1. Screening Geobacillus Strains for Heavy Metal Resistance Heather Wheeler, Sanborn Regional High School, Kingston, New Hampshire Kang Wu, Department of Chemical Engineering, University of New Hampshire Abstract: The resistance of certain extremophile bacteria to heavy metals is of growing interest because of the extreme toxicity of heavy metals in groundwater pollution and the potential for use of bacterial strains in the bioremediation of such pollution. The resistance of 62 strains of Geobacillus to copper, nickel, manganese, cobalt, molybdenum, cadmium, zinc, mercury, and potassium at a high and medium concentration were tested. Of these, 14 strains demonstrated resistance to one or more of these metals and will be the subject of further investigation. Background Geobacillus thermoglucosidasius Geobacillus is a genus of thermophilic bacteria that are isolated from various geothermal environments such as compost, waste water, or oil polluted soil. They can grow over a range of 45-75 °C and are capable of surviving in non-optimal conditions in their sporulated form for long periods of time. Many have shown strong potential for bioremediation, like hydrocarbon degradation, dye detoxification, and heavy metal removal. In this study, we have characterized the resistance of 62 Geobacillus strains to nine heavy metals to screen the ones with high heavy metal tolerance for further investigation of the underlying mechanisms and optimization of strains for heavy metal bioremediation. Methods Individual strains of Geobacillus were grown on solid TBAB medium plates at 60 °C overnight, then a single colony was picked to inoculate 10ml of seed culture in TGP liquid medium. It was cultured for 6-7 hours at 60 °C on a shaker. 50µl of seed culture was used to inoculate an overnight culture in 250µl TGP or LB medium prepared with each metal at the specified concentrations (Table 1). 100µl of each culture was transferred to a 96-well microplate and the Optical Density at 600nm was measured to determine bacterial growth. Table 1. Concentration of Heavy Metals used in this study. Results Figure 1. Growth of two representative Geobacillus strains in presence of each heavy metal compared with that in absence of heavy metal. Table 2. Resistance of 14 Geobacillus strains to each heavy metal. White: limited resistance; light blue: moderate resistance; dark blue: high resistance. Reference Chatterjee, S.k., I. Bhattacharjee, and G. Chandra. "Biosorption of Heavy Metals from Industrial Waste Water by Geobacillus Thermodenitrificans." Journal of Hazardous Materials 175.1-3 (2010): 117-25. Web. Poli, Annarita, Anna Salerno, Giusi Laezza, Paola Di Donato, Stefano Dumontet, and Barbara Nicolaus. "Heavy Metal Resistance of Some Thermophiles: Potential Use of α-amylase from Anoxybacillus Amylolyticus as a Microbial Enzymatic Bioassay." Research in Microbiology 160.2 (2009): 99-106. Web. Poli, Annarita, Ida Romano, Gaetano Caliendo, Giancarlo Nicolaus, Pierangelo Orlando, Antonio De Falco, Licia Lama, Agata Gambacorta, and Barbara Nicolaus. "Geobacillus Toebii Subsp. Decanicus Subsp. Nov., a Hydrocarbon-degrading, Heavy Metal Resistant Bacterium from Hot Compost." The Journal of General and Applied Microbiology J. Gen. Appl. Microbiol. 52.4 (2006): 223- 34. Web. Özdemir, Sadin, Ersin Kilinc, Annarita Poli, Barbara Nicolaus, and Kemal Güven. "Cd, Cu, Ni, Mn and Zn Resistance and Bioaccumulation by Thermophilic Bacteria, Geobacillus Toebii Subsp. Decanicus and Geobacillus Thermoleovorans Subsp. Stromboliensis." World Journal of Microbiology and Biotechnology World J Microbiol Biotechnol 28.1 (2011): 155-63. Web. Zeigler, D. R. "The Geobacillus Paradox: Why Is a Thermophilic Bacterial Genus so Prevalent on a Mesophilic Planet?" Microbiology 160.Pt_1 (2013): 1-11. Web. Acknowledgements – This research was generously supported with funding from the National Science Foundation’s Research Experience for Teachers in Engineering Grant (ENG-1132648) http://www.bbsrc.ac.uk MnCl 2 Co(NO 3 ) 2 Na 2 MoO 4 K 2 Cr 2 O 7 HgCl 2 ZnCl 2 NiSO 4 CuSO 4 CdSO 4 91A1T W9A103 W9A44 W9A72 95A2 W9A14 96A5 W9A85 92A1T W9A86 W9A50 94A1T 96A1T W9A2 MnCl 2 Co(NO 3 ) 2 Na 2 MoO 4 K 2 Cr 2 O 7 HgCl 2 ZnCl 2 NiSO 4 CuSO 4 CdSO 4 Concentration 1 10mM 1mM 1mM 1mM 0.25mM 0.5mM 1mM 1mM 0.5mM Concentration 2 20mM 2mM 2mM 2mM 0.5mM 3mM 3mM 3mM 1mM Future Work Sequence the genome of the selected Geobacillus strains to identify the underlying pathway for heavy metal resistance.