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

Abstract Bacterial biofilms are structured microbial communities that play a big role in diverse processes such as nutrient cycling and bacterial pathogenesis. Biofilms are known for their electron transfer properties which are essential for metabolic processes, microbial survival, and maintaining redox balance. In this study, 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 as well as genetic mutants unable to create them (biofilm-deficient), over three days of growth. 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. In contrast, biofilm-deficient cells showed no corresponding changes in current. Cyclic voltammetry (CV) revealed significant differences between the voltammograms of biofilm-forming and biofilm-deficient cells that were hypothesized to be a result of the reduction of secreted flavodoxin only in biofilm-forming cells. Electrochemical impedance spectroscopy (EIS) was also performed at various intervals and analyzed using an equivalent circuit model. We identified the presence of a charge transfer resistance (R ct ) exclusively in biofilm-forming cells which correlated to the time of increased electrochemical activity measured using choronoamperometry. Finally, through confocal microscopy, we found that the expression of a gene involved in biofilm matrix formation, tasA , was correlated with the time where electrochemical charge transfer was measured. Altogether, 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, a methodology was developed to continuously monitor the stages of biofilm formation through measurement of electrochemical activity, substantiating a correlation between the expression of biofilm genes and their electrochemical or redox activities. These data show 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.