Testing the growth of Nocardioides sp. strain CF8

1. Testing the growth of Nocardioidessp. strain CF8 http://genome.jgi-psf.org/noc_j/noc_j.home.html Kelsey Drewry Dr. Luis Sayavedra-Soto Dr. Daniel Arp HHMI Summer Research 2011

2. Introduction • Fuel Spills and Leaks • Bioremediation http://news.softpedia.com/newsImage/Experts-Produce-Plastic-Without-Fossil-Fuels-2.jpg/ http://newbridgeblog.com/?tag=fuel-spill-liability http://www.thisland.illinois.edu/50ways/50ways_47.html http://www.gwpc.org/CallToAction/UndergroundStorageTanks.aspx

3. CF8’s Genome: The Starting Point Hydrogenase Gene BMO Gene NocardioidesCF8 genome

4. 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

5. Hypothesis Based on the information in its genome, CF8 will be capable of degrading: Hydrogen Toluene Methyl Tertiary Butyl Ether (MTBE) http://www.myclimatechange.net/default.aspx?Page=Article&SubjectId=42&cat=1&sub=1

6. 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

7. CF8 CF8 CF8 CF8 http://ocean.nationalgeographic.com/ocean/photos/ocean-pollution/

8. 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

9. 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

10. Measurement Methods • Optical Density (600 nm) • Unit substrate degraded per µg protein • Gas Chromatography • Hydrogen • Flame Ionization Detector • MTBE

11. 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.

12. Hydrogen Continued H2 H2 H2 O2 O2 O2 CO2 CO2 CO2

13. 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.

14. Hydrogen Continued Killed Cells Butane Only Presence of H2

15. 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.

16. Future Work with Hydrogen • Why does CF8 have the hydrogenase? • Stimulation of growth? • Anaerobic Growth? • Nitrate/nitrite and bicarbonate.

17. 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

18. 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.

19. 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?

20. 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

21. 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

22. MTBE Work Killed Cells Live Cells

23. MTBE Tentative Conclusions • MTBE is degraded by CF8. • The average rate of MTBE degradation is 50 nmol/hr · mg protein

24. 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

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