Nitrous oxide is the main product during nitrate reduction by a novel lithoautotrophic iron(II)-oxidizing culture from an organic-rich paddy soil

Microbial nitrate reduction coupled to iron(II) oxidation (NRFeOx) occurs in paddy soils due to high levels of dissolved iron(II) and regular application of nitrogen fertilizer. However, to date, there is no lithoautotrophic NRFeOx isolate or enrichment culture available from this soil environment. Thus, resulting impacts on greenhouse gas emissions during nitrate reduction (i.e., nitrous oxide [N₂O]) and on toxic metalloid (i.e., arsenic) mobility can hardly be investigated. We enriched a lithoautotrophic NRFeOx culture, culture HP (Huilongpu paddy, named after its origin), from a paddy soil (Huilongpu Town, China), which was dominated by <i>Gallionella</i> (71%). The culture reduced 0.45 to 0.63 mM nitrate and oxidized 1.76 to 2.31 mM iron(II) within 4 days leading to N₂O as the main N-product (62%-88% N₂O-N of total reduced NO₃^--N). Nitrite was present as an intermediate at a maximum of 0.16 ± 0.1 mM. Cells were associated with, but mostly not encrusted by, poorly crystalline iron(III) minerals (ferrihydrite). Culture HP performed best below an iron(II) threshold of 2.5-3.5 mM and in a pH range of 6.50-7.05. In the presence of 100 µM arsenite, only 0%-18% of iron(II) was oxidized. Due to low iron(II) oxidation, arsenite was not immobilized. However, the proportion of N₂O-N of total reduced NO₃^--N decreased from 77% to 30%. Our results indicate that lithoautotrophic NRFeOx occurs even in organic-rich paddy soils, resulting in denitrification and subsequent N₂O emissions. The obtained novel enrichment culture allows us to study the impact of lithoautotrophic NRFeOx on arsenic mobility and N₂O emissions in paddy soils.IMPORTANCEPaddy soils are naturally rich in iron(II) and regularly experience nitrogen inputs due to fertilization. Nitrogen fertilization increases nitrous oxide emissions as it is an intermediate product during nitrate reduction. Microorganisms can live using nitrate and iron(II) as electron acceptor and donor, respectively, but mostly require an organic co-substrate. By contrast, microorganisms that only rely on nitrate, iron(II), and CO₂ could inhabit carbon-limited ecological niches. So far, no isolate or consortium of lithoautotrophic iron(II)-oxidizing, nitrate-reducing microorganisms has been obtained from paddy soil. Here, we describe a lithoautotrophic enrichment culture, dominated by a typical iron(II)-oxidizer (<i>Gallionella</i>), that oxidized iron(II) and reduced nitrate to nitrous oxide, negatively impacting greenhouse gas dynamics. High arsenic concentrations were toxic to the culture but decreased the proportion of nitrous oxide of the total reduced nitrate. Our results suggest that autotrophic nitrate reduction coupled with iron(II) oxidation is a relevant, previously overlooked process in paddy soils.