Nitrate Removal by a Novel Lithoautotrophic Nitrate-Reducing, Iron(II)-Oxidizing Culture Enriched from a Pyrite-Rich Limestone Aquifer

Abstract

Nitrate removal in oligotrophic environments is often limited by the availability of suitable organic electron donors. Chemolithoautotrophic bacteria may play a key role in denitrification in aquifers depleted in organic carbon. Under anoxic and circumneutral pH conditions, iron(II) was hypothesized to serve as an electron donor for microbially mediated nitrate reduction by Fe(II)-oxidizing (NRFeOx) microorganisms. However, lithoautotrophic NRFeOx cultures have never been enriched from any aquifer, and as such, there are no model cultures available to study the physiology and geochemistry of this potentially environmentally relevant process. Using iron(II) as an electron donor, we enriched a lithoautotrophic NRFeOx culture from nitrate-containing groundwater of a pyrite-rich limestone aquifer. In the enriched NRFeOx culture that does not require additional organic cosubstrates for growth, within 7 to 11 days, 0.3 to 0.5 mM nitrate was reduced and 1.3 to 2 mM iron(II) was oxidized, leading to a stoichiometric NO₃^-/Fe(II) ratio of 0.2, with N₂ and N₂O identified as the main nitrate reduction products. Short-range ordered Fe(III) (oxyhydr)oxides were the product of iron(II) oxidation. Microorganisms were observed to be closely associated with formed minerals, but only few cells were encrusted, suggesting that most of the bacteria were able to avoid mineral precipitation at their surface. Analysis of the microbial community by long-read 16S rRNA gene sequencing revealed that the culture is dominated by members of the Gallionellaceae family that are known as autotrophic, neutrophilic, and microaerophilic iron(II) oxidizers. In summary, our study suggests that NRFeOx mediated by lithoautotrophic bacteria can lead to nitrate removal in anthropogenically affected aquifers. IMPORTANCE Removal of nitrate by microbial denitrification in groundwater is often limited by low concentrations of organic carbon. In these carbon-poor ecosystems, nitrate-reducing bacteria that can use inorganic compounds such as Fe(II) (NRFeOx) as electron donors could play a major role in nitrate removal. However, no lithoautotrophic NRFeOx culture has been successfully isolated or enriched from this type of environment, and as such, there are no model cultures available to study the rate-limiting factors of this potentially important process. Here, we present the physiology and microbial community composition of a novel lithoautotrophic NRFeOx culture enriched from a fractured aquifer in southern Germany. The culture is dominated by a putative Fe(II) oxidizer affiliated with the Gallionellaceae family and performs nitrate reduction coupled to Fe(II) oxidation leading to N₂O and N₂ formation without the addition of organic substrates. Our analyses demonstrate that lithoautotrophic NRFeOx can potentially lead to nitrate removal in nitrate-contaminated aquifers.

Keywords: NRFeOx; aquifer; geomicrobiology; groundwater; iron metabolism; iron oxidizers; nitrate; pyrite.

FIG 1

Relative abundance of taxa (A) found in the autotrophic NRFeOx enrichment culture based on long-read 16S rRNA gene sequences, where sequence similarities to cultured representatives at family and genus level ranged from 96% to 100% (query coverage >99%), based on SINAsearch (86) using the SILVA 132 database. Neighbor-joining phylogenetic tree (B) constructed showing the relation of the most dominant bacterium in the enrichment culture (blue background, bold and underlined) and representative microaerophilic Fe(II) oxidizers related to Gallionella spp. and Sideroxydans sp. including the most abundant representative of Gallionellaceae family present in culture KS (blue background). Mariprofundus ferrooxydans PV-1, a microaerophilic Fe(II) oxidizer, was included as outgroup. The scale bar corresponds to 0.02 nucleotide substitutions per site. At the branches, high-confidence (>50) bootstrap values (from 1,000 replications) are shown. GenBank accession numbers are shown in parentheses next to the organism names. Squares indicate bacteria enriched from aquifers, and circles indicate bacteria originating from mineral springs.

FIG 2

Concentrations of dissolved NO₃⁻, NO₂⁻, and Fe²⁺ in three consecutive transfers of the enriched autotrophic nitrate-reducing, Fe(II)-oxidizing culture. All data points are mean values of samples from three replicate bottles; error bars represent standard deviations. The transfers on days 0, 7, and 17 are indicated by arrows.

FIG 3

X-ray diffractogram (A) and Mössbauer spectrum (B) of the minerals formed during oxidation of 2 mM Fe(II) by the Fe(II)-oxidizing, nitrate-reducing enrichment culture. Samples were taken after 11 days. The diffraction reflexes at 51.0°2θ and 65.9°2θ belong to Si-wafer (sample holder). In the Mössbauer spectrum, the black circles indicate raw data, the brown area represents short-range ordered Fe(III) oxyhydroxide, likely ferrihydrite, and the blue-shaded represents Fe(II).

FIG 4

Overlay of fluorescence and transmission light microscopic pictures of the autotrophic NRFeOx culture enriched in this study. Cells were stained with the LIVE/DEAD stain (green, alive; red, dead) (A). Scanning electron micrographs of the culture after 11 days of incubation showing nonencrusted (B) and encrusted (C) cells. Arrows indicate cells.