Ocean Worlds (2019) 6025.pdf METAGENOMIC PROFILING OF THE METHANE-RICH ANOXIC WATERS OF LAKE UNTERSEE AS AN OCEAN WORLDS ANALOG. N. Y. Wagner1, A. S. Hahn2,3, D. Andersen4, C. Roy5, M. B. Wilhelm6, M. Vanderwilt1, S. S. Johnson1, 1 Johnson Biosignatures Lab, Georgetown University, 2 Department of Microbiology and Immunology, University of British Columbia, 3 Koonkie Cloud Services Inc., 4 Carl Sagan Center, SETI Institute, 5 Department of Geography, McGill Uni- versity, 6NASA Ames Research Center. Untersee, Central Dronning Maud Land, East Antarcti- Introduction: Under ocean worlds conditions on En- ca.” Limnology and Oceanography, vol. 51, no. 2, 2006, celadus, it has been shown that biological methane produc- pp.1180–1194, [4] Bevington, James, et al. “The Thermal tion may be possible [1]. Also, the possibility of methano- Structure of the Anoxic Trough in Lake Untersee, Antarcti- genesis on Europa has been hypothesized [2]. Given the po- ca.”Antarctic Science, vol. 30, no. 6, 2018, pp. 333–344. tential for methanogenesis on the icy moons of Saturn and Jupiter, we are exploring the range of life capable of survival in an extremely methane-rich terrestrial analog. Lake Untersee as an Ocean Worlds Analog: Lake Un- tersee is located in Queen Maud Land, East Antarctica. It is perennially covered in 3 meters of ice and closed off from the outside world by the Anuchin glacier. The lake contains Figure 1. Depth profile of the aerobic and anoxic basins of an aerobic basin and anoxic basin (Figure 1). The aerobic Lake Untersee [4]. basin has been measured to be up to 169m deep with a con- stant temperature of 0.25˚C. It contains a well-mixed water column. The anoxic basin has a maximum depth of 100m. While the top 50m of the anoxic basin are well-mixed, simi- lar to the aerobic basin [4], the level of dissolved oxygen (DO) drops from 70-75m. Below this suboxic region is a uniformly anoxic environment (Figure 2). These anoxic waters comprise one of the most methane-rich naturally- occurring aquatic ecosystems on Earth, with CH4 levels reaching as high as 21.8 ± 1.4 mmol L-1 [3]. Using Lake Untersee as an analog allows us to study the chemosynthetic pathways used by life forms that dwell in an extreme environment that bears strong similarities to ocean worlds that are characterized by bodies of water permanently covered in ice, with low temperature, a lack of oxygen, and the presence of methane as a potential energy source. Sample Collection: From October to December of 2018, we collected samples from Lake Untersee using sterile tech- niques. Microbes were concentrated from approximately 100mL of water into ~200µL of Tris using a CP Select (In- novaPrep). DNA and RNA were extracted in triplicate using custom protocols from samples collected at ~20m in depth (the aerobic zone), ~75m in depth (the oxycline zone), ~86m, ~92m and ~99m in depth (the anoxic zone) (Fig. 1), as well as a sediment sample from the bottom of the water column (~100m in depth). Figure 2. The depth profile of dissolved oxygen in the anox- Genomic Characterization: Library preparation and se- ic basin of Lake Untersee. The pink dots indicated the points quencing will soon be complete for these samples. 16S rRNA in the water column that were sampled. The purple lines amplicon sequences will be used to compare the prokaryotic indicate the sampling depths [4]. profiles of these depths whereas metagenomic analysis will identify potential gene clusters and emergent pathways de- veloped as adaptation mechanisms to this cold, methane-rich environment. Metatranscriptomics will give a detailed look at gene expression, enabling analyses of metabolic coupling among microbes living in the water column of this lake. References: [1] Taubner, Ruth-Sophie, et al. “Biological Methane Production under Putative Enceladus-like Condi- tions.” Nature Communications, vol. 9, no. 1, 27 Feb. 2018,. [2] Seckbach, J. Life in the Universe: from the Miller Exper- iment to the Search for Life on Other Worlds: Proceedings of the Seventh Conference on Chemical Evolution and the Origin of Life, Trieste, Italy, September 15-19, 2003. Kluwer Academic Publishers, 2004. [3] Wand, Ulrich, et al. “Bioge- ochemistry of Methane in the Permanently Ice-Covered Lake .