Oxidation of Fe(II)-EDTA by nitrite and by two nitrate-reducing Fe(II)-oxidizing Acidovorax strains.

The enzymatic oxidation of Fe(II) by nitrate-reducing bacteria was first suggested about two decades ago. It has since been found that most strains are mixotrophic and need an additional organic co-substrate for complete and prolonged Fe(II) oxidation. Research during the last few years has tried to determine to what extent the observed Fe(II) oxidation is driven enzymatically, or abiotically by nitrite produced during heterotrophic denitrification. A recent study reported that nitrite was not able to oxidize Fe(II)-EDTA abiotically, but the addition of the mixotrophic nitrate-reducing Fe(II)-oxidizer, Acidovorax sp. strain 2AN, led to Fe(II) oxidation (Chakraborty & Picardal, 2013). This, along with other results of that study, was used to argue that Fe(II) oxidation in strain 2AN was enzymatically catalyzed. However, the absence of abiotic Fe(II)-EDTA oxidation by nitrite reported in that study contrasts with previously published data. We have repeated the abiotic and biotic experiments and observed rapid abiotic oxidation of Fe(II)-EDTA by nitrite, resulting in the formation of Fe(III)-EDTA and the green Fe(II)-EDTA-NO complex. Additionally, we found that cultivating the Acidovorax strains BoFeN1 and 2AN with 10 mM nitrate, 5 mm acetate, and approximately 10 mM Fe(II)-EDTA resulted only in incomplete Fe(II)-EDTA oxidation of 47-71%. Cultures of strain BoFeN1 turned green (due to the presence of Fe(II)-EDTA-NO) and the green color persisted over the course of the experiments, whereas strain 2AN was able to further oxidize the Fe(II)-EDTA-NO complex. Our work shows that the two used Acidovorax strains behave very differently in their ability to deal with toxic effects of Fe-EDTA species and the further reduction of the Fe(II)-EDTA-NO nitrosyl complex. Although the enzymatic oxidation of Fe(II) cannot be ruled out, this study underlines the importance of nitrite in nitrate-reducing Fe(II)- and Fe(II)-EDTA-oxidizing cultures and demonstrates that Fe(II)-EDTA cannot be used to demonstrate unequivocally the enzymatic oxidation of Fe(II) by mixotrophic Fe(II)-oxidizers.

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.

Nitrate-reducing, Fe(II)-oxidizing bacteria were suggested to couple with enzymatic Fe(II) oxidation to nitrate reduction. Denitrification proceeds via intermediates (NO2 -, 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 O(2) , 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 N(2) O as electron acceptor. When adding Fe(II), no Fe(II) oxidation was observed, suggesting that the absence of reactive N intermediates (NO2 -, 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.