Urea Hydrolysis by Biofilms composed of urease producing bacteria are of primordial interest in several fields ¹. The ureolysis at circumneutral pH requires water and a proton producing two molecules of ammonium ions and one molecule of bicarbonate ion. The consumption of one proton increases the pH and carbonate mineral precipitation (biomineralization) may proceed when calcium or other divalent cations are present ¹. This biochemical process can be useful for applications such as building materials, soil stabilization, and contaminant remediation; it is also of interest to industrial, agricultural and wastewater treatment ¹. To increase the ureolysis rate new methods should be sought to maintain bacterial cells adhered to different solid surfaces However, biofilms are generally studied by imaging, rheological and physical techniques which are difficult to implement and do not represent an efficient form of “metabolic” evaluation, thus it is necessary to develop alternative techniques that allow the direct study of the biofilms properties.

In light of this scenario, the present work proposes the selection of a microbial consortium from cow dung and the study of the formation and growth of an ureolytic biofilm on graphite surfaces by Electrochemical Impedance Spectroscopy (EIS) without noticeable modifications of the biofilm. EIS spectra were obtained at open circuit potential (OCP) (without additional electric perturbation apart from sinusoidal perturbation ±10 mV), during the time of immersion of graphite in the culture medium, in the absence (Control) and presence of bacteria (Biotic).

The results (figure 1) indicate that the spectra of both control and biotic conditions exhibit just one time constant. Moreover, spectra adjustment to equivalent electric circuits indicates that the biofilm adhesion is directly related to the modification of the capacitance, while the generation of ammonium ions directly influences the decrease in electrical resistance of the culture medium. The biofilm maturation and its interaction with the graphite surface induces lower charge transfer resistance, which can be applied as a novel technology for the urea hydrolysis. SEM and CLSM images confirm the presence of ureolytic biofilm and confirm the interpretations obtained by EIS.

Figure 1. Evolution of Bode plots obtained on a graphite surface (0.283 cm²) during the time of immersion. Solid symbols identify frequency where the impedance was obtained. The lines are simulated EIS diagrams constructed with the electric parameter values obtained from the best fit of EIS experimental spectra. The study systems: a) and c) Control (without bacteria), and b) and d) Biotic: with microbial consortium inoculum.

Funding

This research was supported by the National Council for Science and Technology (CONACyT) México and for the scholarship 328072.

References

1. J. M. Connolly, B. Jackson, A. P. Rothman, I. Klapper, and R. Gerlach, npj Biofilms Microbiomes, 1, 15014 (2015) http://www.nature.com/articles/npjbiofilms201514.