New microbial sources of 2- and 3-methylhopanoids from anoxic, acidic, sulfidic hydrothermal springs

<p>Biomarkers are membrane lipids that can record microbial inputs to the sedimentary rock record and can provide a longstanding record of microbial life on Earth. Hopanoids, cyclic triterpenoid membrane lipids, are some of the most abundant bacterial lipid biomarkers on Earth. Previously, we identified the production of 3-methylhopanoids (3-MeBHPs) in <em>Rhodopila</em> sp. LVNP - a novel purple non-sulfur (PNS) anoxygenic phototroph we isolated from an acidic (pH 3.9) sulfidic spring in Lassen Volcanic National Park. We also detected 3-MeBHPs in a closely related microbe (<em>Rhodopila globiformis</em>) isolated from a similar spring in Yellowstone National Park (pH 3.4). We went back to the environments from which these cultures were isolated and performed lipid analyses on microbial consortia and sediments <em>in situ</em> to link the laboratory studies to source organisms in the natural environment. We used liquid chromatography mass spectrometry (LC-MS) analyses to characterize the intact bacteriohopanepolyol (BHP) profiles, 16S rRNA microbial community composition, and metagenomic evidence of methylated hopanoid production potential in the two acidic springs. Using the combination of these techniques, we detected a multitude of novel source organisms for both 3-MeBHPs and 2-methylhopanoids (2-MeBHPs). 3-MeBHPs are traditionally associated with oxygen requiring methanotrophic and acetic acid bacteria and have long been considered a signal for aerobic methanotrophy in the rock record. 2-MeBHPs are traditionally associated with cyanobacteria and the origins of oxygenic photosynthesis. Previous studies have found organisms that are exceptions to this but are considered not environmentally relevant outside of laboratory studies. Our work shows production of these geologically important lipid biomarkers in an anoxic natural environment where no cyanobacteria and no methanotrophs are present. This work combines laboratory studies and environmental analyses, which led to the discovery of new source organisms for methylated hopanoids. New evidence of non-traditional source organisms in these anoxic environments may have implications for re-interpretations of the sedimentary rock record.</p>