Nitrate reduction and iron (II) oxidation by subsurface bacteria from the Iberian Pyritic Belt: Insights into the biogeochemical cycles in this environment.

Nitrogen and iron are central to Earth's biogeochemical cycles. The Iberian Pyrite Belt (IPB), a vast iron reservoir, hosts extremophilic microorganisms adapted to high metal and sulfur concentrations. This study investigates two isolates from the deep IPB subsurface, Citrobacter telavivensis T1.2D-1 and Stutzerimonas stutzeri T2.31D-1, evaluating their nitrate-reducing capacity and the role of iron in denitrification, with emphasis on nitrate-dependent ferrous iron oxidation (NDFO). Both species could reduce nitrate in sucrose-, lactate-, or acetate-fed medium, while their consortium enhanced nitrate consumption and biomass production. Genome analysis revealed no genes for iron (II) oxidation, yet NDFO likely occurs, as proposed, via a heterotrophic-lithotrophic mechanism: enzymatic nitrate reduction to nitrite, followed by abiotic iron (II) oxidation that converts nitrite to nitric oxide and then to nitrous oxide. This is particularly relevant for C. telavivensis, which can perform only of the first reduction step. Experimentally, iron sometimes inhibited nitrate consumption, likely because it accumulated in crusts on bacterial cells. However, extracellular polymeric substances (EPS) production could reduce iron toxicity and allow iron to act beneficially by supporting microbial activity, as observed with sucrose supplementation. Ecologically, nitrate reducers may significantly influence IPB subsurface cycles. Even lacking iron (II) oxidation genes, these bacteria could contribute to pyrite bioleaching through: (i) chemical attack by NDFO-generated iron (III), (ii) organic acids produced by metabolism, or (iii) yet undescribed mechanisms. Overall, these findings highlight the adaptive strategies and potential environmental roles of these nitrate-reducing microorganisms in the IPB subsurface.