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 http://upload.wikimedia.org/wikipedia/commons/9/9d/2Y6Q.jpg
Hypothesis HydrogenMethyl 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 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) 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 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