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

Abstract

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 and 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 Fe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled andC-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 (, ) increased in abundance under acetate-amended conditions, whereas genomes related to and 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. 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 , 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.

Investigating the Composition and Metabolic Potential of Microbial Communities in Chocolate Pots Hot Springs

“…Raw sequences were processed using QIIME following the protocol for 454 pyrosequencing data or Illumina MiSeq 16S rRNA gene amplicon sequencing data, according to previously published methods (Fortney et al, 2016 , 2018 ).…”

“…mtoABCD ; Rhodobacter ferrooxidans, foxEZY ; and Rhodopseudomonas palustris, pioABC ; Ilbert and Bonnefoy, 2013 , and references therein) using command-line BLAST and the BLASTp function in IMG. Genes coding for putative EET systems, which are not homologous to known models, were identified according to previously published methods (Fortney et al, 2018 ). MAGs encoding putative outer-membrane porins, and multiheme c- type cytochromes ( c- cyts) with predicted extracellular and periplasmic locations, as well as other supplemental genes predicted to be involved in Fe transformation pathways, are hereafter referred to as pcc -like EET systems.…”

“…For example, natural amorphous Fe(III)-oxides from CP were shown to host communities containing known and putative FeRB (Fortney et al, 2016 ). Subsequent incubation experiments combined with stable isotope probing (SIP) experiments using 13 C-labeled acetate identified putative FeRB under acetate-stimulated and unamended incubation conditions (Fortney et al, 2018 ). However, constraints on the spatial distribution of FeRB within the sediment column of CP were not examined in detail.…”

A review of the mechanisms of mineral-based metabolism in early Earth analog rock-hosted hydrothermal ecosystems

“…The iron (hydr)oxide mats that form at Chocolate Pots (pH 5.8 to 6.0 at source), a vapor influenced mildly acidic spring just south of Norris Geyser Basin (Allen and Day 1935), is perhaps the most well studied iron (hydr)oxide mat in a hot spring in YNP if not the world. This spring has been studied extensively from both the mineralogical [e.g., (Wade et al 1999;Wu et al 2013)] and the biological perspectives [e.g., (Fortney et al 2016;Fortney et al 2018a;Fortney et al 2018b;Pierson and Parenteau 2000;Trouwborst et al 2007)]. Current understanding suggests that Fe(II) in the source vent waters of Chocolate Pots is rapidly oxidized by O2 that is in supersaturating concentrations due to the activity of oxygenic Cyanobacteria.…”

“…Homologs of putative porin-cytochrome complexes, which have been hypothesized to be involved in EET and iron reduction in Geobacter (Liu et al 2014), have been identified in iron oxide mats recovered from Chocolate Pots hot spring in YNP (Fortney et al 2016;Fortney et al 2018a;Fortney et al 2018b). These homologs were related to divergent Ignavibacterium-and Thermodesulfovibrio-like populations, among others.…”