TESTING THE GROWTH OF NOCARDIOIDES SP. STRAIN CF8 Kelsey Drewry Dr. Luis Sayavedra-Soto Dr. Daniel Arp HHMI Summer Research

Introduction  Fuel Spills and Leaks  Bioremediation

Nocardioides CF8 genome 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

Hypothesis HydrogenMethyl Tertiary Butyl Ether (MTBE) Toluene Based on the information in its genome, CF8 will be capable of degrading:

Objectives  Explore the range of substrates that can be oxidized by CF8.  Further explore CF8’s potential in bioremediation.

CF8

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 H 2 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 H 2 ) centrifuged and resuspended to an OD of approx. 2.0  1 ml aliquots into 8 ml vials, 100 µl H 2 added.  Gas chromatography used to measure degradation of hydrogen.

Hydrogen Continued H2H2 H2H2 H2H2 CO 2 O2O2 O2O2 O2O2

Hydrogen Tentative Conclusions Hydrogenase is expressed by CF8. Cells grown in butane+H 2 : consume more H 2 than cells grown in butane alone. Cells grown in butane alone: consume more O 2 than cells grown in butane+H 2. produce more CO 2 than cells grown in butane+H 2.

Hydrogen Continued Killed Cells Presence of H 2 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)

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

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)

MTBE Work 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