1. Physiological Characterization of tertiary Butyl Alcohol-Degrading Bacteria Samantha Zelin Advisor: Dr.Michael Hyman North Carolina State University, Department of Microbiology, Raleigh NC 27695 Abstract Tertiary butyl alcohol (TBA) is a metabolic byproduct of the degradation of the gasoline oxygenate methyl tertiary butyl ether (MTBE). Both MTBE and TBA are recognized as important ground water pollutants. The overall aim of this project has been to establish the growth substrate range of two TBA-utilizing isolates (S1B1 and G2B2) and to investigate their respective pathways of TBA degradation. Understanding the pathway of TBA degradation could help identify treatment approaches that could be used to remediate TBA-contaminated water. In a typical experiment a colony of each strain was inoculated into mineral salts medium (10mL) in a sealed culture tube. Each substrate (5µl) was added and the tubes were incubated in a rotary incubator at 3O o C. After 7 d, the bacteria growth in each test tube was analyzed using a spectrophotometer and any significant growth is recorded. Each substrate is tested at least twice and each experiment included a TBA-grown culture as a positive control. Strain S1B1 grew on a wide range of 1 o n- alcohols (C 2 -C 10 ) whereas strain G2B2 did not utilize these compounds. Neither organism grew on 2 o alcohols, including 2-propanol, a putative intermediate in the MTBE and TBA degradation pathway. In addition to growth on TBA, strain S1B1 also grew on another branched alcohol, tertiary amyl alcohol. This activity was not observed with strain G2B2. Overall, our results show the two bacterial strains have significantly different growth substrate ranges and may have significantly different TBA degradation pathways. Materials and Methods A colony of each strain (S1B1 and G2B2) was inoculated into 10mL of mineral salts medium Five micro liters of the substrate in question were then added to the medium The test tube was sealed off and placed horizontally in a rotary incubator at 30 o C After 7 days the bacteria growth in each test tube was analyzed using a spectrophotometer and significant growth was recorded Each substrate was tested at least twice to account for any errors A positive TBA control was prepared every week LB plates were prepared each week from randomly selected test tubes that contained growth to test for contamination Results Significant growth was determined to be > 0.1 The table to the right exhibits the extent of growth of G2B2 and S1B1 on some of the substrates in question. NSG stands for no significant growth. The values on the table are the higher of the two results from the spectrophotometer analysis. Table 1: Growth of G2B2 and S1B1 on Selected Substrates Bacteria Strain SubstratesS1B1G2B2 Ethanol0.318NSG Propanol0.472NSG Butanol0.420NSG Pentanol0.478NSG Hexanol0.325NSG HeptanolNSG OctanolNSG Nonanol0.286NSG Decanol0.204NSG 2-pentanone0.227NSG TAA0.632NSG DIOL0.2440.537 HIBA0.101NSG Hydroxyacetone0.332NSG TBA (control)0.5120.448 Discussion and Conclusion References and Acknowledgments Dr. Michael Hyman Kimberly Glover Alan House In loving memory of Joel Zelin. Always questioning; always learning; forever inspiring. Introduction As stated in the abstract, TBA is a direct product of MTBE degradation and is now recognized as a potent groundwater contaminant.