Anaerobic Neutrophilic Pyrite Oxidation by a Chemolithoautotrophic Nitrate-Reducing Iron(II)-Oxidizing Culture Enriched from a Fractured Aquifer

Neutrophilic microbial pyrite (FeS₂) oxidation coupled to denitrification is thought to be an important natural nitrate attenuation pathway in nitrate-contaminated aquifers. However, the poor solubility of pyrite raises questions about its bioavailability and the mechanisms underlying its oxidation. Here, we investigated direct microbial pyrite oxidation by a neutrophilic chemolithoautotrophic nitrate-reducing Fe­(II)-oxidizing culture enriched from a pyrite-rich aquifer. We used pyrite with natural abundance (NA) of Fe isotopes (^NAFe-pyrite) and ⁵⁷Fe-labeled siderite to evaluate whether the oxidation of the more soluble Fe­(II)-carbonate (FeCO₃) can indirectly drive abiotic pyrite oxidation. Our results showed that in setups where only pyrite was incubated with bacteria, direct microbial pyrite oxidation contributed ca. 26% to overall nitrate reduction. The rest was attributed to the oxidation of elemental sulfur (S⁰), present as a residue from pyrite synthesis. Pyrite oxidation was evidenced in the ^NAFe-pyrite/⁵⁷Fe-siderite setups by maps of ⁵⁶FeO and ³²S obtained using a combination of SEM with nanoscale secondary ion MS (NanoSIMS), which showed the presence of ⁵⁶Fe­(III) (oxyhydr)­oxides that could solely originate from ⁵⁶FeS₂. Based on the fit of a reaction model to the geochemical data and the Fe-isotope distributions from NanoSIMS, we conclude that anaerobic oxidation of pyrite by our neutrophilic enrichment culture was mainly driven by direct enzymatic activity of the cells. The contribution of abiotic pyrite oxidation by Fe³⁺ appeared to be negligible in our experimental setup.