In order to assess the importance of nitrate-dependent Fe(II) oxidation and its impact on the growth physiology of dominant Fe oxidizers, we counted these bacteria in freshwater lake sediments and studied their growth physiology. Most probable number counts of nitrate-reducing Fe(II)-oxidizing bacteria in the sediment of Lake Constance, a freshwater lake in Southern Germany, yielded about 10(5) cells mL(-1) of the total heterotrophic nitrate-reducing bacteria, with about 1% (10(3) cells mL(-1)) of nitrate-reducing Fe(II) oxidizers. We investigated the growth physiology of Acidovorax sp. strain BoFeN1, a dominant nitrate-reducing mixotrophic Fe(II) oxidizer isolated from this sediment. Strain BoFeN1 uses several organic compounds (but no sugars) as substrates for nitrate reduction. It also reduces nitrite, dinitrogen monoxide, and O(2), but cannot reduce Fe(III). Growth experiments with cultures amended either with acetate plus Fe(II) or with acetate alone demonstrated that the simultaneous oxidation of Fe(II) and acetate enhanced growth yields with acetate alone (12.5 g dry mass mol(-1) acetate) by about 1.4 g dry mass mol(-1) Fe(II). Also, pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans strains can oxidize Fe(II) with nitrate, whereas Pseudomonas fluorescens and Thiobacillus denitrificans strains did not. Our study demonstrates that nitrate-dependent Fe(II) oxidation contributes to the energy metabolism of these bacteria, and that nitrate-dependent Fe(II) oxidation can essentially contribute to anaerobic iron cycling.