Effect of pH on the Electrochemical Behavior of Hydrogen Peroxide in the Presence of Pseudomonas aeruginosa

Abstract

The influence of pH on the electrochemical behavior of hydrogen peroxide in the presence of Pseudomonas aeruginosa was investigated using electrochemical techniques. Cyclic and square wave voltammetry were used to monitor the enzymatic activity. A modified cobalt phthalocyanine (CoPc) carbon electrode (OPG), a known catalyst for reducing O₂ to H₂O₂, was used to detect species resulting from the enzyme activity. The electrolyte was a sterilized aqueous medium containing Mueller-Hinton (MH) broth. The open-circuit potential (OCP) of the Pseudomonas aeruginosa culture in MH decreased rapidly with time, reaching a stable state after 4 h. Peculiarities in the E / I response were observed in voltammograms conducted in less than 4 h of exposure to the culture medium. Such particular E/I responses are due to the catalase's enzymatic action related to the conversion of hydrogen peroxide to oxygen, confirming the authors' previous findings related to the behavior of other catalase-positive microorganisms. The enzymatic activity exhibits maximum activity at pH 7.5, assessed by the potential at which oxygen is reduced to hydrogen peroxide. At higher or lower pHs, the oxygen reduction reaction (ORR) occurs at higher overpotentials, i.e., at more negative potentials. In addition, and to assess the influence of bacterial adhesion on the electrochemical behavior, measurements of the bacterial-substrate metal interaction were performed at different pH using atomic force microscopy.

Keywords: catalase; cobalt phthalocyanines; hydrogen peroxide; oxygen reduction; pseudomonas aeruginosa.

FIGURE 1

Open circuit potentials obtained in the absence (Control/black line) and presence (red line) of P. aeruginosa in 100 ml of Mueller-Hinton broth at pH 7 in a solution previously saturated with O₂.

SCHEME 1

Features of the force extension curve used for the estimation of adhesion work, W_adh. Inset: Sketch of the colloidal probe inseminated with bacteria, showing the principle employed for the testing of the bacteria-alloy adhesion.

FIGURE 2

Open circuit potentials obtained in 100 ml of Mueller-Hinton broth with 1 ml of bacterial culture at different pHs in a previously saturated with O₂.

FIGURE 3

Potentiodynamic response of OPG bare (black line) and CoPc modified OPG electrode in Mueller Hinton in the absence (red line) and presence (blue line) of P. aeruginosa a pH 7.0: (A) Cyclic voltammograms recorded at 100 mV/s; (B) Square waves voltammograms. Experimental SWV parameters: initial potential -0.2 V, end potential 1.0V, step potential 5 mV, amplitude 20 mV, frequency 25 Hz, equilibration time 5 s.

FIGURE 4

Potentiodynamic response of CoPc-modified OPG electrodes in Mueller Hinton broth, in the presence of P. aeruginosa at different pHs. (A) Cyclic voltammograms recorded at 100 mV/s; (B) Square waves voltammograms. Experimental SWV parameters: initial potential -0.2 V, end potential 1.0V, step potential 5 mV, amplitude 20 mV, frequency 25 Hz, equilibration time 5 s.

FIGURE 5

Typical AFM images of bacteria adhered to aluminum alloys surfaces obtained in the tapping mode (Left panels: height, amplitude, and phase, respectively. Rightmost panels: three-dimensional profiles of bacteria). (A) Set of images at 2 distinct locations of alloy surface AA 2024 inseminated by P. aeruginosa bacteria. (B) Same for alloy AA 6063. All images were obtained at pH = 7.5.

FIGURE 6

Typical force curves obtained at pH 6.0 for AA2024 and AA6063 alloys reveal bacterial adhesion. Single rupture bonds (A, B). Multiple force peaks (C–G) forming sawtooth patterns often superimposed onto constant force plateaus (D): as a pilus segment is pulled, a typical nonlinear extension curve characteristic of entropic elasticity (C–G) is revealed, while fast unloading signatures the rupture of a pilus bonds at the surface. Zipper-like adhesion reflected by the sequential detachment of multiple pili-surface bonds leading to force plateaus (D), sometimes these plateaus superimpose onto one another (F–H).

FIGURE 7

Histograms for (A) adhesion forces, Fadh. (B) adhesion work, Wadh, and (C) adhesion length, Ladh, for AA2024 and AA6063 aluminum alloy surfaces as function of pH.

FIGURE 8

Average work of adhesion for AA2024 and AA6063 aluminum alloy surfaces as a pH function. A clear maximum on the adhesion work is observed for pH near 6.