American Society of Microbiology North Central Branch Meeting October 21-22, 2016 Department of Geological and Atmospheric Sciences Nick Lambrecht¹, Elizabeth Swanner¹, Chad Wittkop², Cody Sheik³, Sergei Katsev³ ¹Department of Geological and Atmospheric Sciences, Iowa State University, Ames, IA; ²Department of Chemistry and Geology, Minnesota State University Mankato, Mankato, MN; ³Large Lakes Observatory, University of Minnesota Duluth, Duluth, MN The Isolation of a Novel Photoferrotroph from Brownie Lake Provides a Mechanism for Studying the Iron Biogeochemical Cycle of a Pre-Oxic Earth Put numbers on poster Introduction Anoxygenic phototrophic Fe(II)-oxidizing bacteria (“photoferrotrophs”) potentially thrived in early Earth oceans whose chemistry lacked oxygen and was dominated by dissolved Fe(II) (i.e. “ferruginous”). Due to the lack of analogous chemical conditions in the oceans today, it is not well understood how iron biogeochemistry functions in such a system, where photoferrotrophy may have been the predominant pathway for primary productivity. Brownie Lake is a chemically stratified lake in Minnesota with oxygen contents dropping to <10µM at 5m below the lake surface and dissolved Fe(II) reaching 650µM in the bottom waters. The shallow depth of the chemocline in Brownie Lake should promote a robust community of photoferrotrophs. We seek to: Detect bigeochemical signatures of photoferrotrophy by documenting lake geochemistry with depth Assess overall diversity and biogeochemical pathways with 16S shotgun sequencing of environmental samples Demonstrate photoferrotrophy in environmental organisms detected by sequencing through isolation Methods Analyze Brownie Lake biogeochemistry Measure Fe(II), nutrients, light, oxygen, and conductivity at discrete depths Obtain DNA from environmental samples for PCR and 16S Shotgun sequencing to assess who is present in the bacterial community Filter chemocline waters to collect cells on filters Extract DNA from filters via Lever et al. 2015 protocol Culture a photoferrotroph by inoculating Fe(II)-rich medium with Brownie Lake chemocline water under: Anoxic conditions Using a light filter to allow only wavelengths >700 nm (excludes organisms utilizing chlorophyll a) Incubation at 20°C Figure 1. Ideal schematic of a ferruginous system, showing the chemocline between oxygen and Fe(II). This chemocline is illuminated, driving primary productivity and Fe(III) production by photoferrotrophic organisms. III. Results (1) Brownie Lake Biogeochemistry (2) PCR and 16S Shotgun Sequencing (3) Culturing a Photoferrotroph IV. Conclusions Brownie Lake is a ferruginous lake with the appropriate chemical and physical conditions to harbor photoferrotrophs Bacterial orders known to include photoferrotrophs are present in Brownie Lake An Fe(II)-oxidizing enrichment culture grew from Brownie Lake V. Future Directions Sequence extracted DNA from the culture Physiologically characterize isolate: Electron donors beside Fe(II)? Optimal growing conditions (temp, pH, light)? Innate antibiotic resistance? (3) Identify putative Fe(II) oxidation genes in isolate Figure 3. PCR indicating the presence of green sulfur bacteria using primer set GSB532F-GSB822R. Figure 4. PCR indicating the presence of purple phototrophic bacteria using primer set pufM557F-pufM750R. Figure 6. Fe(II)-rich culture tubes exhibiting putative photoferrotroph growth after inoculation by Brownie Lake chemocline water. Figure 5. Abundance of known photoferrotroph-containing orders in Brownie chemocline. Figure 2. A composite schematic of biogeochemical data from Brownie Lake. Acknowledgements: I would like to thank Elizabeth Swanner, Chad Wittkop, and Cody Sheik for providing figures. Lever, M. A. et al. (2015). A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types. Frontiers in Microbiology, 6(MAY).