Electrogenic dynamics of biofilm formation: Correlation between genetic expression and electrochemical activity in Bacillus subtilis

Abstract

Biofilms are structured microbial communities, known for their electron transfer properties, which are essential for metabolic processes and microbial survival. Here, we investigated the electrogenic properties of Bacillus subtilis, a bacterial producer of electron-donating biofilms. Interdigitated gold electrodes were utilized to continuously measure the electrochemical activity of biofilm-forming B. subtilis cells and genetic mutants unable to create them (biofilm-deficient). The formation of extracellular polymeric substances (EPS) and filamentous appendages was monitored via scanning electron microscopy (SEM). Chronoamperometry was used to assess electrochemical activity, which showed fluctuations in electrical current at specific time points in biofilm-forming cells. Cyclic voltammetry (CV) revealed significant differences between the voltammograms of biofilm-forming and biofilm-deficient cells, hypothesized to be a result of the reduction of secreted flavodoxin. Electrochemical impedance spectroscopy (EIS) was also performed at various intervals and analyzed using an equivalent circuit. We identified the presence of a charge transfer resistance (R_ct) exclusively in biofilm which correlated to the time of increased electrochemical activity measured using chronoamperometry. Finally, through confocal microscopy, we found that the expression of a gene involved in biofilm matrix formation, tasA, was correlated with the time when charge transfer was measured. These results indicate that electrochemical activity is primarily present in biofilm-forming cells rather than in biofilm-deficient mutants. By combining electrochemical and microscopic methods, we developed a methodology to continuously monitor the stages of biofilm formation and showed that electrochemical activities within biofilms vary over time and there is a temporal relationship between these processes and the expression of genes responsible for biofilm development.