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Genigraphics® has been producing output from PowerPoint® longer than anyone in the industry; dating back to when we helped Microsoft® design the PowerPoint® software. US and Canada: 1-800-790-4001 Email: info@genigraphics.com [This sidebar area does not print.] Characterization of Hydrocarbon Degradation by Thermophilic Geobacillus Strains Michael Pappas and Kang Wu Chemical Engineering Department, University of New Hampshire Michael Pappas University of New Hampshire Chemical Engineering Email: mcq92@wildcats.unh.edu Contact 1.Chemical Engineering Dept, UNH 2. Dr. Kang Wu 3. Erin Drufva Acknowledgements Geobacillus is a genus of thermophilic bacteria that grow at 45-75 °C. They are isolated from various geothermal environments, such as hot springs, soil, wastewater, and compost, and due to these harsh conditions of their habitats, many of them have shown great potential for bioremediation. In this study, we characterized the ability of five Geobacillus strains isolated from oil contaminated soils and oilfields to degrade crude oil and selected one strain that is able to degrade n- alkanes with a wide spectrum of carbon numbers. Background Results Figure 1. Growth of strains W9A95 (Black square), W9A101 (Green diamond), W9A104 (Red square), W9A111 (Purple circle) and 92A2 (Blue triangle) in minimal media with 1% (w/v) of crude oil as sole carbon source. Cells were shaken and grown at 60°C. Methods -Strains W9A95, W9A101, W9A104, W9A111 and 92A2 were grown overnight on TBAB plates at 60°C -A single colony was selected from each plate and grown in overnight TGP media and shaken at 60°C -Cells are then washed with minimal media and added to 100 mL of minimal media until the OD of the media was 0.05. 1% (w/v) of either crude oil or hydrocarbons are then added to the flask. For hydrocarbons greater than C22, 0.001% (w/v) PLYsurf was added as a dispersant. The innoculated media is then shaken at 60°C for six days with OD readings taken every day. -GC-MS and GC-FID tests to quantify the degradation of n-alkanes -Directed evolution of the strain W9A104 to enhance its abilities to degrade hydrocarbons -Sequence both wildtype W9A104 and evolved W9A104 to elucidate the pathway of n-alkanes metabolism and identify novel enzymes involved in hydrocarbon degradation. StrainC8-C16C18-C24C28-C44Cyclic Compounds W9A95 ---- W9A101 -+-- W9A104 ++ +- W9A111 ++ -- 92A2 +++-- Figure 2. Predicted pathways for alkane degradation via alkB and ladA genes. [4] [1] Feng, L., et. al. “Isolation and characterization of a novel thermophilic Bacilus strain degrading long-chain n-alkanes” Extremophiles 2006 Aug; 10(4): 347-56 [2] Shen, B., et. Al. “ Molecular characterization of the alkB gene in thermophilic Geobacillus sp, strain MH-1” Research in Microbiology 160 (2009) 560-566 [3]Geobacillus Thermoglucosidasius Y41MC1. Digital Image. Geobacillus Enzymes. C56 Technologies, 2016. Web. [4]S, Kira, and Mathais V. Digital image. N.p., 10 July 2010. Web. [5] Feng, L., et. al. “Proteomics analysis of aromatic catabolic pathways in thermophilic Geobacillus thermodenitrificans NG80-2” Journal of Proteomics 75 (2012) 1201-1210 Future Work Discussion References Table 1. Displays + positive or – negative growth for strains W9A95, W9A101, W9A104, W9A111 and 92A2 in minimal media with 1% (w/v) of hydrocarbons as the sole carbon source. Cells were grown in a flask at 60°C with vigorous shaking.