Oxidation of Fe(II) by nitrate-reducing Fe(II)-oxidizers enriched from Fe(III)-rich microbial mats of an extreme acidic river (Río Tinto, Spain)

ABSTRACT Nitrate-reducing Fe(II)-oxidizing (NRFeOx) microorganisms have been detected in the subsurface of the Iberian Pyrite Belt (Spain), where the Río Tinto originates. However, it is not known whether NRFeOx microorganisms are present in the Fe(III)-rich microbial mats that developed within the surface waters where the river originates, and the geochemical conditions progressively shift from circumneutral to highly acidic. Moreover, it is not known what the main (microbial) Fe(II) oxidation pathway is under in situ pH conditions. To answer these questions, we (i) analyzed the in situ bacterial community and Fe mineralogy of the Fe(III)-rich microbial mats, (ii) set up NRFeOx enrichment cultures (either with NO 3 − /Fe 2+ aq or acetate/NO 3 − /Fe 2+ aq ), and (iii) evaluated denitrification and Fe(II) oxidation capabilities of the NRFeOx enrichments under in situ pH conditions. We found that the microbial mat was composed mainly of poorly crystalline minerals, such as ferrihydrite and schwertmannite, with the microaerophilic Fe(II)-oxidizing genus Gallionella (18.3% ± 4.9% DNA-based and 10.8% ± 1.9% RNA-based relative 16S rRNA [gene] abundances) being the main bacteria present and active. NRFeOx was only observed in the enrichment culture amended with acetate/NO 3 − /Fe 2+ aq , where Rhodoblastus (between 73.1% and 91.0% DNA-based) was the main enriched genus. The production of nitrite in the Rhodoblastus-dominated culture suggested that chemodenitrification, i.e., the abiotic oxidation of Fe(II) by nitrite, was the main Fe(II) oxidation pathway at pH between 6.2 and 7. However, under more acidic conditions (pH 5.8), nitrate reduction still occurred without Fe(II) oxidation. Overall, this study suggests that NRFeOx microorganisms present in the Río Tinto microbial mats may couple NO 3 − reduction to Fe(II) oxidation in anoxic circumneutral microniches where nitrate and organic carbon are also available. IMPORTANCE The potential role of nitrate-reducing Fe(II)-oxidizing (NRFeOx) microorganisms in subsurface pyrite weathering and the formation of acid mine drainage environments, such as the Río Tinto river, is still poorly understood. By focusing on Fe(III)-rich microbial mats formed at the interface between anoxic Fe(II)-rich groundwater and oxic conditions, this study addresses how NRFeOx microorganisms contribute to nitrogen, iron, and carbon cycling in this acidic environment. Moreover, identifying and enriching NRFeOx microorganisms adapted to low pH can deepen our understanding of their metabolic versatility and resilience. These insights extend beyond characterizing the origin of Río Tinto by informing models of acidic rock drainage generation, guiding bioremediation strategies for the simultaneous removal of nitrate and heavy metals from contaminated waters, and providing analogs for potential microbial life in iron-rich, anoxic extraterrestrial environments such as early Mars.