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Abby

Grade 10 | Iowa

Significantly Increasing the Concentration of Cellulosic Ethanol Using Cedecea davisae

Introduction
Imagine a world with extreme heat waves every other year, and intense droughts, floods and hurricanes devastating every part of the biome. In this land, one third of the animals are soon to be extinct, with millions of square miles of forest gone. Diseases are widespread, with unstoppable epidemics of malaria. No one in this place has ever heard of coral reefs, icebergs, glaciers or coastal cities like Miami, Boston and New York. I call this world “the future,” the year 2100, to be exact. Your children and your grandchildren will be living in this society unless the rapid increase of global warming is stopped.

Background Research
The effects of excessive carbon dioxide emissions are becoming increasingly evident in today’s society. Due to the extensive burning of fossil fuels, the concentration of carbon dioxide in the air has doubled from an amount that had been stable for 800,000 years, and is estimated to double again by 2100 (“Global Warming Statistics”). In fact, a fifth of these emissions originate from the United States (“11 Facts About Global Warming”). All the data scientists have been collecting show that due to the increasing carbon dioxide, temperatures have been rising since 1850, with a yearly increase of 0.6ºC (1.3ºF) since 1990 (“11 Facts About Global Warming”). 

If this rise continues, there will be dramatic changes in future weather patterns, including intense rain, hurricanes, flooding, droughts, wildfires and heat waves. For example, in 2012, Iowa experienced its fifth driest summer in 140 years. On the other hand, parts of Minnesota were flooded all summer, with some towns receiving 10 inches of rain in one day. These dramatic differences illustrate how drastic the weather will become within a few years.  Additionally, heat waves are estimated to occur every other year by 2100, as opposed to once every 20 years, which will result in an additional 150,000 deaths yearly from the increased spread of diseases (“11 Facts About Global Warming”). Oceans, icebergs, animals and other habitats will also be greatly affected.  

A simple, cost-effective and beneficial solution to all of these problems is cellulosic ethanol. Cellulose is a strong fibrous material of repeating glucose strands that is found in switchgrass, cornstalks, wheat straw and grass. This organic matter can be made into ethanol through fermentation. The National Commission on Energy Policy predicts that it will be able to blend more than 30 percent of gasoline with cellulosic ethanol and sell it for under a dollar per gallon (“The Facts About Biofuels”), which would help the economy. Using cellulosic ethanol will also lower greenhouse gas emissions by 85 percent, while corn ethanol only has the potential to lower carbon dioxide emissions by 18 to 29 percent (“Fast Facts”). Additionally, this resource is widely abundant and renewable, as opposed to the oil expected to sustain the world for only 40 more years (“Researchers State Only 40 Years of Oil Left—65 Years of Gas”). In fact, to do this experiment I used my very own lawn clippings, which emphasizes the simplicity.

I have conducted these experiments to provide information on how to produce substantially higher yields of ethanol than from conventional methods. It is my hope that the results from my experiment can bring cellulosic ethanol even closer to becoming a prominent fuel source.

Experiment Introduction
Last year, I conducted a series of experiments dealing with the fermentation of grass into ethanol utilizing cellulase. Cellulase is an enzyme that catalyzes the hydrolysis of cellulose, meaning that the cellulase causes the water to react with the cellulose and break it up into individual glucose molecules. Other organisms can then consume this sugar to produce ethanol. It is a common belief that yeast is a vital microorganism in this process; however, the experiment I conducted proved that there must be a naturally occurring microorganism in grass that aids in the fermentation process, since the grass was able to ferment without yeast. The goal of this experiment was to discover what this organism is, and if it could be concentrated and added into the sample to increase ethanol production in fermentation.

Hypothesis
If I isolate a species of bacterium naturally existing in grass, concentrate it, and inoculate it into a container consisting of grass, water and cellulase, then a significantly greater yield of ethanol will be produced. This will occur because the microorganism will consume the glucose in the mixture and increase fermentation.

Materials

This is a completed grass sample.


Kentucky bluegrass, Carolina Biological fermentation containers, Carolina Biological cellulase, distilled water, balance, test tubes, incubator, autoclave, centrifuge, 0.45-micrometer cellulose acetate membrane and a gas chromatograph-mass spectrometer (GC-MS).

Sample Size
I was limited in the number of samples I could test due to the expense of the GC-MS. I had a relatively small sample size, yet I had enough containers to eliminate any outliers.

 

Control and Variables
In each experiment I conducted, I compared the samples using a control. This control was a container with 10 grams grass and 70 grams water during the first step to determine which bacteria best ferments grass. In the second step of the experiment, the control was 10 grams grass, 70 grams water and 5 grams cellulase when compared to the samples containing the isolated bacteria. Although I used a different control for different experiments, I only changed one variable in the test samples and kept the rest constant, including temperature, fermentation time, and type and amount of grass.

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These are pictures of me working in the Microbiology Laboratory at the University of Iowa.


Procedure
I designed this procedure to create the grass mixtures I used in each step of this experiment.

  1. Chop Kentucky bluegrass blades up into one-centimeter fragments. This will assist the cellulase in breaking down the cellulose.
  2. Mash 10 grams of grass and add this to a sealed container.
  3. Add 70 milliliters of distilled water.
  4. Add 5 grams of cellulase to selected samples, and allow it to dissolve in the mixture.
  5. Leave samples in an incubator at 32ºC. Shake the container daily to prevent layering.
  6. Release any accumulating pressure every 12 hours by quickly venting the containers.
  7. After a week, pour the liquid out of the containers and into separate test tubes.
  8. Place test tubes in a centrifuge and spin them at a rate of 3,800 rotations per minute to allow any debris in the solution to settle to the bottom of the test tube.
  9. Remove any bacterial contamination by filtering out the supernatant through a 0.45-micrometer cellulose acetate membrane filter.
  10. Once the mixture has been properly filtered, test the samples using a GC-MS to determine the concentration of ethanol.

Step 1: Isolating the Bacteria

In order to isolate the species of bacteria responsible for fermentation, I produced four grass mixtures using the previously described procedure. I created two of these samples with 5 grams cellulase and two without. After allowing them to ferment for five days, I observed 2 milliliters of each mixture under a microscope at 1,000 times magnification.

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(Left) This culture contains a greater amount of bacillus shape species.  The next step was to see if one of these bacteria colonies can be added to increase the rate of fermentation.

(Right) This sample consists of fewer individual coccus bacteria, illustrating that different bacteria were able to grow in the presence of cellulase.


Next, I plated the test and control samples on five different Luria Bertani (LB) agar plates. I set these plates in an incubator at 32ºC. After two days, I observed the growth of the samples. There were three bacteria identified in the test samples, including a colony of small white bacteria, one with large white bacteria, and one with yellow bacteria. The control had one dominant bacteria colony that looked like a tan smear. I named these colonies A, B, C and D, respectively.

These are the isolated bacteria colonies that I allowed to grow in separate test tubes to later be inoculated into the grass samples. The pictured plates from left to right are C, B, and A with D in the front


In order to isolate the separate bacteria colonies, I used a sterile wooden rod to transport a sample of each colony into separate test tubes containing 5 milliliters of nutrient-rich LB broth. I then placed each test tube in a heated shaking incubator to increase their growth rate.

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These are the test tubes with the grown bacteria colonies.


After shaking overnight, I could tell by the turbidity of the liquid in the test tubes that the bacteria had grown considerably. Next, I tested the fermentation ability of each isolated bacteria. To do this, I created four new grass and water samples, using the amounts listed in the procedure above. I then purified them in a pressurized autoclave so the only bacteria species present in each sample was the one I  added. Subsequently, I inoculated one milliliter of each isolated bacteria into a different grass container, labeled A, B, C and D, the last acting as a control. Each day for a week, I recorded observations of these samples in a data table.

Table One - Bacteria Observations
  Odor Observations Prediction
Bacteria A
  • Repulsive
  • Rotting grass
  • No bubbles
  • Assimilation of grass and water
Not undergoing fermentation
Bacteria B
  • Fruity
  • Similar to alcohol
  • About half an inch of bubbles
  • Release of gas when venting
Actively fermenting
Bacteria C
  • Fruity
  • Similar to alcohol
  • Nearly an inch of bubbles
  • Separation into layers
  • Audible release of pressure when venting
Actively fermenting
Bacteria D
  • Slightly grassy
  • Few bubbles
  • No layers
Not undergoing fermentation

These test tubes contain liquid from the separate grass samples. The solid particulate settled at the bottom of the container after being spun on the centrifuge. This allows for easier filtration.


To verify which samples fermented, I filtered the samples and brought them to the State Hygienic Lab at the University of Iowa. There the samples were tested using a GC-MS, an apparatus that tests the concentration of any molecule in a liquid. A GC-MS is a special oven with a coil of copper tubing in which the liquid mixture is shot through with helium gas, which acts as a solvent. The temperature is then increased, and when the boiling points of different compounds within the mixture are met, they shoot through the tubing and produce a signal whose strength depends on the concentration of each molecule within the liquid. The concentration of ethanol in the samples was then recorded.

 

Table Two - Concentration of Ethanol Inoculated with Different Bacteria
  Container A Container B Container C Container D

Concentration of

Ethanol (mg/L)

Not Detectable 4600 5600 Not Detectable
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Step 2: Identifying the Bacteria

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The four pure cultures of isolated bacteria from the grass samples were then tested at University of Iowa to determine the identity of the organism. The first step was to grow an overnight culture in LB broth. The genomic DNA was then harvested using Qiagen DNA genomic kit. Next, the polymerase chain reaction (PCR) process was performed to amplify the specific 16s rDNA section. Using the amplified 16s rDNA fragment, DNA sequencing was performed at the University of Iowa DNA core using the ABI 3730 automated sequencer. Once I received the DNA sequence, I determined the identity of the unknown bacteria by using the database NCBI Blast. After doing this, I researched facts about each bacterium.

It is significant to note that the control, which was not inoculated with the auxiliary cellulase, only detected a banal bacteria strain, while the samples augmented with cellulase allowed rare bacterium to grow, which in turn causes the fermentation process to occur.       

 

Table Three - Characteristics of Bacteria
  Name Appearance Commonly Found Ethanol Produced (mg/L)
Bacteria A Enteroccus mundtii White, rod-shaped bacillus
  • Very rare in the environment
N/A
Bacteria B Cedecea davisae White, rod-shaped
  • Rare
  • Encountered in wounds, infections, and fluids of mammals
  • Detected in gas emitted from glucose
4600
Bacteria C Failed Reaction ---------------- ----------------------------------------------------- 5600
Bacteria D Acinetobacter calcoacetius Tan, rod-shaped
  • Common
  • Found in flora of mammals' mouths
N/A

 

Step 3: Inoculating the Samples with Cedecea davisae

Knowing that Cedecea davisae is able to aid in the fermentation of grass, I wanted to test if the ethanol yield would be greater with a higher concentration of this bacterium. In order to test this, I created six samples containing 10 grams of grass, 70 milliliters of water and 5 grams of cellulase. None of these samples were autoclaved, meaning that all the bacteria naturally occurring in grass will be present. Therefore, since there is a small concentration of all the catalyzing bacteria in all the samples, each should produce ethanol. Next, I inoculated 1 milliliter of the Cedecea davisae strain I had isolated into three of the samples. The other three samples I did not add anything other than cellulase to act as the control. I let these samples sit for a week at room temperature and vented them regularly. I noticed that the controls had a fruity odor, while the test samples had a much more subtle alcohol scent.

After one week, I filtered the samples using the mentioned procedure and brought them to the State Hygienic Lab at the University of Iowa and used their GC-MS.

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(Left) I viewed the liquid from the control sample and tested sample under the microscope at 1,000 time magnification. This is a picture from the control, with small specks of bacteria and larger blobs of grass particulate.

(Right) The miniscule, bacillus shaped bacteria are the Cedecea davisea.


Results
The results were recorded in a data table. I named the three samples without extra bacteria added Control 1, 2 and 3, and the three samples inoculated with 1 milliliter of Cedecea davisae Bacteria 1, 2 and 3.

Table Four - Concentration of Ethanol with and without Cedecea davisae
  Control 1 Control 2 Control 3 Bacteria 1 Bacteria 2 Bacteria 3

Ethanol

Concentration

mg/L

1,655 ±

75.03

1,513 ±

75.03

1,542 ±

75.03

24,168 ±

9827.3

26,905 ±

9827.3

42,392 ±

9827.3

abby_graph_3

As shown by these numbers, the addition of the bacteria Cedecea davisae greatly increased the concentration of ethanol in the grass, water and cellulase mixture. In fact, this bacterium resulted in an average of 31,155 mg/L, or 3.1 percent ethanol. By comparison, the control samples without the added bacteria only produced an average of 1,570 mg/L, or 0.16 percent ethanol. In other words, the use of Cedecea davisae increased the yield of ethanol by 19 fold, or 1,900 percent.

Using statistics, I found the results to have a P-value of 0.0065. Since any null hypothesis less than 0.05 is considered valid, these results are considered highly significant.

                                                                                            

Overall Conclusion
This experiment has concluded with several interesting results. First of all, Kentucky bluegrass has a dominant bacterium that alone cannot produce ethanol. However, once cellulase is added and the reaction with water and cellulose is catalyzed, three main bacteria strands are enriched and allowed to grow. Two of these bacteria species are able to ferment cellulose, one of them being Cedecea davisae. After isolating and inoculating this bacterial colony into the grass sample, the concentration of ethanol increased by 1,900 percent.

This is significant because in the future, people could mow their lawn, place their grass clippings in a large container with water, add cellulase, inoculate with concentrated Cedecea davisae and let the mixture ferment for a week. After the grass was allowed to ferment, a distillation company could distill everybody’s mixture into pure ethanol. Done on a large scale, this will help to counteract some of the world’s problems. First of all, as mentioned in the introduction, cellulosic ethanol burns more cleanly than gasoline, meaning less greenhouse gases are emitted. If cars’ harmful emissions were decreased, pollution and global warming would be slowed. This could prevent drastic weather changes, the extinction of arctic animals, the melting of icebergs, and rising sea levels. Secondly, the world’s oil reserves are limited and will be depleted within this generation if an alternative is not found. Ethanol made from grass would be a perfect alternative because it is abundant, reusable, renewable, easily obtained and unutilized. All of these facts indicate that cellulosic ethanol made from grass would be an ideal alternative energy source.

This experiment could help the future because I have isolated a bacteria species that greatly increases the cellulosic ethanol yield. If we begin to use cellulosic ethanol in place of gasoline, we could solve many of the current problems and change the world.

Further Research
Although this experiment produced positive results, there are a few things I would change if I were to conduct it again. First of all, I would have liked to perform the DNA identification test again to identify the second bacteria that increased the rate of fermentation (Bacteria C). I would also plate the bacteria found in grass from other places, to compare the flora of my yard to others.  In addition, I would like to further experiment on how to increase the rate of fermentation. Would different temperatures, time frames, amounts of cellulase, or added bacteria produce a higher yield of ethanol? I wish to continue experimenting with this potential alternative fuel source so that one day we all can enjoy the benefits of cellulosic ethanol.

Acknowledgments
I am very grateful to my mentor, Dr. Jonathan Willet, who helped me conduct this experiment. I would like to thank Dr. John Kirby, who permitted me to work on this experiment in the Department of Microbiology at the University of Iowa. In addition, I would like to thank the Hygienic Lab at the University of Iowa for testing my samples with its GC-MS and my mentor Terrance Cain, who helped me analyze these graphs and results. I would also like to thank my family for making this experiment possible. Without them, I never could have gotten transportation to the lab or support while writing the report. My dad was especially helpful by helping me to understand the PCR, DNA sequencing, and statistical significance. My mom was also very generous by reviewing my paper numerous times.

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