Targeted Formation of Biofilms on the Surface of Graphite Electrodes as an Effective Approach to the Development of Biosensors for Early Warning Systems

Abstract

Biofilms based on bacteria Pseudomonas veronii (Ps. veronii) and Escherichia coli (E. coli) and yeast Saccharomyces cerevisiae (S. cerevisiae) were used for novel biosensor creation for rapid biochemical oxygen demand (BOD) monitoring. Based on the electrochemical measurement results, it was shown that the endogenous mediator in the matrix of E. coli and Ps. veronii biofilms and ferrocene form a two-mediator system that improves electron transport in the system. Biofilms based on Ps. veronii and E. coli had a high biotechnological potential for BOD assessment; bioreceptors based on such biofilms had high sensitivity (the lower limits of detectable BOD₅ concentrations were 0.61 (Ps. veronii) and 0.87 (E. coli) mg/dm³) and high efficiency of analysis (a measurement time 5-10 min). The maximum biosensor response based on bacterial biofilms has been observed in the pH range of 6.6-7.2. The greatest protective effect was found for biofilms based on E. coli, which has high long-term stability (151 days for Ps. veronii and 163 days for E. coli). The results of the BOD₅ analysis of water samples obtained using the developed biosensors had a high correlation with the results of the standard 5-day method (R² = 0.9820, number of tested samples is 10 for Ps. veronii, and R² = 0.9862, number of tested samples is 10 for E. coli). Thus, biosensors based on Ps. veronii biofilms and E. coli biofilms could be a novel analytical system to give early warnings of pollution.

Keywords: BOD biosensors; Escherichia coli; Pseudomonas veronii; Saccharomyces cerevisiae; biofilms.

Figure 1

The approach used in this work for the formation of biosensor early warning systems.

Figure 2

The formation of working electrodes based on a suspension of microorganisms and in the form of a biofilm.

Figure 3

Microscopic studies of the formation of a biofilm of P. veronii DSM 11331^T: (A) a photograph of the optical microscopy for immobilized microorganisms of; (B) a photograph of the optical microscopy for the biofilm after 47 h of growth. The results of the respiratory MTT assay during biofilm formation: (C) E. coli, (D) P. veronii, and (E) S. cerevisiae.

Figure 4

Microscopic studies of the formation of biofilms: (A) elemental distribution of the biofilm made from P. veronii DSM 11331^T at the GPE; (B) elemental distribution of a pure GPE; (C) the surface of a clean GPE, obtained by SEM; (D) E. coli biofilm, obtained by SEM; (E) P. veronii biofilm, obtained by SEM; (F) an integral image of the electrode surface for the P. veronii biofilm, obtained by the LCSM method; (G) an image of the electrode surface in the Nomarski contrast mode for the P. veronii biofilm, obtained by the LCSM method; (H) an image of the electrode surface in the SYTO 11 dye display mode mode for the P. veronii biofilm, obtained by the LCSM method; (I) an image of the electrode surface in conA dye display mode for the P. veronii biofilm, obtained by the LCSM method.

Figure 5

Determination of the interaction constant of the ferrocene mediator with microorganisms in a biofilm through cyclic voltammetry. (A) A voltammogram for the S. cerevisiae biofilm; (B) the dependence of the ratio of limiting currents in the presence and in the absence of a substrate on the reciprocal of the root of the scan rate 1/ν^1/2 for calculating the rate constant of the interaction between the S. cerevisiae biofilm and the ferrocene mediator; (C) electrochemical impedance spectroscopy for the suspension and the biofilms made from Ps. Veronii and E. coli at 0.25 V compared to Ag/AgCl. The lines show equivalent circuit fitting.

Figure 6

The mechanism of electron transfer in the studied biosensor systems. A—in the system “GPE—ferrocene—immobilized microorganisms”; B—in the “GPE—ferrocene—biofilm of microorganisms” system.

Figure 7

(A) A comparative diagram of the substrate specificity of receptor elements based on biofilms and activated sludge; (B,C) calibration dependencies of the sensor response on BOD₅ for receptor systems based on immobilized microorganisms and biofilms of the studied microorganisms; (D) the long-term stability of P.veronii for biofilms; (E) the long-term stability of P.veronii for suspension; (F) the operational stability of P.veronii for suspension; (G) the operational stability of P.veronii for biofilms.

Figure 8

The dependence of the biosensor response based on free-living microorganisms and bacterial biofilms (A,B) on the presence of heavy metals; (C) biosensor response to the pH of the medium.

Figure 9

The linear dependence of the results of the BOD₅ analysis obtained by using the standard method and the developed biosensors based on biofilms.