BACTERIAL
REMEDIATION OF
OIL POLLUTION
JP SENTNER
9 TH GRADE
CENTRAL CATHOLIC HIGH SCHOOL
RATIONALE
•  Aquaspirillum serpens consumes oil.
•  Not tested on synthetic oil.
•  Effect of synthetic oil on bacteria
unknown.
•  Cellular Model- Aquaspirillum serpens
•  Petroleum Based Motor Oil- Castrol GTX
SAE 5W-30 Motor Oil
•  Synthetic Motor Oil- Castrol Edge SAE
5W-30 Full Synthetic Motor Oil
PETROLEUM BASED
MOTOR OIL
•  Petroleum goes through oil refining to
become a lubricating oil.
•  Petroleum is heated and separated based
upon the length of the hydrocarbon
chain.
•  After the distilled oil is treated and cooled
it becomes a lubricating oil.
SYNTHETIC MOTOR
OIL
•  Made in process called gasification.
•  Made using coal, water, and oxygen.
•  Main product is synthetic oil
•  Combination of CO and H2
•  By Products:
•  Ash
•  Steam
•  CO2
•  H2S
Aquaspirillum serpens
•  Prokaryotic, aerobic, marine
bacteria.
•  Is often used for cleaning oil spills.
PURPOSE
•  Determine if synthetic oil affects
the growth of Aquaspirillum
serpens differently than
petroleum based motor oil.
•  Determine if the difference in oil
concentration affects the growth
of Aquaspirillum serpens.
HYPOTHESES
•  Null Hypothesis:
•  Synthetic oil and petroleum based
motor oil will not affect the growth of
Aquaspirillum serpens.
•  Alternate Hypothesis:
•  Synthetic oil and petroleum based
motor oil will reduce the growth of
Aquaspirillum serpens.
MATERIALS
•  Aquaspirillum serpens
•  Luria Broth (LB) Media
•  Castrol GTX SAE 5W-30
•  1% tryptone
Motor Oil
•  0.5% yeast extract
•  Castrol Edge SAE 5W-30
•  1% NaCl
Full Synthetic Motor Oil
•  Sterile Dilution Fluid
•  Test tubes
(SDF)
•  Micro pipettes and tips
•  10 mM KH2PO4
•  Sterile Macro Pipette
•  10 mM K2HPO4
•  Carolina
•  1 mM MgSO4
Spectrophotometer
•  0.1 mM CaCl2
•  Incubator
•  100 mM NaCl
•  Wax paper
•  Test tube rack
PROCEDURE
1.  Aquaspirillum serpens was grown
overnight (density was approximately
108 cells per mL) in the LB media.
2.  40 test tubes were set up, 20 for each oil
and 5 replicates for each solution.
3.  The bacteria, the oils, the LB Media, and
the SDF were combined to make a 5ml
solution of 0%, 0.1%, 1%, and 10% oil.
CONCENTRATION
TABLE
Percent of
Oil
0%
0.1%
1%
10%
Aquaspirillum
serpens
1mL
1mL
1mL
1mL
Synthetic/
Petroleum Oil
0mL
0.005mL
0.05mL
0.5mL
LB Media
2mL
2mL
2mL
2mL
SDF
2mL
1.995mL
1.95mL
1.5mL
Total
5mL
5mL
5mL
5mL
PROCEDURE
4.  A blanking solution of 3mL LB Media and 1mL SDF was
also made.
5.  The spectrophotometer was blanked.
6.  The solutions were tested in the spectrophotometer at a
wavelength of 600.
7.  The bacteria tubes were incubated at 30°C throughout the
experiment.
8.  Absorbances were recorded every hour.
9.  One final absorbance reading was recorded the next
morning after 450 mins.
0.4
0.35
0.3
OIL EFFECTS
[0%], Control
[0.1%], Synth.
[1%], Synth.
[0.1%], Perto.
[1%], Petro.
[10%], Petro.
[10%], Synth.
0 min. p-value: 0.160906 345 min. p-value: 0.317807
Absorbance
525 min. p-value: 0.147201 975 min. p-value: 1.83E-08
0.25
0.2
0.15
0.1
0.05
0
0 min.
165
min.
225
min.
285
345
405
465
min.
min.
min.
min.
Time Elapsed (minutes)
525
min.
975
min.
DUNNETT’S TEST (975
MINUTES)
T-crit: 3.73
Type of Oil
Concentration
Synthetic
0.1%
Synthetic
1%
Synthetic
10%
Petroleum
Based
0.1%
Petroleum
Based
1%
Petroleum
Based
10%
T-value
Sig or Not Sig
3.451273269 Not sig
6.250803946 Sig
5.613873686 Sig
Sig
4.186952406 Sig
5.475625258 Sig
5.845933549 CONCLUSIONS
•  Null hypothesis accepted for all time periods
except for the longest exposure (975 Mins.)
•  There is significant variation between the control
and all the subjects except the synthetic 0.1%
after 975 mins.
•  The results indicate that petroleum based oil and
a high concentration of synthetic oil can affect
the survivorship of Aquaspirillum serpens over a
long exposure.
LIMITATIONS AND
EXTENSIONS
LIMITATIONS
•  The sterilization of the
blank might not have
been perfect, effecting
spectrophotometer
readings.
•  The bacteria might not
have been given
enough time to grow.
•  Limited concentrations
of oil.
•  Oil exposure?
•  Cell health (Dead or
dying cells recorded)?
EXTENSIONS
•  Longer growth
period.
•  Other brands and
types of oil.
•  Investigate cell
health.
•  Cell counts
(hemacytometer)
SOURCES
•  http://library.thinkquest.org/CR0215471/oil_spills.htm
•  http://www.synthetic-oil-technology.info
•  https://www.purdue.edu/discoverypark/energy/assets/
pdfs/cctr/outreach/Basics1-CoalGasification-Jun07.pdf
•  http://science.howstuffworks.com/environmental/energy/
oil-refining.htm
•  http://www.biology-online.org/dictionary/Aquaspirillum
•  http://www.connecticutvalleybiological.com/bacillusmegaterium-on-nutrient-agar-tube-pi-11142.html