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.

Hematite enhances microbial autotrophic nitrate removal in carbonate and phosphate-rich environments by increasing Fe(II) activity.

Groundwater contamination by nitrates presents significant risks to both human health and the environment. In groundwater characterized as oligotrophic-low in organic carbon, but abundant in carbonate and phosphate-chemolithoautotrophic bacteria, including nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB), play a vital role in denitrification. The chemoautotrophic nitrate reduction is sensitive to environmental factors, including widespread iron oxides like hematite in nature. However, the specific mechanisms of this influence remain unclear. We examined the mechanism of how hematite impacts autotrophic nitrate reduction in a model NRFeOB community known as culture KS. We found that hematite enhances the rate of autotrophic nitrate reduction by promoting Fe(II) oxidation. Mössbauer spectroscopy detected a significant amount of adsorbed Fe(II) when hematite was present, leading to a reduction in dissolved ferrous iron. In conjunction with XRD data, it can be inferred that the formation of vivianite decreased, thereby increasing the Fe(II) activity in the reaction system. Within the culture KS bacterial consortium, hematite fosters the proliferation of autotrophic microorganisms, specifically Gallionellaceae, and amplifies the presence of denitrifying microbes, notably Rhodanobacter. This dual enhancement improves Fe(II) utilization and nitrate reduction capabilities. Our findings highlight intricate interactions between hematite and a model NRFeOB community, offering insights into groundwater nitrate removal mechanisms and the ecological strategies of autotrophic bacteria in mineral-rich environments.