Exp 6 - Oil Degrading Properties of Marine Bacteria
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Page 1 of 3 Experiment 6 –
Oil Degrading Properties of Marine Bacteria PURPOSE
To examine the bioemulsification properties of Acinetobacter venetianus
RAG-1 on used and unused motor oil. INTRODUCTION
Oceanic oil spills have a predictable progression. Usually the oil spill is crude oil, which contains volatile substances that have low boiling points. These substances evaporate immediately, reducing the spill by 25%, but releasing toxic substances into the atmosphere. The remaining oil is very thick and sticky, and adheres to everything, including aquatic life. Some of the oil is degraded by naturally occurring marine bacteria. Some species have been genetically engineered to increase the amount of oil they will degrade. Acinetobacter venetianus
RAG-1 is a marine bacteria which can utilize the hydrocarbons in oil as a source of carbon. RAG-1 releases an emulsan which is a polysaccharide that will emulsify oil. PREPARATION VIDEOS Please watch all associated videos on Canvas prior to the start of your lab session. PRE-LAB QUIZ
The Pre-lab quiz for this experiment is available Canvas and must be completed before the start of your lab session to receive marks. Additionally Prepare data table 1 in your lab notebook as shown in the lab manual. REAGENTS
Cultures of RAG-1 (
Acinetobacter venetianus
) Used and fresh motor oil Carbon minimal salts culture media (also called carbon deficient media which contains the following): 2.2 g K
2
HPO
4
.3H
2
O 0.73g KH
2
PO
4
1 g (NH
4
)
2
SO
4 30 g NaCl dH
2
O to 1L 0.2 g MgSO
4
pH adjusted to 7.0 with NaOH, autoclaved and cooled to room temperature. (This is already prepared for you.)
Page 2 of 3 PROCEDURE
1. Turn on the spec 20 spectrophotometer and allow warm up for 20 minutes. 2. Number six 10 ml culture tubes with caps 1 to 6. 3. Add 3 ml carbon minimal salts media to each tube. 4. Label Tube 1 as “
blank
”
. 5. Prepare the remaining tubes 2 –
6 as follows: Tube 2 - add 60 μl of new motor oil
Tube 3 - add 60 μl of used motor oil
Tube 4 - inoculate the tube with one ml of an overnight growth of RAG-1 grown in LB broth. Use proper aseptic technique. Tube 5 - inoculate the tube with 1 ml of an overnight growth of RAG-1 grown in LB broth. Use proper aseptic technique. Add 60 μl of new motor oil
Tube 6 - inoculate the tube with one ml of an overnight growth of RAG-1 grown in LB broth. Use proper aseptic technique. Add 60 μl of used motor oil
6. Calibrate the spec 20 as follows: a. Set wavelength to 500 nm b. Place blank tube (Tube 1) in the cuvette holder c. Pres the blank button on the spec and wait. This will display a value of 0. 7. Take an initial reading of tubes 2 through 6. Record in the data Table 1. 8. Set up a shaking water bath for 30°C and 140 rpm. 9. Place tubes 2 –
6 in the shaker. Make sure to open the lids of the culture tubes every 24 hrs
to allow oxygen to enter the cultures!!
10. Take and record three absorbance
readings (every 24 hours if possible).
Page 3 of 3 Table 1: Absorbance Values at 500 nm. Tube Number A500 at 0 hrs A500 at __ hrs A500 at __ hrs A500 at __ hrs 2 3 4 5 6 DISCUSSION Before leaving this lab session please discuss the following with your instructor: 1. Is there a correlation between optical density taken of the bacteria and presence of oil? Explain. 2. Do you hypothesize there will be a difference in the results between the used and new motor oil (Tube 5 vs Tube 6)? What do you predict will happen? POST LAB - QUIZ No lab report is required for this lab, however there is a post-lab quiz on Canvas that must be completed one week after the completion of this experiment.
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1.59 x 10^-5
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0.14
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0.10-
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0.02-
0.00+
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0 2 4 6
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Cl₂
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CO
COCI₂
8 10 12 14 16 18
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