Nitrate-reducing, Fe(II)-oxidizing bacteria were suggested to couple with enzymatic Fe(II) oxidation to nitrate reduction. Denitrification proceeds via intermediates (NO � , NO) that can oxidize Fe(II) abiotically at neutral and particularly at acidic pH. Here, we present a revised Fe(II) quantification protocol preventing artifacts during acidic Fe extraction and evaluate the contribution of abiotic vs. enzymatic Fe(II) oxidation in cultures of the nitrate-reducing, Fe(II) oxidizer Acidovorax sp. BoFeN1. Sulfamic acid used instead of HCl reacts with nitrite and prevents abiotic Fe(II) oxidation during Fe extraction. Abiotic experiments without sulfamic acid showed that acidification of oxic Fe(II) nitrite samples leads to 5.6-fold more Fe(II) oxidation than in anoxic samples because the formed NO becomes rapidly reoxidized by O2, therefore leading to abiotic oxidation and underestimation of Fe(II). With our revised protocol using sulfamic acid, we quantified oxidation of approximately 7 mM of Fe(II) by BoFeN1 within 4 days. Without addition of sulfamic acid, the same oxidation was detected within only 2 days. Additionally, abiotic incubation of Fe(II) with nitrite in the presence of goethite as surface catalyst led to similar abiotic Fe(II) oxidation rates as observed in growing BoFeN1 cultures. BoFeN1 growth was observed on acetate with N2O as electron acceptor. When adding Fe(II), no Fe(II) oxidation was observed, suggesting that the absence of reactive N intermediates (NO � , NO) precludes Fe(II) oxidation. The addition of ferrihydrite [Fe(OH)3] to acetate/nitrate BoFeN1 cultures led to growth stimulation equivalent to previously described effects on growth by adding Fe(II). This suggests that elevated iron concentrations might provide a nutritional effect rather than energy-yielding Fe(II) oxidation. Our findings therefore suggest that although enzymatic Fe(II) oxidation by denitrifiers cannot be fully ruled out, its contribution to the observed Fe(II) oxidation in microbial cultures is probably lower than previously suggested and has to be questioned in general until the enzymatic machinery-mediating Fe(II) oxidation is identified.

Abiotic oxidation of Fe(II) by reactive nitrogen species in cultures of the nitrate‐reducing Fe(II) oxidizer Acidovorax sp. BoFeN1 – questioning the existence of enzymatic Fe(II) oxidation

Microorganisms have been observed to oxidize Fe(II) at neutral pH under anoxic and microoxic conditions. While most of the mixotrophic nitrate-reducing Fe(II)-oxidizing bacteria become encrusted with Fe(III)-rich minerals, photoautotrophic and microaerophilic Fe(II) oxidizers avoid cell encrustation. The Fe(II) oxidation mechanisms and the reasons for encrustation remain largely unresolved. Here we used cultivation-based methods and electron microscopy to compare two previously described nitrate-reducing Fe(II) oxidizers ( Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002) and two heterotrophic nitrate reducers (Paracoccus denitrificans ATCC 19367 and P. denitrificans Pd 1222). All four strains oxidized ∼8 mM Fe(II) within 5 days in the presence of 5 mM acetate and accumulated nitrite (maximum concentrations of 0.8 to 1.0 mM) in the culture media. Iron(III) minerals, mainly goethite, formed and precipitated extracellularly in close proximity to the cell surface. Interestingly, mineral formation was also observed within the periplasm and cytoplasm; intracellular mineralization is expected to be physiologically disadvantageous, yet acetate consumption continued to be observed even at an advanced stage of Fe(II) oxidation. Extracellular polymeric substances (EPS) were detected by lectin staining with fluorescence microscopy, particularly in the presence of Fe(II), suggesting that EPS production is a response to Fe(II) toxicity or a strategy to decrease encrustation. Based on the data presented here, we propose a nitrite-driven, indirect mechanism of cell encrustation whereby nitrite forms during heterotrophic denitrification and abiotically oxidizes Fe(II). This work adds to the known assemblage of Fe(II)-oxidizing bacteria in nature and complicates our ability to delineate microbial Fe(II) oxidation in ancient microbes preserved as fossils in the geological record.

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Potential Role of Nitrite for Abiotic Fe(II) Oxidation and Cell Encrustation during Nitrate Reduction by Denitrifying Bacteria

Phylogenetically diverse species of bacteria can catalyze the oxidation of ferrous iron [Fe(II)] coupled to nitrate (NO3−) reduction, often referred to as nitrate-dependent iron oxidation (NDFO). Very little is known about the biochemistry of NDFO, and though growth benefits have been observed, mineral encrustations and nitrite accumulation likely limit growth. Acidovorax ebreus, like other species in the Acidovorax genus, is proficient at catalyzing NDFO. Our results suggest that the induction of specific Fe(II) oxidoreductase proteins is not required for NDFO. No upregulated periplasmic or outer membrane redox-active proteins, like those involved in Fe(II) oxidation by acidophilic iron oxidizers or anaerobic photoferrotrophs, were observed in proteomic experiments. We demonstrate that while “abiotic” extracellular reactions between Fe(II) and biogenic NO2−/NO can be involved in NDFO, intracellular reactions between Fe(II) and periplasmic components are essential to initiate extensive NDFO. We present evidence that an organic cosubstrate inhibits NDFO, likely by keeping periplasmic enzymes in their reduced state, stimulating metal efflux pumping, or both, and that growth during NDFO relies on the capacity of a nitrate-reducing bacterium to overcome the toxicity of Fe(II) and reactive nitrogen species. On the basis of our data and evidence in the literature, we postulate that all respiratory nitrate-reducing bacteria are innately capable of catalyzing NDFO. Our findings have implications for a mechanistic understanding of NDFO, the biogeochemical controls on anaerobic Fe(II) oxidation, and the production of NO2−, NO, and N2O in the environment.

Fe(II) Oxidation Is an Innate Capability of Nitrate-Reducing Bacteria That Involves Abiotic and Biotic Reactions

Microbial Fe(II) oxidation using NO3- as the terminal electron acceptor (nitrate-dependent Fe(II) oxidation; NDFO) has been studied for over 15 years. Although there are reports of autotrophic isolates and stable enrichments, many of the bacteria capable of NDFO are known organotrophic NO3- -reducers that require the presence of an organic, primary substrate, e.g., acetate, for significant amounts of Fe(II) oxidation. Although the thermodynamics of Fe(II) oxidation are favorable when coupled to either NO3- or NO2- reduction, the kinetics of abiotic Fe(II) oxidation by NO3- are relatively slow except under special conditions. NDFO is typically studied in batch cultures containing millimolar concentrations of Fe(II), NO3-, and the primary substrate. In such systems, NO2- is often observed to accumulate in culture media during Fe(II) oxidation. Compared to NO3-, abiotic reactions of biogenic NO2- and Fe(II) are relatively rapid. The kinetics and reaction pathways of Fe(II) oxidation by NO2- are strongly affected by medium composition and pH, reactant concentration, and the presence of Fe(II)-sorptive surfaces, e.g., Fe(III) oxyhydroxides and cellular surfaces. In batch cultures, the combination of abiotic and microbial Fe(II) oxidation can alter product distribution and, more importantly, results in the formation of intracellular precipitates and extracellular Fe(III) oxyhydroxide encrustations that apparently limit further cell growth and Fe(II) oxidation. Unless steps are taken to minimize or account for potential abiotic reactions, results of microbial NDFO studies can be obfuscated by artifacts of the chosen experimental conditions, the use of inappropriate analytical methods, and the resulting uncertainties about the relative importance of abiotic and microbial reactions. In this manuscript, abiotic reactions of NO3- and NO2- with aqueous Fe2+, chelated Fe(II), and solid-phase Fe(II) are reviewed along with factors that can influence overall NDFO reac

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Abiotic and Microbial Interactions during Anaerobic Transformations of Fe(II) and NOX

The link between biomineralization and fossilization of bacteria: Insights from field and experimental studies

Iron-oxidizing bacteria are important actors of the geochemical cycle of iron in modern environments and may have played a key role all over Earth's history. However, in order to better assess that role on the modern and the past Earth, there is a need for better understanding the mechanisms of bacterial iron oxidation and for defining potential biosignatures to be looked for in the geologic record. In this study, we investigated experimentally and at the nanometre scale the mineralization of iron-oxidizing bacteria with a combination of synchrotron-based scanning transmission X-ray microscopy (STXM), scanning transmission electron microscopy (STEM) and cryo-transmission electron microscopy (cryo-TEM). We show that the use of cryo-TEM instead of conventional microscopy provides detailed information of the successive iron biomineralization stages in anaerobic nitrate-reducing iron-oxidizing bacteria. These results suggest the existence of preferential Fe-binding and Fe-oxidizing sites on the outer face of the plasma membrane leading to the nucleation and growth of Fe minerals within the periplasm of these cells that eventually become completely encrusted. In contrast, the septa of dividing cells remain nonmineralized. In addition, the use of cryo-TEM offers a detailed view of the exceptional preservation of protein globules and the peptidoglycan within the Fe-mineralized cell walls of these bacteria. These organic molecules and ultrastructural details might be protected from further degradation by entrapment in the mineral matrix down to the nanometre scale. This is discussed in the light of previous studies on the properties of Fe-organic interactions and more generally on the fossilization of mineral-organic assemblies.

Preservation of protein globules and peptidoglycan in the mineralized cell wall of nitrate‐reducing, iron(II)‐oxidizing bacteria: a cryo‐electron microscopy study

N Boisset

Papers

Preservation of protein globules and peptidoglycan in the mineralized cell wall of nitrate‐reducing, iron(II)‐oxidizing bacteria: a cryo‐electron microscopy study