Identifying and Quantifying the Intermediate Processes during Nitrate-Dependent Iron(II) Oxidation

Abstract

Microbially driven nitrate-dependent iron (Fe) oxidation (NDFO) in subsurface environments has been intensively studied. However, the extent to which Fe(II) oxidation is biologically catalyzed remains unclear because no neutrophilic iron-oxidizing and nitrate reducing autotroph has been isolated to confirm the existence of an enzymatic pathway. While mixotrophic NDFO bacteria have been isolated, understanding the process is complicated by simultaneous abiotic oxidation due to nitrite produced during denitrification. In this study, the relative contributions of biotic and abiotic processes during NDFO were quantified through the compilation and model-based interpretation of previously published experimental data. The kinetics of chemical denitrification by Fe(II) (chemodenitrification) were assessed, and compelling evidence was found for the importance of organic ligands, specifically exopolymeric substances secreted by bacteria, in enhancing abiotic oxidation of Fe(II). However, nitrite alone could not explain the observed magnitude of Fe(II) oxidation, with 60-75% of overall Fe(II) oxidation attributed to an enzymatic pathway for investigated strains: Acidovorax ( A.) strain BoFeN1, 2AN, A. ebreus strain TPSY, Paracoccus denitrificans Pd 1222, and Pseudogulbenkiania sp. strain 2002. By rigorously quantifying the intermediate processes, this study eliminated the potential for abiotic Fe(II) oxidation to be exclusively responsible for NDFO and verified the key contribution from an additional, biological Fe(II) oxidation process catalyzed by NDFO bacteria.

Figure 1.

Chemodenitrification rates for experiments by Klueglein et al. (left); Klueglein and Kappler (middle); Jones et al. (right) (used in simulations S1a, S1b, and S1c, respectively). Experiment 4 mM NO₂—8 mM Fe(II) is replotted on the left panel with 4 mM NO₂—8 mM Fe(II)-EPS for reference. Chemodenitrification kinetics were described using eq 14. Observed Fe(II) concentrations are presented as a percentage of initial concentration (C/C₀).

Figure 2.

Simulation and experimental results for three Acidovorax strains BoFeNl, 2AN, and TPSY as well as Paracoccus denitrificans strain Pd 1222 and Pseudogulbenkiania strain 2002. Symbols represent observed concentrations for total Fe(II) (red circle), nitrate (blue down triangle), acetate (yellow up triangle), nitrite (yellow square), and biomass (filled green diamonds) from Klueglein and Kappler, Chakraborty et al., and Carlson et al. Simulations Sla (solid black line), Slb (dashed black line), and Slc (dash dot black line) were compared against S2 (solid red/gold line). All model results are similar for parameters presented in rows 2 and 4 to 6 and are therefore excluded to improve figure clarity. The fifth row presents the kinetic factors F_A and F_D (red and blue, respectively) and the thermodynamic potential factor F_T (green). The sixth row presents the contribution of chemodenitrification (red hatches) and enzymatic NDFO (blue hatches) to overall Fe(II) oxidation. Biomass subject to encrustation is provided (solid black line) as well as uninhibited biomass for reference (solid green line).

Figure 3.

Conceptual models of NDFO with varying levels of complexity. Model (3a) represents the processes included in the biogeochemical model developed for this study. Dashed gray arrows represent processes that are either unknown or potential processes active in NDFO cultures that require ongoing investigation. (1) A dedicated Fe(II) oxidoreductase exclusively responsible for NDFO. (2) Respiratory complexes catalyze NDFO (enzymatic NDFO) with additional Fe(II) oxidation occurring extracellularly via chemodenitrification. (3a) Identical to (2) but with the inclusion of green rust oxidation by nitrite, producing goethite and ammonium. (3b) Identical to (3a) but including species-specific rates for Fe(II) carbonate/organic complexes; enhanced abiotic Fe(II) oxidation within the periplasm due to low pH.