Metabolic Performance and Fate of Electrons during Nitrate-Reducing Fe(II) Oxidation by the Autotrophic Enrichment Culture KS Grown at Different Initial Fe/N Ratios.

Autotrophic nitrate-reducing Fe(II)-oxidizing (NRFeOx) microorganisms fix CO2 and oxidize Fe(II) coupled to denitrification, influencing carbon, iron, and nitrogen cycles in pH-neutral, anoxic environments. However, the distribution of electrons from Fe(II) oxidation to either biomass production (CO2 fixation) or energy generation (nitrate reduction) in autotrophic NRFeOx microorganisms has not been quantified. We therefore cultivated the autotrophic NRFeOx culture KS at different initial Fe/N ratios, followed geochemical parameters, identified minerals, analyzed N isotopes, and applied numerical modeling. We found that at all initial Fe/N ratios, the ratios of Fe(II)oxidized to nitratereduced were slightly higher (5.11 to 5.94 at Fe/N ratios of 10:1 and 10:0.5) or lower (4.27 to 4.59 at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1) than the theoretical ratio for 100% Fe(II) oxidation being coupled to nitrate reduction (5:1). The main N denitrification product was N2O (71.88 to 96.29% at Fe/15N ratios of 10:4 and 5:1; 43.13 to 66.26% at an Fe/15N ratio of 10:1), implying that denitrification during NRFeOx was incomplete in culture KS. Based on the reaction model, on average 12% of electrons from Fe(II) oxidation were used for CO2 fixation while 88% of electrons were used for reduction of NO3- to N2O at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1. With 10 mM Fe(II) (and 4, 2, 1, or 0.5 mM nitrate), most cells were closely associated with and partially encrusted by the Fe(III) (oxyhydr)oxide minerals, whereas at 5 mM Fe(II), most cells were free of cell surface mineral precipitates. The genus Gallionella (>80%) dominated culture KS regardless of the initial Fe/N ratios. Our results showed that Fe/N ratios play a key role in regulating N2O emissions, for distributing electrons between nitrate reduction and CO2 fixation, and for the degree of cell-mineral interactions in the autotrophic NRFeOx culture KS. IMPORTANCE Autotrophic NRFeOx microorganisms that oxidize Fe(II), reduce nitrate, and produce biomass play a key role in carbon, iron, and nitrogen cycles in pH-neutral, anoxic environments. Electrons from Fe(II) oxidation are used for the reduction of both carbon dioxide and nitrate. However, the question is how many electrons go into biomass production versus energy generation during autotrophic growth. Here, we demonstrated that in the autotrophic NRFeOx culture KS cultivated at Fe/N ratios of 10:4, 10:2, 5:2, and 5:1, ca. 12% of electrons went into biomass formation, while 88% of electrons were used for reduction of NO3- to N2O. Isotope analysis also showed that denitrification during NRFeOx was incomplete in culture KS and the main N denitrification product was N2O. Therefore, most electrons stemming from Fe(II) oxidation seemed to be used for N2O formation in culture KS. This is environmentally important for the greenhouse gas budget.

'Candidatus ferrigenium straubiae' sp. nov., 'Candidatus ferrigenium bremense' sp. nov., 'Candidatus ferrigenium altingense' sp. nov., are autotrophic Fe(II)-oxidizing bacteria of the family Gallionellaceae.

Iron(II) [Fe(II)] oxidation coupled to denitrification is recognized as an environmentally important process in many ecosystems. However, the Fe(II)-oxidizing bacteria (FeOB) dominating autotrophic nitrate-reducing Fe(II)-oxidizing enrichment cultures, affiliated with the family Gallionellaceae, remain poorly taxonomically defined due to lack of representative isolates. We describe the taxonomic classification of three novel FeOB based on metagenome-assembled genomes (MAGs) acquired from the autotrophic nitrate-reducing enrichment cultures KS, BP and AG. Phylogenetic analysis of nearly full-length 16S rRNA gene sequences demonstrated that these three FeOB were most closely affiliated to the genera Ferrigenium, Sideroxydans and Gallionella, with up to 96.5%, 95.4% and 96.2% 16S rRNA gene sequence identities to representative isolates of these genera, respectively. In addition, average amino acid identities (AAI) of the genomes compared to the most closely related genera revealed highest AAI with Ferrigenium kumadai An22 (76.35-76.74%), suggesting that the three FeOB are members of this genus. Phylogenetic analysis of conserved functional genes further supported that these FeOB represent three novel species of the genus Ferrigenium. Moreover, the three novel FeOB likely have characteristic features, performing partial denitrification coupled to Fe(II) oxidation and carbon fixation. Scanning electron microscopy of the enrichment cultures showed slightly curved rod-shaped cells, ranging from 0.2-0.7 µm in width and 0.5-2.3 µm in length. Based on the phylogenetic, genomic and physiological characteristics, we propose that these FeOB represent three novel species, 'Candidatus Ferrigenium straubiae' sp. nov., 'Candidatus Ferrigenium bremense' sp. nov. and 'Candidatus Ferrigenium altingense' sp. nov. that might have unique metabolic features among the genus Ferrigenium.

Presence of Fe(II) and nitrate shapes aquifer-originating communities leading to an autotrophic enrichment dominated by an Fe(II)-oxidizing Gallionellaceae sp.

Autotrophic nitrate reduction coupled to Fe(II) oxidation is an important nitrate removal process in anoxic aquifers. However, it remains unknown how changes of O2 and carbon availability influence the community structure of nitrate-reducing Fe(II)-oxidizing (NRFeOx) microbial assemblages and what the genomic traits of these NRFeOx key players are. We compared three metabolically distinct denitrifying assemblages, supplemented with acetate, acetate/Fe(II) or Fe(II), enriched from an organic-poor, pyrite-rich aquifer. The presence of Fe(II) promoted the growth of denitrifying Burkholderiaceae spp. and an unclassified Gallionellaceae sp. This Gallionellaceae sp. was related to microaerophilic Fe(II) oxidizers; however, it did not grow under microoxic conditions. Furthermore, we explored a metagenome and 15 metagenome-assembled genomes from an aquifer-originating, autotrophic NRFeOx culture. The dominant Gallionellaceae sp. revealed the potential to oxidize Fe(II) (e.g. cyc2), fix CO2 (e.g. rbcL) and perform near-complete denitrification leading to N2O formation (e.g. narGHJI,nirK/S and norBC). In addition, Curvibacter spp.,Methyloversatilis sp. and Thermomonas spp. were identified as novel putative NRFeOx taxa. Our findings provide first insights into the genetic traits of the so far only known autotrophic NRFeOx culture originating from an organic-poor aquifer, providing the genomic basis to study mechanisms of nitrate removal in organic-poor subsurface ecosystems.

Anaerobic Neutrophilic Pyrite Oxidation by a Chemolithoautotrophic Nitrate-Reducing Iron(II)-Oxidizing Culture Enriched from a Fractured Aquifer.

Neutrophilic microbial pyrite (FeS2) oxidation coupled to denitrification is thought to be an important natural nitrate attenuation pathway in nitrate-contaminated aquifers. However, the poor solubility of pyrite raises questions about its bioavailability and the mechanisms underlying its oxidation. Here, we investigated direct microbial pyrite oxidation by a neutrophilic chemolithoautotrophic nitrate-reducing Fe(II)-oxidizing culture enriched from a pyrite-rich aquifer. We used pyrite with natural abundance (NA) of Fe isotopes (NAFe-pyrite) and 57Fe-labeled siderite to evaluate whether the oxidation of the more soluble Fe(II)-carbonate (FeCO3) can indirectly drive abiotic pyrite oxidation. Our results showed that in setups where only pyrite was incubated with bacteria, direct microbial pyrite oxidation contributed ca. 26% to overall nitrate reduction. The rest was attributed to the oxidation of elemental sulfur (S0), present as a residue from pyrite synthesis. Pyrite oxidation was evidenced in the NAFe-pyrite/57Fe-siderite setups by maps of 56FeO and 32S obtained using a combination of SEM with nanoscale secondary ion MS (NanoSIMS), which showed the presence of 56Fe(III) (oxyhydr)oxides that could solely originate from 56FeS2. Based on the fit of a reaction model to the geochemical data and the Fe-isotope distributions from NanoSIMS, we conclude that anaerobic oxidation of pyrite by our neutrophilic enrichment culture was mainly driven by direct enzymatic activity of the cells. The contribution of abiotic pyrite oxidation by Fe3+ appeared to be negligible in our experimental setup.

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

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.