ResultsOver the course of our research

program, we’ve started to isolate several strains of bacteria.

Turtle Water Samples in Solid Media

Vivian spread water from her pet turtle’s tank onto a petri dish with biodiesel medium. After 3 days, many different bacterial colonies can be seen on the dish. We labeled several colonies A-E. Then, after inoculating each of them into a rich liquid media, we streaked each sample onto 2 petri dishes.

Of the petri dishes, those with samples B and C were chosen for further isolation, because they had the most growth. As you can see, we’ve worked with samples B and C into the 4th generation.

We’re also interested in future work in Sample E. Although it had slower growth, it was a pink colony…and colored bacteria are exciting!

Turtle Water Samples A –E (Biodiesel Media)

Turtle Water Sample B(Biodiesel

Media)

Turtle Water Sample B (Biodiesel

Media)

Turtle Water Sample B in Biodie

sel Media

Turtle Water Sample B in Rich Medi

a

Turtle Water Sample C(Biodiesel

Media)

Turtle Water Sample C (Biodiesel

Media)

Turtle Water Sample C in Biodie

sel Media

Turtle Water Sample C in Rich Medi

a

Turtle Water Sample E(Biodiesel

Media)

Gen I

Gen II

Gen III

Gen IV

Other Environmental Samples in Solid Media

We’ve also began isolating several colonies from our other environmental samples. However, we haven’t had the time to work with those bacteria beyond the third generation.

Casting Pond Water Samples A-B

Casting Pond Water Sample B

Casting Pond Water Sample B

Gen I

Gen II

Gen III

Fish Bowl Samples A-E

Fish Bowl Sample D

Gen I

Gen II

Turtle Shell Samples A-D

Turtle Shell Sample A

Turtle Shell Sample D

Gen I

Gen II

Arroyo River Samples A-D

Arroyo River Sample A

Arroyo River Sample C

Gen I

Gen II

Jar Samples in Liquid Media

We had several jars containing mid-processed biodiesel that we thought might contain bacteria. We inoculated several samples from the jars into a rich liquid media. Here is the optical density of these liquid samples over the course of several days.

Note: W is a fast-growing bacteria used frequently in the lab. Here, we used it as a positive control.

Growth of Bacteria in Jar Samples

0 1 2 3 4 5 6 7 80

0.5

1

1.5

2

2.5

Negative Control (LB broth only)

Positive Control (W)

Sample 1

Sample 2

Sample 3

Sample 4

Time [Days]

Op

tica

l D

en

sity

@ 6

00

nm

Turtle Water Samples in Liquid MediaWhen we inoculated the Generation III bacteria into tubes with rich liquid media, we measured the optical density of the tubes every hour.

Once the optical density reached 0.5-0.7, the bacteria is in log phase. This is when we streaked them onto Generation IV petri dishes.

Growth of Bacteria in Turtle Water Samples

1 2 3 4 5 6 7 80

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Bacteria Log Phase

Series1

Series2

Series3

Series4

Time (hours)

Op

tic

al D

en

sit

y (

OD

)

Sample B1Sample B2

Sample C1

Sample C2

Importance

As the search for an alternative energy source …

What is Biodiesel Fuel?• It is an alternative fuel that is made up of renewable resources and emits lower CO2 emissions than

petroleum diesel. • The process of making biodiesel fuel is called transesterification. It involves using an oil, such as

vegetable oil or waste vegetable oil, and mixing it with an alcohol, such as methanol. Then, using lye as a catalyst, biodiesel fuel is created with glycerin as a byproduct. The next steps include separating the glycerin from the biodiesel and then washing the biodiesel to remove any contaminants.

This is a picture of mid-processed biodiesel fuel. The top yellowish layer is biodiesel fuel, and the bottom caramel colored layer is the byproduct glycerin. This sample of biodiesel fuel was made from virgin soy oil, methanol, and lye.

What are some problems with Biodiesel?• With the uprising of biodiesel fuel plants, there is the potential of

biodiesel fuel spills, either from the transportation or the production of biodiesel fuel. On March 16, 2008, an article was released about an Alabama biodiesel fuel plant and its negative effects on the Black Warrior River. The article claimed that the biodiesel fuel plant was polluting the river and that the spills were harming the wildlife near that river.

• Another problem that comes from dealing with biodiesel fuel is unwanted microbial growth. Because of biodiesel fuel’s organic characteristics, it can be a good food source for some microorganisms, which is problematic in the storage of biodiesel. The growth of microorganisms in biodiesel fuel poses several storage problems. At gas stations, the fuel is stored beneath the ground, which would be difficult to clean if growth were to occur. Also, growth in fuel tanks could potentially damage the interior of vehicles.

http://www.sfgate.com/cgi-bin/article.cgi?f=/c/a/2008/03/16/MNGJVHEHO.DTL

Bioremediation• Def: the use of microorganisms to restore a contaminated environment

to its original condition [get link]• Off the shore of Alaska, there was a large petroleum diesel oil spill.

Bacteria was then brought to the spill site, and the bacteria released certain enzymes. These enzymes then break down the harmful hydrocarbons found in petroleum oil to less harmful substances. The Exxon Valdez Oil spill was one of the first times that the method of bioremediation was used to clean up an oil spill of that size.

• Our goal for this project is to identify and culture a bacteria that can be used for the bioremediation of biodiesel fuel.

http://www.bact.wisc.edu/Microtextbook/index.php?name=Sections&req=viewarticle&artid=160&page=1

Conclusion

Conclusions• Cultures can be grown directly from mid-processed biodiesel fuel, but they were slow- growing.

– Many of the samples we used that were directly from our samples grew but they were very slow growers.– After we saw they were slow growers we decided to take a side project and look in environmental places

and hope for growth in our Petri dishes. • Bacteria from different habitats can be enriched successfully with biodiesel fuel as the sole carbon

source.– We each decided to look in different places that we thought would have the most bacteria. – We looked in a total of about 14 places and most of the samples of our Petri dishes had growth except for a

few.• Biodiesel fuel is challenging to incorporate into a media.

– It’s very difficult to work with because in a liquid media the biodiesel separates away from the solution.– In a solid media the biodiesel clumps up into droplets so the biodiesel tends to emulsify.

• Biodiesel fuel droplets on Petri dishes do not stop the growth of bacteria.– In our experiment we used Petri dishes that contained agar media but since we wanted our bacteria to

grow on biodiesel we had to spread the biodiesel on top of the agar in order to see if the bacteria could grow.

– In one of our experiments we did a comparison to see if the bacteria grew the same in a nutrient broth and in a biodiesel media. The bacteria grew in the nutrient broth as we thought it would. In the biodiesel media it did grow but the bacteria were smaller.

Techniques

Aseptic Techniques

• During our project, it was very important that we used sterile or “aseptic” techniques in order to avoid contamination in our samples. We worked in a special hood called a Laminar flow cabinet , which uses a HEPA filter to blow air towards the user. Everything that goes inside of this hood must be sterilized before it enters. We used a bottle of 70% ethanol to spray down our (gloved) hands and samples going into the hood. We also used a Bunsen burner, which produces a single open gas flame and is used to sterilize tools.

Streaking Streaking is a technique of isolating pure colonies, often from bacteria. We used this

technique in order to identify, characterize , and test the bacteria . After sterilizing our inoculation loops using the Bunsen burner, we obtained some bacteria from our samples. We used petri dishes which had media on their surfaces. A media is a special gel or liquid that supports the growth of bacteria.

SpreadingSpreading is similar to streaking, but instead of using an inoculation loop, a sterilized

spreader (using 70% ethanol and the Bunsen burner) is used to spread the sample evenly across the plate. The plate is first placed on a small turn table. The user must simultaneously turn the table while controlling the spreader.

Gram StainingGram Staining is used to determine gram status to classify bacteria.

SmearingSmearing is a technique where a sample is spread onto a slide before being stained for

viewing under the microscope. The Bunsen burner is used throughout the smearing process to keep our tools and our environment sterile, so that we do not contaminate our samples. The purpose of smearing is to fix the bacteria onto the slide.

Equipment: SpectrophotometerA Spectrophotometer is a machine that let us measure the optical densities, or how

much red light was passing through our tube samples. If a sample was more turbid, meaning that it was cloudy or hazy, it told us that there was growth in that particular sample.

Hi, we're a group of students chosen to participate in Caltech's Summer Research Connection Program. We did our laboratory work at Oak Crest Institute of Science. Our summer research project was focused on microbiology in biodiesel fuel.

Veronica Garcia - Veronica Garcia is a senior at John Muir High School in Pasadena. She hopes to become a pediatrician one day. Vivian Lam - Vivian is a senior at Gabrielino High School in San Gabriel. She also wants to be a pediatrician. Carter McGee - Carter is a senior at the Webb School of California in Claremont. He is considering becoming an engineer. Breana Powell - Breana Powell is a senior at John Muir High School. She is thinking about majoring in Film or Biology, Fanny Xu - Fanny is a senior at Gabrielino High School. She is going to major in Biology, and is considering becoming a researcher. Our Teachers: Michael Winters - Mr. Winters is currently a teacher at Gabrielino High School. He teaches Environmental Technology, Drafting, Modern

Technology,... Doss Jones - Mrs. Jones is currently a teacher at John Muir High School. She teaches Biotechnology,... Our Mentors: Mark M. Baum - see http://www.oak-crest.org/oakcrest/pages/research_faculty/m_baum_faculty.htmlSherry Tsai - see http://www.oak-crest.org/oakcrest/pages/research_faculty/s_tsai_faculty.html