Coupling of Fe and N cycles by nitrate-reducing Fe(II)-oxidizing microorganisms in the tidal sediments of an extreme acidic river (Río Tinto, Spain)

Río Tinto (Huelva, Spain) is an acid rock drainage-affected fluvial estuarine system where Fe(II)-oxidizing microorganisms were shown to be active both in the water column and in the top sediment layer, contributing to Fe mineral accumulation (up to 30 % of dry sediment weight) in the estuarian sediment. However, it is still unknown if the Fe(II)-oxidizing microorganisms thriving in the upper sediment layer are also capable of using nitrate present in the river (0.05–0.5 mM) as electron acceptor. We performed sediment incubations amended with either lactate, acetate-/NO3−-/Fe2+aq or NO3−/Fe2+aq to evaluate Fe-cycling processes and, specifically, if nitrate reduction coupled to Fe(II) oxidation (NRFeOx) can occur in the sediment under anoxic conditions. Geochemical data showed that in the NO3−-/Fe2+aq-amended setup, NO3− (from 1.5 to 0 mM), Fe2+aq (from 1.5 to 0 mM) and pH (from 6.0 to 5.4) decreased, while the poorly crystalline Fe(III) mineral pool increased by 6.9 % during 114 days of incubation. In the acetate-/NO3−-/Fe2+aq-amended setup, nitrate reduction rates (0.31 mM NO3−/day) were 10 times faster compared to the NO3−-/Fe2+aq-amended setup (0.03 mM NO3−/day). pH and poorly crystalline Fe(II) mineral content increased due to Fe(III) reduction after amendment with only lactate (0.26 ± 0.03 mM Fe2+aq/day) but also after amendment with acetate-/NO3−-/Fe2+aq (0.16 ± 0.02 mM Fe2+aq/day) suggesting that acetate-stimulated Fe(III) reduction superimposed Fe(II) oxidation coupled to nitrate reduction. Sequencing data showed that upon addition of nitrate, members of the genus Rhodanobacter increased by ⁓10 % in relative DNA-based 16S rRNA gene abundances in both acetate-/NO3−-/Fe2+aq- and NO3−-/Fe2+aq-amended setups. However, the Rhodanobacter RNA-based 16S rRNA relative gene abundance was higher in the acetate-/NO3−-/Fe2+aq-amended setup (11.15 ± 2.24 %) than in the NO3−-/Fe2+aq-amended one (5.40 ± 0.31 %). Combining geochemical and sequencing data obtained from anoxic sediment incubations, we conclude that NRFeOx processes, potentially catalyzed by the genus Rhodanobacter, can play a role in Fe cycling in this extreme acid rock drainage affected river, under low organic carbon (OC) conditions. At higher OC levels, NRFeOx microorganisms seem to become more active but their net effects on Fe(II) oxidation can be diminished due to the simultaneous activity of Fe(III)-reducers.