Fe(II) oxidation and nitrate reduction by a denitrifying bacterium, Pseudomonas stutzeri LS-2, isolated from paddy soil

Abstract

Ferrous iron (Fe(II)) oxidation and nitrate (NO3 −) reduction are commonly observed in environments with denitrifying bacteria. The intermediate nitrite (NO2 −) from denitrification can chemically oxidize Fe(II). However, it is difficult to distinguish how chemical and biological reactions are involved. Pseudomonas stutzeri LS-2, a denitrifying bacterium isolated from paddy soil in southern China, was used in this study to investigate the chemical and biological reactions contributing to Fe(II) oxidation and NO3 − reduction under denitrifying conditions. Concentrations of dissolved Fe(II), NO3 −, NO2 −, and nitrous oxide (N2O) over time were quantified to investigate the kinetics of Fe(II) oxidation and NO3 −/NO2 − reduction in different treatments (i.e., microbial treatments: Cell + NO3 − and Cell + NO2 −, chemical treatment: Fe(II) + NO2 −, and combined treatments: LS-2 + Fe(II) + NO3 − and LS-2 + Fe(II) + NO2 −). Stable isotope fractionations of δ15N-N2O in different treatments were also determined over time. Fe(III) minerals and cell-mineral precipitates formed due to Fe(II) oxidation after 6 days of incubation were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). P. stutzeri LS-2 could completely reduce NO3 − or NO2 − within 2 days in the microbial treatment of Cell + NO3 − or Cell + NO2 −. The presence of Fe(II) resulted in a decrease of NO3 − or NO2 − reduction rates and an increase in the amount of nitrous oxide (N2O) production in the combined treatments of Cell + Fe(II) + NO3 − and Cell + Fe(II) + NO2 −. Fe(II) oxidation was only observed in the two combined treatments and the chemical treatment of Fe(II) + NO2 −. Lepidocrocite was formed due to Fe(II) oxidation after 6 days of incubation, which fully covered the bacterial cell surfaces in both combined treatments. Encrustation occurred in the periplasm and on the cell surface. The δ15N-N2O were 7.8 to − 10‰ in both microbial treatments during incubation, while those were − 23 to − 15‰ in the Fe(II) + NO2 − and Cell + Fe(II) + NO2 − treatments. In the Cell + Fe(II) + NO3 − treatment, however, the δ15N in N2O were − 37 to − 25‰, which were different from the microbial and chemical treatments. This difference is probably due to the accelerated reaction between Fe(II) and NO3 −/NO2 − by lepidocrocite. Our results indicate that once NO3 − was reduced to NO2 − by the denitrifying bacterium P. stutzeri LS-2, the NO2 − chemically reacted with Fe(II), and the concomitant Fe(III) oxide formation and cell encrustation led to an inhibition to denitrification. The stable isotope fractionation technique in combination with the transformation kinetics analyses is useful to distinguish the chemical and biological reactions involved in Fe(II) oxidation and nitrate reduction by denitrifying bacteria.