Electrochemical Characterization of electroactive Biomolecules Isolated from Novel Fungal Sources Applied in Bioelectrodes for Microbial Fuel Cell

Recent researches have promoted the progressive application and substitution of expensive metallic and abiotic catalysts used in the cathodic reduction of oxygen  by oxidasic enzymes as well as the use of better electron transportation systems to improve the current deficiencies and to diminish the implementation costs of the bioelectrodes for biofuel cells (BFC). Nevertheless, most of these biocatalysts still have serious problems, regarding use and stabilization. In contrast, the use of electronic mediators remains restricted due to their price and toxicity. As an attractive and promising alternative we studied the in-situ production of oxidasic enzymes as well the use of novel fungal biomolecules applied as electron shuttles. As such, we initiated the screening with 12 Brazilian indigenous fungal strains. At the end, three fungi were identified as sources of oxidasic enzymes (mono or polyphenol oxidases): Aspergillus sp. (SIS-18), Penicillium sp. (SIS-21), and Rhyzopus microsporus var. microsporus (SIS-31), and four strains as sources of pigments with high redox capacity: Aspergillus sp. (red), Penicillium sp. (green), Talaromyces sp. (orange) and Penicillium sp. (yellow). The production of these fungal biomolecules was controlled by the used of different carbon substrates including glucose and glycerol. The In-situ production was studied by chronoamperometric analysis in fungal air-cathodes and compared with the profiles obtained for Laccase from T. versicolor in terms of coulumbic efficiencies and maxima current densities. The BFC system was composed by an anodic compartment with a graphite electrode submerged in 20 mmol L^-1 Potassium Ferrocyanide and a fungal cathodic compartment with Pt-Black carbon or free Pt graphite electrodes submerged in the culture medium. A saline bridge of saturated KCl was used as cation exchange system. Fungal BCF were studied in parallel with submerged cultures during 7 days at 28^oC, in terms of substrate consumption, enzyme production and biomass production. Novel fungal pigments were obtained in separate experiments from submerged cultures after 15 days. Crude extracts were characterized by chromatography and spectrometry. Cyclovoltammetric analyses were compared with well-known mediators as triphenylmethane dyes and tiazinic dyes, and also in the presence of bioelectrodes composed by glucose oxidase (GOx) or laccase (LAC). The electrochemical analyses were performed in a 25 mL cell using graphite as working electrode, Pt as auxiliary electrode and Ag|AgCl in saturated KCl as reference in 10 mmol L^-1 potassium phosphate buffer pH 5.0 or in 10 mmol L^-1 KCl. Although the highest oxidasic activity was found for the strain SIS-21 (4600 UI.mL^-1), the strain SIS-31 showed the highest current density value of 125.27 mA.cm^-2. In terms of coulumbic efficiencies, using glucose as substrate, the obtained values were of 23.8%, 46.5% e 56.5%, for SIS-18, SIS-21 and SIS-31, respectively.  Cyclovoltammograms of the enzymatic extracts in the presence of 10 mmol mL^-1 or in 100 mmol L^-1 of potassium phosphate buffer pH 5.0, showed similar profiles for  SIS-18 and SIS-31 with two oxidation peaks and one reduction peak, whereas for SIS-21 two oxidation peaks and two reduction peaks were observed. This observation could indicate the presence of monoxidases in the case of SIS-18 and 31; and a polyphenoloxidase for SIS-21. On the other hand, Preliminary analyses by cyclicvoltammetry of the pigments showed in general similar profiles, being observed two oxidation peaks and three reduction peaks.  Also increases on current densities for GOx in the presence of 1 mmol mL^-1 glucose and for LAC in the presence of 1 mmol mL^-1 pyrogallol were observedwhen the pigments  were evaluated along with these enzymes. Currently, we are studying the mechanisms for electron transportation of these pigments based on the structure-activity analyses and the concomitant in-situ used of pigment producer strains with the ones with proven oxidasic activity.