Fabrication and Characterization of Biofilm-Based Hybrid Matrices in Bioelectrocatalysis

We exploited unique properties of biofilms, i.e. polymeric aggregates of microorganisms, in which cells adhere to each other on the electrode surfaces, and they are characterized by of extracellular electron transfers involving c-type cytochromes (heme-containing proteins). Although aqueous suspensions of gold, silver and certain transition metal oxide (TiO₂ and ZrO₂) nanoparticles tended to inhibit formation of biofilms produced by Pseudomonas aeruginosa, Staphylococcus aureus and Yersinia enterocolitic bacteria, application of composite matrices of inorganic nanostructures within porous conducting polymer layers, e.g. of poly(3,4-ethylenodioxythiophene (PEDOT), facilitated growth of robust and mature bacterial biofilms on glassy carbon electrodes. Independent diagnostic electroanalytical experiments showed that biofilms grown by the following bacteria, P. aeruginosa ATCC 9027, Y. enterocolitica Ye9, Y. enterocolitica AR4, L. monocytogenes 10403S and L. monocytogenes 1115, on inert carbon substrates exhibited by themselves electrocatalytic properties towards oxygen and hydrogen peroxide reductions in neutral media. The processes were found to be further enhanced by introduction of multi-walled carbon nanotubes (MCNTs) that had been modified with ultra-thin layers of organic (e.g. 4-(pyrrole-l-yl) benzoic acid. We expect here attractive electrostatic interactions between carboxyl-group containing anionic adsorbates and positively charged domains of the biofilm with cytochrome enzymatic sites. Co-existence of the above components leads to synergistic effect that was evident from positive shift of the oxygen reduction voltammetric potentials  and significant increase of voltammetric currents. The film exhibited high activity towards reduction of hydrogen peroxide. Most likely, the reduction of oxygen was initiated at cobalt porphyrin redox centers, and the undesirable hydrogen peroxide intermediate was further at the biofilm's cytochrome sites. Comparative measurements were also performed using metal nanoparticles (e.g. Au-Pt), conventional enzymes (e.g. laccase), molecular systems (e.g. metalloporphyrins) in the presence and absence of selected bacterial biofilms.

Development of the biofilm and enzyme based anodes was considered too. To facilitate electron transfers between the electrode surface and the redox protein centers, the concept of co-deposition of MCNTs within the bioelectrocatalytic film was also pursued here. First, MCNTs were modified with ultra-thin layers of tetrathiafulvalene (TTF) or poly(dimethyldiallylammonium chloride) (PDDA). The presence of TTF or PDDA was expected to mediate effectively flow of electrons from enzyme active sites through biofilm to the electrode surface. Combination of derivatized MWCNTs with biofilm matrtices and appropriate enzymes produced biocatalytic systems capable of effective oxidation of glucose or ethanol in neutral buffer solution.

Technical help of W. Lotowska,  E. Szaniawska, E. Seta, B. Kowalewska, M. Gierwatowska,  I. A. Rutkowska and S. Zoladek (Faculty of Chemistry, University of Warsaw),  as well as collaboration with K. Brzostek and A. Raczkowska (Faculty of Biology, University of Warsaw) is highly appreciated.