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

Microorganisms have been observed to oxidize Fe(II) at neutral pHunder anoxic andmicrooxic conditions.Whilemost of themix-otrophic nitrate-reducing Fe(II)-oxidizing bacteria become encrustedwith Fe(III)-richminerals, photoautotrophic andmicroaero-philic Fe(II) oxidizers avoid cell encrustation. The Fe(II) oxidationmechanisms and the reasons for encrustation remain largely unre-solved.Herewe used cultivation-basedmethods and electronmicroscopy to compare twopreviously described nitrate-reducing Fe(II) oxidizers (Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002) and two heterotrophic nitrate reducers (Para-coccus denitrificans ATCC 19367 and P. denitrificans Pd 1222). All four strains oxidized8 mM Fe(II) within 5 days in the pres-ence of 5 mM acetate and accumulated nitrite (maximum concentrations of 0.8 to 1.0 mM) in the culture media. Iron(III) miner-als, 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 denitrifi-cation and abiotically oxidizes Fe(II). This work adds to the known assemblage of Fe(II)-oxidizing bacteria in nature and compli