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Chocolate Pots hot springs (CP) is a circumneutral-pH Fe-rich geothermal feature located in Yellowstone National Park. Previous Fe(III)-reducing enrichment culture studies with CP sediments identified close relatives of known dissimilatory Fe(III)-reducing bacterial (FeRB) taxa, including Geobacter and Melioribacter However, the abundances and activities of such organisms in the native microbial community are unknown. Here, we used stable isotope probing experiments combined with 16S rRNA gene amplicon and shotgun metagenomic sequencing to gain an understanding of the in situ Fe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled and 13 C-labeled acetate to target active FeRB. We searched reconstructed genomes for homologs of genes involved in known extracellular electron transfer (EET) systems to identify the taxa involved in Fe redox transformations. Known FeRB taxa containing putative EET systems ( Geobacter , Ignavibacteria ) increased in abundance under acetate-amended conditions, whereas genomes related to Ignavibacterium and Thermodesulfovibrio that contained putative EET systems were recovered from incubations without electron donor. Our results suggest that FeRB play an active role in Fe redox cycling within Fe-Si oxide-rich deposits located at the hot spring vent. IMPORTANCE The identification of past near-surface hydrothermal environments on Mars emphasizes the importance of using modern Earth environments, such as CP, to gain insight into potential Fe-based microbial life on other rocky worlds, as well as ancient Fe-rich Earth ecosystems. By combining stable carbon isotope probing techniques and DNA sequencing technology, we gained insight into the pathways of microbial Fe redox cycling at CP. The results suggest that microbial Fe(III) oxide reduction is prominent in situ , with important implications for the generation of geochemical and stable Fe isotopic signatures of microbial Fe redox metabolism within Fe-rich circumneutral-pH thermal spring environments on Earth and Mars.

Stable Isotope Probing for Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park