Slide 1 - Summer Research Connection

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22 Φεβ 2013 (πριν από 4 χρόνια και 8 μήνες)

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Results

Over 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 4
th

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!





Gen IV

Gen III

Gen II

Gen I

Turtle Water
Samples A

E

(Biodiesel Media)

Turtle Water
Sample B

(Biodiesel Media)

Turtle Water
Sample B
(Biodiesel Media)

Turtle Water
Sample B in
Biodiesel Media

Turtle Water
Sample B in Rich
Media

Turtle Water
Sample C

(Biodiesel Media)

Turtle Water
Sample C
(Biodiesel Media)

Turtle Water
Sample C in
Biodiesel Media

Turtle Water
Sample C in Rich
Media

Turtle Water
Sample E

(Biodiesel Media)

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.




Gen III

Gen II

Gen I

Casting Pond
Water
Samples A
-
B

Casting Pond
Water
Sample B

Casting Pond
Water
Sample B

Gen
II

Gen I

Fish Bowl
Samples A
-
E

Fish Bowl
Sample D

Gen II

Gen I

Turtle Shell
Samples A
-
D

Turtle Shell
Sample A

Turtle Shell
Sample D

Gen II

Gen I

Arroyo River
Samples A
-
D

Arroyo River
Sample A

Arroyo River
Sample C

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
0.5
1
1.5
2
2.5
0
1
2
3
4
5
6
7
8
Optical Density @ 600 nm

Time [Days]

Negative Control
(LB broth only)
Positive Control (W)
Sample 1
Sample 2
Sample 3
Sample 4
Turtle Water Samples in Liquid Media


When 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

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1
2
3
4
5
6
7
8
9
Optical Density (OD)

Time (hours)

Bacteria Log Phase

Series1
Series2
Series3
Series4
Sample
B1

Sample
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.


Spreading

Spreading
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 Staining

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

Smearing

Smearing
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: Spectrophotometer

A

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 wo
rk
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.html

Sherry Tsai
-

see http://www.oak
-
crest.org/oakcrest/pages/research_faculty/s_tsai_faculty.html