Autotrophic denitrification coupled to Fe(II)-oxidation driven by aquifer-originating bacteria

Autotrophic denitrification coupled to pyrite oxidation is considered a natural nitrate attenuation process in aquifers limited in organic carbon. The process was hypothesized to be one of the possible nitrate removal mechanisms occurring in the pyrite-rich aquifer in Ammer catchment, southwestern Germany. However, autotrophic nitrate-reducing Fe(II)-oxidizing (NRFeOx) bacteria have never been enriched from this type of habitat, and as such, there are no model cultures to study the identity, physiology, and genetic repertoire of the microbial key players. Moreover, the lack of evidence for direct enzymatic oxidation of pyrite by autotrophic NRFeOx bacteria and the poor solubility of this mineral at circumneutral pH raises a critical question about the oxidation mechanism. First, crushed pyrite-rich rock particles were subjected to long-term groundwater exposure in one of the low-nitrate monitoring wells in Ammer valley, resulting in an enrichment of an autotrophic NRFeOx community. The novel enrichment culture was then demonstrated to be able to reduce nitrate and oxidize dissolved Fe(II) without the addition of organic co-substrates, leading to the formation of N2 and N2O and short-range ordered Fe(III) (oxyhydr)oxides. Next, the culture was incubated with pyrite and siderite to evaluate the rates, and the extent of nitrate reduction when solid sources of Fe(II) are supplied. Combined results of the chemical analysis, NanoSIMS analysis, SEM imaging and reaction-modeling evidenced that the bacteria are capable of direct oxidation of structural Fe(II) coupled to the reduction of nitrate. Further, the identity and genetic potential of the enriched community were evaluated by applying 16S rRNA gene and metagenome sequencing. The culture was shown to be dominated (ca. 62% relative abundance) by a so-far uncultured species belonging to the family Gallionellaceae. Metagenome-assembled genome analysis of these bacteria revealed the presence of a gene encoding cytochrome c Cyc2, putatively involved in extracellular electron transfer from Fe(II), and the presence of a complete set of genes (narGHJI, nirKS, and norBC) necessary to reduce NO3- to N2O. The Gallionellaceae sp. lacks, however, a gene mediating the last step of denitrification (nosZ), suggesting that complete NRFe oxidation in the pyrite-containing aquifer requires multiple inter-species metabolic hand-offs. These findings highlight the importance of Gallionellaceae spp. in linking biogeochemical cycles of N and Fe in aquifers and support the potential of autotrophic nitrate reduction coupled to Fe(II) oxidation to be the possible mechanism leading to nitrate removal in the aquifer in the Ammer catchment. The results have implications for predicting the fate of nitrate in freshwater ecosystems poor in organic carbon where Fe(II)-minerals are present