TESTING THE GROWTH OF
NOCARDIOIDES SP. STRAIN CF8
Kelsey Drewry
http://genome.jgi-psf.org/noc_j/noc_j.home.html
Dr. Luis Sayavedra-Soto
Dr. Daniel Arp
HHMI Summer Research 2011
Introduction


Fuel Spills and Leaks
Bioremediation
http://newbridgeblog.com/?tag=fuel-spill-liability
http://www.thisland.illinois.edu/50ways/50ways_47.html
http://www.gwpc.org/CallToAction/UndergroundStorageTanks.aspx
http://news.softpedia.com/newsImage/Experts-Produce-Plastic-Without-Fossil-Fuels-2.jpg/
CF8’s Genome:
The Starting Point
Hydrogenase Gene
BMO Gene
Nocardioides CF8
genome
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
Based on the information in its genome, CF8 will be capable of degrading:
Hydrogen
http://www.myclimatechange.net/default.aspx?Page=Article&SubjectId=42&cat=1&sub=1
Toluene
Methyl Tertiary Butyl Ether
(MTBE)
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
CF8
CF8
CF8
CF8
http://ocean.nationalgeographic.com/ocean/photos/ocean-pollution/
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
CF8 Grown in Butane
CF8 Grown in Butane + H2
2500
Killed Cells
2500
2500
H2
H2
2000
2000
2000
O2
O2
O2
1500
Peak Area
Peak Area
1500
Peak Area
1500
CO2
H2
1000
1000
1000
CO2
500
500
500
CO2
0
0
0
0
50
100
150
Time After Innoculation (min)
200
0
50
100
150
Time After Innoculation (min)
200
0
50
100
150
Time After Innoculation
200
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
Degradation of Hydrogen by CF8
110
Killed Cells
100
µmol Hydrogen in Vial
90
80
70
60
Control (Killed Cells)
50
Cells Grown in Presence of H2
Cells Grown on Butane Alone
40
30
20
10
0
0
2
4
6
Hours After Addition of Hydrogen
8
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
Degradation of MTBE by CF8
20
Killed Cells
19
18
17
µmoles MTBE
16
Live Cells
15
Control (Killed Cells)
Mean Live Cells
14
13
12
11
10
0
1
2
3
Hours After Addition of MTBE
4
5
6
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
Acknowledgements






Howard Hughes Medical Institute
Cripps Fund
Luis Sayavedra-Soto
Dr. Daniel J. Arp
Dr. Kevin Ahern
Members of Arp Lab