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/

Nocardioides CF8genome

Hydrogenase Gene

BMO Gene

CF8’s Genome: The Starting Point

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

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

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

Acknowledgements

Howard Hughes Medical Institute Cripps Fund Luis Sayavedra-Soto Dr. Daniel J. Arp Dr. Kevin Ahern Members of Arp Lab