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Denitrification driven by pyrite oxidation can play a major role in the removal of nitrate from groundwater systems. As yet, limited information is available on the interactions between the micro-organisms and aqueous and mineral phases in aquifers where pyrite oxidation is occurring. In this study, we examine the groundwater and sediment composition along a well-characterized redox gradient in a heavily nitrate-polluted pyritic sandy aquifer in Oostrum (the Netherlands) to identify the sequence of steps involved in denitrification coupled to pyrite oxidation. Multi-isotope analyses (δ¹⁵N-NO₃⁻, δ¹⁸O-NO₃⁻, δ³⁴S-SO₄²⁻, δ¹⁸O-SO₄²⁻ and δ³⁴S_pyrite) confirm that pyrite is the main electron donor for denitrification at this location. Enrichment factors derived from the observed changes in nitrate isotopic composition range from −2.0 to −10.9‰ for ε¹⁵N and from −2.0 to −9.1‰ for ε¹⁸O. The isotopic data indicate that …