Pyrite (FeS ) is the most abundant sulfide mineral on Earth and represents a significant reservoir of reduced iron and sulfur both today and in the geologic past. In modern environments, oxidative transformations of pyrite and other metal sulfides play a key role in terrestrial element partitioning with broad impacts to contaminant mobility and the formation of acid mine drainage systems. Although the role of aerobic micro-organisms in pyrite oxidation under acidic-pH conditions is well known, to date there is very little known about the capacity for aerobic micro-organisms to oxidize pyrite at circumneutral pH. Here, we describe two enrichment cultures, obtained from pyrite-bearing subsurface sediments, that were capable of sustained cell growth linked to pyrite oxidation and sulfate generation at neutral pH. The cultures were dominated by two Rhizobiales species (Bradyrhizobium sp. and Mesorhizobium sp.) and a Ralstonia species. Shotgun metagenomic sequencing and genome reconstruction indicated the presence of Fe and S oxidation pathways in these organisms, and the presence of a complete Calvin-Benson-Bassham CO fixation system in the Bradyrhizobium sp. Oxidation of pyrite resulted in thin (30-50 nm) coatings of amorphous Fe(III) oxide on the pyrite surface, with no other secondary Fe or S phases detected by electron microscopy or X-ray absorption spectroscopy. Rates of microbial pyrite oxidation were approximately one order of magnitude higher than abiotic rates. These results demonstrate the ability of aerobic microbial activity to accelerate pyrite oxidation and expand the potential contribution of micro-organisms to continental sulfide mineral weathering around the time of the Great Oxidation Event to include neutral-pH environments. In addition, our findings have direct implications for the geochemistry of modern sedimentary environments, including stimulation of the early stages of acid mine drainage formation and mobilization of pyrite-associated metals.

e Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).