Reduction Kinetic of Water Soluble Metal Salts by Geobacter sulfurreducens: Fe2+/Hemes Stabilize and Regulate Electron Flux Rates

Abstract

Geobacter sulfurreducens is a widely applied microorganism for the reduction of toxic metal salts, as an electron source for bioelectrochemical devices, and as a reagent for the synthesis of nanoparticles. In order to understand the influence of metal salts, and of electron transporting, multiheme c-cytochromes on the electron flux during respiration of G. sulfurreducens, the reduction kinetic of Fe³⁺, Co³⁺, V⁵⁺, Cr⁶⁺, and Mn⁷⁺ containing complexes were measured. Starting from the resting phase, each G. sulfurreducens cell produced an electron flux of 3.7 × 10⁵ electrons per second during the respiration process. Reduction rates were within ± 30% the same for the 6 different metal salts, and reaction kinetics were of zero order. Decrease of c-cytochrome concentrations by downregulation and mutation demonstrated that c-cytochromes stabilized respiration rates by variation of their redox states. Increasing Fe²⁺/heme levels increased electron flux rates, and induced respiration flexibility. The kinetic effects parallel electrochemical results of G. sulfurreducens biofilms on electrodes, and might help to optimize bioelectrochemical devices.

Keywords: Geobacter sulfurreducens; bioelectrochemistry; c-cytochrome; reaction kinetic; remediation.

FIGURE 1

Synthesis of Ag nanoparticles (AgNPs) during respiration of G. sulfurreducens with water soluble Ag⁺ ions. The c-type cytochromes at the inner cell membrane (Imc), in the periplasm (Ppc), and at the outer cell membrane (Omc) transport electrons from intracellular electron donors like NADH to Ag⁺/Omc complexes, which leads to AgNPs, attached to the outer cell membrane (Chabert et al., 2020). In recent cell growth experiments with CrO₄^2– (Gong et al., 2018), Cr³⁺ reduction products could be detected within the cells, if the bacteria reacted for several hours with the chromium salts. Therefore, our reduction experiments of CrO₄^2– might also occur partly inside of the cells, although the reaction conditions are different.

FIGURE 2

Analytical tools for the analysis of extracellular metal ion, Fe²⁺/heme, and cell concentrations. (A) Reduction of oxidizing metals salts by G. sulfurreducens by time dependent UV/Vis spectroscopy at wavelengths where product absorptions are negligible. CrO₄^2– respiration is shown as an example. (B) ICP analysis of Cr³⁺ ions outside of the cell (blue) and at the cell membrane (orange) after reduction of CrO₄^2–. Percentage data for reduced metal salts in the supernatant are given. (C) Changes of Fe²⁺/heme concentration during respiration using the areas of Q-bands. (D) Increase of UV/Vis absorptions during bacteria growth with fumarate as intracellular oxidant. The insert shows scattering increases at 600 nm (OD₆₀₀) during 2 days of cell growth. (E) UV/Vis absorptions during cell growth using low chromate concentrations for G. sulfurreducens respiration, starting from the resting state.

FIGURE 3

Kinetic experiments of G. sulfurreducens with extracellular metal salts. (A) Reduction of 6 different oxidizing metal salt solutions (blue, 0.05–0.15 mM) with G. sulfurreducens solutions in the resting state (8.9 ± 0.3 pM). Their linear time dependences of the metal salt concentrations demonstrate zero kinetic order of the reduction process. At the start of the experiments, all iron ions of the bacterial multiheme cytochromes were in the Fe²⁺ state (red). They were rapidly oxidized upon addition of extracellular metal salts, their levels remained low during reduction of the metal salts, and increased again after major consumption of the oxidants. (B) Experiments could be repeated several times. Shown are Fe²⁺/heme levels upon three consecutive additions of CrO₄^2–. (C) A closer look at the Fe²⁺/heme (red) and CrO₄^2– (blue) concentrations. The Fe²⁺ /heme levels increased after major part of CrO₄^2– was reduced, which obviously stabilized EET rates at low concentrations of the extracellular metal salts. (D) Zero order reduction rates per cell for the 6 different metal salts at 30°C and pH = 7.4.

FIGURE 4

CrO₄^2– induce respiration of G. sulfurreducens, starting from the log phase. (A) Fumarate- respiring growth of G. sulfurreducens. Bacteria after 30 and 36 h growth, respectively, were used for metal induced respiration of G. sulfurreducens in the log phase. (B) Fe²⁺/heme levels during CrO₄^2– induced respiration of bacteria starting from log phases 1 and 2, as well as the resting (lag) phase. (C) Reduction of CrO₄^2– (0.05 mM) by bacteria (0.63 ± 0.2 pM) in the log phase 1 and the lag phase. (D) Reduction of CrO₄^2– (0.1 mM) by bacteria (0.83 ± 0.2 pM) in the log phase 2 and the lag phase.

FIGURE 5

Changes of Fe²⁺/heme levels during metal induced respiration of G. sulfurreducens. (A) Linear correlation (red line) between Fe²⁺/heme concentrations (Q-bands) and G. sulfurreducens concentrations (OD₆₀₀ data). The data (red points) were gained by dilution and different cell growth experiments in medium A. Measurement (magenta point) with G. sulfurreducens, grown in medium B, where c-cytochromes are downregulated. (B) Fe²⁺/heme levels during CrO₄^2– induced respiration (0.05 mM) of cells grown in media A (0.63 pM) and B (0.73 pM). (C) Fe²⁺/heme levels (red) and CrO₄^2– concentrations (blue) during CrO₄^2– induced respiration (0.05 mM) of mutants lacking OmcBEST (0.76 pM) grown in medium A. (D) Fe²⁺/heme levels of experiments with G. sulfurreducens (0.74 pM), grown in medium B, with [Fe(edta)] ^– (0.15 mM) and CrO₄^2– (0.05 mM), respectively.

FIGURE 6

Increase of cell concentration and decrease of oxidants [Fe(CN)₆]^3– and CrO₄^2– during G. sulfurreducens respiration in grow medium A, lacking fumarate, and stating from the lag phase. (A) Linear increase of the bacterial growth with 0.15 mM [Fe(CN)₆]^3–, R² = 0.98. (B) Linear increase of the bacterial growth with 0.05 and 0.03 mM CrO₄^2– (insert), R² = 0.98. (C) Linear CrO₄^2– decrease (blue, R² = 0.99), and linear bacterial growth (brown, R² = 0.98) of the same respiration experiment.