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TESTING THE GROWTH OF NOCARDIOIDES SP. STRAIN CF8
Kelsey Drewry
Dr. Luis Sayavedra-Soto
Dr. Daniel Arp
HHMI Summer Research 2011
http://genome.jgi-psf.org/noc_j/
noc_j.home.html
Introduction
Fuel Spills and Leaks Bioremediation
http://newbridgeblog.com/?tag=fuel-spill-liability http://www.gwpc.org/CallToAction/UndergroundStorageTanks.aspxhttp://www.thisland.illinois.edu/50ways/50ways_47.html http://news.softpedia.com/newsImage/Experts-Produce-Plastic-Without-Fossil-Fuels-
2.jpg/
Introduction
CF8 uses monooxygenase to break down C(2) to C(16) alkanes
Monooxygenase has potential to degrade a variety of substrates, including various environmental toxins
http://upload.wikimedia.org/wikipedia/commons/9/9d/2Y6Q.jpg
Hypothesis
Hydrogen Methyl Tertiary Butyl Ether(MTBE)
Toluene
Based on the information in its genome, CF8 will be capable of degrading:
http://www.myclimatechange.net/default.aspx?
Page=Article&SubjectId=42&cat=1&sub=1
Objectives
Explore the range of substrates that can be oxidized by CF8.
Further explore CF8’s potential in bioremediation.
http://harrisonstatham.com/start-your-own-
website
Methods
Preparation of basic growth media Growth and maintenance of stock
culture Cells grown on media (50 or 250 ml)
in 150 or 500 ml bottles at 36 ˚C with shaking
Stock cells grown on butane (20-30% headspace)
Cells grown in presence of hydrogen have additional 5 ml H2 gas
Methods Continued
Testing of enzyme activities after substrate addition Addition of substrate, allowance for growth,
measurement of substrate degradation using appropriate methods
Measurement of substrate degradation
Measurement Methods
Optical Density (600 nm) Unit substrate degraded per µg protein
Gas Chromatography Hydrogen
Flame Ionization Detector MTBE
Hydrogen
Why hydrogen? Hydrogenase is indicated in genome Is it expressed? Does presence of hydrogen stimulate cell growth?
Cells (grown with and without presence of H2) centrifuged and resuspended to an OD of approx. 2.0
1 ml aliquots into 8 ml vials, 100 µl H2 added. Gas chromatography used to measure degradation
of hydrogen.
Hydrogen Continued
0
500
1000
1500
2000
2500
CF8 Grown in Butane
Time After Innoculation (min)
Peak A
rea
0
500
1000
1500
2000
2500
CF8 Grown in Butane + H2
Time After Innoculation (min)
Peak A
rea
0 20 40 60 80 100
120
140
160
180
200
0
500
1000
1500
2000
2500Killed Cells
Time After Innoculation
Peak A
rea
H2H2
H2
CO2
O2O2
O2
CO2
CO2
Hydrogen Tentative Conclusions
• Hydrogenase is expressed by CF8.• Cells grown in butane+H2:
• consume more H2 than cells grown in butane alone.
• Cells grown in butane alone:• consume more O2 than cells grown in butane+H2.
• produce more CO2 than cells grown in butane+H2.
Hydrogen Continued
0 1 2 3 4 5 6 7 80
102030405060708090
100110
Degradation of Hydrogen by CF8
Control (Killed Cells)
Cells Grown in Presence of H2
Cells Grown on Butane Alone
Hours After Addition of Hydrogen
µm
ol H
ydro
gen
in V
ial
Killed Cells
Presence of H2
Butane Only
Hydrogen Tentative Conclusions
Cells grown in presence of hydrogen degrade hydrogen at a rate of 0.45 µmol/hr · mg protein
Cells grown in butane alone degrade hydrogen at a rate of 0.32 µmol/hr · mg protein
These results suggest that the hydrogenase must be induced by the presence of hydrogen to become effective.
Future Work with Hydrogen
Why does CF8 have the hydrogenase? Stimulation of growth? Anaerobic Growth?
Nitrate/nitrite and bicarbonate.
Toluene
Used as octane enhancer in gasoline, also in paint, paint thinners, rubber, etc.
Leaches into soil and groundwater when disposed, and with underground leaks (especially during transfer and storage of fuel)
http://www.gwpc.org/CallToAction/UndergroundStorageTanks.aspx
Toluene Investigation
Cells grown with 5 mM toluene for about 7 days No butane or alternate substrate, cells grow on
toluene alone. OD equivalent to cells grown on butane.
Since CF8 grows on toluene alone, it must have a toluene oxidizing enzyme, possibly a toluene monooxygenase.
Toluene Investigation
Is the enzyme induced by presence of toluene? Comparison of growth of cultures on toluene
inoculated with butane grown and toluene grown cultures.
What biochemical pathway is CF8 using to break down toluene? Growth of CF8 on intermediates of known pathways
What are the products of toluene degradation by CF8? Are they less harmful to the environment than toluene?
MTBE
Methyl Tertiary Butyl Ether (MTBE) Highly produced gasoline additive (200,000
barrels per day in 1999) Helps oxygenate gas, burn more completely
and reduces harmful tailpipe emissions FDA studies indicate MTBE may be a
carcinogen Affects taste/smell of water at 5-15 µg/L (5
ppb) Travels faster and further through water
supply than many other chemicals due to solubility
http://upload.wikimedia.org/wikipedia/commons/a/a9/MTBE-2D-skeletal.png
MTBE Work
Cells grown on butane for about 2 days
Washed and resuspended with media to an OD of approx. 2.0
Small, varying concentrations of MTBE added
Degradation measured with FID (2 µl injections)
http://www.gchplc.com/gas-chromatography.html
MTBE Work
0 1 2 3 4 5 610
11
12
13
14
15
16
17
18
19
20
Degradation of MTBE by CF8
Control (Killed Cells)Mean Live Cells
Hours After Addition of MTBE
µm
ole
s M
TB
E
Killed Cells
Live Cells
MTBE Tentative Conclusions
MTBE is degraded by CF8. The average rate of MTBE degradation is 50
nmol/hr · mg protein
Conclusions
CF8 can degrade hydrogen, toluene and MTBE. CF8 can use toluene for growth. CF8 has potential in bioremediation.
With Further Research: Products and toxicity of toluene and MTBE
degradation
Recommended