)
o Graph with the rate of fermentation for all three biomasses plus controls

Expectations

1. Project Design: In your project design you need to achieve a balance between number
of treatments and number of replicates. You will be assessed on good use of lab time
ie- if you plan an overly simple experiment with inadequate treatments or replicates,
you will not score well on this component. But you are also limited by time, so mapping
out your project with reasonable timelines is important.

2. Personal Contribution: You must contribute equally to the project. We have a few
checks and balances in place to ensure all team members are contributing a fair amount
of work (ex: GANTT chart and Google Data Sheet that are shared with your lab
instructor).

3. Portfolio Assignment: You will present the results from your project in a Portfolio
assignment. The assignment will include presenting your data (including statistical
analysis), conclusions, significance of research, identifying sources of variation,
application of lecture knowledge, a reflection on the dynamics of working in your team,
in addition to other things. Your portfolio is 12% of your grade, therefore it is important
you design a meaningful project.

All work for the portfolio assignment is INDIVIDUAL work. Group collaboration on the
portfolio is NOT permitted. Your work with the group is to design the experiment, divide
the labour equitably and share data.

Suggested Workflow:

Week 1:
● Plan Project and sign team contract (90 min)
○ Create the tables you need to record your data over the next two weeks
● Practice measuring OD of yeast culture
● Get a good microscope image of yeast for Portfolio assignment (individual work)
● Practice Durham tube set up
Week 2:

A. Determine the rate of fermentation for Biomass 1

● Cellulase treatment of Biomass 1.

● OD of culture
● Glucose and pH measurements
● Rate of fermentation for biomass 1 (plus controls)

Week 3:
Determine the rate of fermentation for Biomass 2 and 3:

● Cellulase treatment of Biomass 2 and 3.

● Glucose and pH measurements
● OD of culture
● Rate of fermentation for biomass 2 and 3 (plus controls)

Procedures
Yeast Culture Procedure
Add 25 g of yeast to 400 ml of tap water, cover with parafilm and stir on the stir plate at 30
degrees.

Microscopy Image Procedure

Each individual person needs their own microscope image of yeast cells , with a scale bar, so be
sure to take your picture through the eyepiece that has the ocular micrometer and record what
objective you used to take the picture . You will likely need to make a 1/10 dilution of the yeast
to be able to see individual cells.

Optical Density Procedure

Measuring the OD (= optical density) of a culture gives you an approximate idea of how
concentrated a culture is. An OD reading is similar to how you used the spec to measure
absorbance: you will measure the yeast absorbance at 600 nm. The difference in this case is
that you are actually measuring the turbidity of the culture: the yeast cells themselves don’t
absorb 600 nm of light, but cause light scattering. The more yeast cells = more light scattered =
less light detected = higher absorbance values. Because the degree of light scattering will
depend on the size and shape of the cells, and is specific to each spectrophotometer, it is
difficult to draw a relationship between OD reading and cell concentration without making a
specific standard curve for each spectrophotometer and cell type used. Therefore you will use
OD readings in your portfolio, rather than converting to cell concentration. You can assume that
a culture with an OD reading of 1.5 has more cells than a culture with an OD reading of 0.8*.

You are using yeast to ferment one biomass one week, and a different culture will be made the
second week for Biomasses 2 and 3. These cultures might not be the same concentration each
week, so you will measure the OD each week and include this information in your Portfolio. You
do not need replicates for the OD, you can assign people on your team each week to measure
the OD and share the OD data.

* You will most likely need to do a 1/10 dilution of the yeast in a large test tube to get an
absorbance value that isn’t flashing.

Biomass Extracts Procedure

Please make the extracts at home and bring them to the lab in the appropriate week.

Each person in the group can make their own, or one person can make enough for everyone. If
each person is making their own, be sure to agree on the amount of biomass you will use (ex: if
two people make banana peel extract, and one person makes an extract with an entire banana
peel, and the other person uses half a banana peel, this will lead to variation). You need 15 ml
of this extract/ fermentation reaction, so blending the biomass to create 250 ml will give you
plenty. The extract needs to be liquid enough that you can use it in your Durham tubes (ex: if
you blend a banana peel and it is really thick, then it won’t work well in the fermentation part.)
Try to filter out the chunky debris. You can use a coffee filter if you have one or let it sit for a
while so the debris settles to the bottom, and you can pour out the less chunky part.

Remember to bring this in on the day of your lab! Do this at home before you come to lab!
Measuring the pH and Glucose content Procedure
Because you are using second generation biomasses, it is necessary to treat the extracts with
the cellulase enzyme before you measure fermentation. As you know from the enzymes
experiment, enzymes work at an optimal pH:
1. Use the pH strips to measure the pH of the biomass (one person can do this and share
the data with the team).
2. Use the glucose strips to measure the glucose content in the extract that was not
treated with cellulase and in the extract that was treated with cellulase. (one person can
do this and share the data with the team).

Cellulase Procedure
Because you are using cellulosic biomasses, it is necessary to treat the biomass
with an enzyme called cellulase. The cellulase enzyme will break down cellulose
into glucose, so that there is glucose for the yeast to ferment to produce ethanol.
Yeast do not have the enzymes necessary to cleave the bonds that link the
glucose molecules that make up cellulose, therefore you are using a cellulase
enzyme that we purchased from a biological supply company. The high costs of
cellulase enzymes, along with difficulties of pre-treating the biomass are a
significant barrier to using cellulosic biomass in the biofuels industry.

The cellulase you are using in this lab comes from fungi. The optimal conditions for this enzyme
are between 50-55C at pH 5. The complex matrix of the microfibril (shown below) makes it
difficult for the cellulase enzymes to access the cellulose. In order for the cellulase enzyme to
access the cellulose, it is necessary to first pre-treat the biomass (ex: mechanical shearing and
heat) prior to adding cellulase.

1. Heat your extract in the microwave to help disrupt the microfibril structure on the plant
cell walls. 30-60 seconds is likely sufficient for the volumes you have. Be careful not to
burn yourself when removing your extracts from the microwave. Allow your extracts to
cool down before the next step.
2. For the extracts you are treating with cellulase: Put 15 ml of the extract in a large test
tube and add 5 ml of the cellulase enzyme.
 For the extracts that are not treated with cellulase, put 15 ml of the extract in a large
test and add 5 ml of water.
(you need 20 ml / fermentation reaction)
3. Place the tubes in the 50 C water bath (optimal temperature for cellulase) for 15
minutes. During this time, the cellulase enzyme will break down cellulose into glucose.
4. After the 15 minutes, measure the glucose content for both the cellulase treated and
not- cellulase treated extracts (only one person needs to do this and share this data with
the team)
5. Now your extracts are ready to use in the fermentation experiment, and you have an
idea of how much glucose is in each extract.

Fermentation Procedure
You will follow the same procedure as in Lab 3, where you mix 4 ml of yeast with 20 ml of your
extracts in a Durham tube and measure the amount of C02 at the top of the small inner test
tube (keep the fermentation reactions at 30 degrees C). Like in Lab 3, you will measure CO2
every 5 minutes so you can determine the rate of fermentation. You will need 3 replicates for
each of the following:

Glucose control
No substrate control
Biomass 1 that was not treated with cellulase
Biomass 1 that was treated with cellulase
Biomass 2 that was not treated with cellulase
Biomass 2 that was treated with cellulase
Biomass 3 that was not treated with cellulase
Biomass 3 that was treated with cellulase

- Each person needs to determine the rate of fermentation (as we did in Lab 3)
- Record your data in your team spreadsheet as rate of C02/min
- Be sure that everyone is sharing their data in the team spreadsheet, as you will need
this data to graph your findings (means with confidence interval error bars) and do
statistical analysis. You are working as a team to collect the data, but the analysis
(graphs and statistics) are individual work.
Clean-Up
Rinse all test tubes and large pipettes and leave test tubes upside down in the
racks so they dry for the next group
Return all equipment and reagents to the correct location
Rinse the flask of yeast out
Rinse all slides so that they can be re-used all week, but coverslips can go in
the glass waste