BRPI0902426A2 - carbon tissue treatment process to obtain biocathode or bioanode - Google Patents

Abstract

CARBON TISSUE TREATMENT PROCESS FOR BIOCATODO OR BIOTODO. The present invention relates to the process of treating refluxed nitric acid carboxylated carbon tissues (TCs) previously for the preparation of methylene blue monolayer or its derivative (azure A, B or C) adsorbed or carbodimide-bonded, followed of enzyme deposition and immobilization with glutaraldehyde in the presence of polar solvent. In this invention the enzymatic extracts of Shiitake mushroom laccase (Lentinula edodes), peroxidase (Curcubita pepo) and yeast alcohol dehydrogenase (Saccharomices cerevisae) are used. The energy density of the fuel cell is preferably between 0.1 and 5.0mW.cm-2 ^ and the electric current density is preferably between 0.1 and 20.0mA.cm--2 ^.

Description

CARBON TISSUE TREATMENT PROCESS FOR DEBIOCATODE OR BIOANODO

FIELD OF INVENTION

The present invention relates to the treatment of previously carboxylated carbon tissues (TCs) in reflux nitric acid, followed by enzyme deposition and immobilization to obtain biofuels or biofuels of biofuels.

BACKGROUND OF THE INVENTION

The worldwide need for new renewable sources of energy is causing an evolution in the area of fuel cells using platinum or other rare metals. Although they are efficient generators of electricity from chemical processes, this type of fuel cell has some difficult problems to solve which are: 1) platinum cost, 2) platinum poisoning with CO and H2S, 3) non-selectivity of platinum and 4) High cost and low amount of platinum ore.

The fuel cell is a special class of fuel cells where a biocatalyst, such as microorganisms or enzymes, is used, rather than inorganic metals as a reaction catalyst. In this type of biocell, the catalyst is directly involved in the oxide-reduction reaction or in the electricity generation chain. Preferably, in biocatalysts, enzymes are immobilized on electrodes to facilitate repeated use of the catalysts. The fuel is enzymatically oxidized by the anode, producing protons and electrons. In the cathode, the oxidant (usually oxygen or peroxides) reacts with electrons and protons. A critical challenge in the development of this type of direct cell is the inefficient conduction of electrons between the ocatalyst and electrodes, and proton membrane protons to the biocatalyst.

Enzymatic fuel cell cells have the following characteristics: (1) Enzymes are totally renewable catalysts, and their production may meet a growing demand for fuel cell cells; (2) The mass production of these enzymes can reduce much more than platinum; (3) Electroenzymes cannot be poisoned by contaminants such as carbon monoxide (CO) and hydrogen sulfide (H2S) present in cheap fuels; (4) These enzymes only catalyze their specific reactions, the unavoidable leakage of gases or reagents on the opposite side of the fuel cell does not reduce their efficiency; and (5) there are in these enzymes a few electron-daring entry sites, as well as substrate (s) and product (s) to reach the center of the enzyme reaction. Enzymes or biocatalysts prefer mainly at room temperature whereas reactions of metal catalysts in a fuel cell generally require high temperatures (> 80 ° C).

Direct electron transfer (DET) between an enzyme and the electrode can be observed in various enzymes such as cytochrome c, laccase, hydrogenase and numerous peroxidases, including microperoperoxidases (Varfolomeev et al., 1996; Ghindilis et al., 1997). Schuhmann et al., 2003). Close contact of the enzyme sites with the electrode surface is critical for DET. For laccase to directly catalyze the oxygen in reduction, the critical distance between the active enzyme sites and the electrode surface should be around 20Â ° (Yaropolov et al., 1981). A separation greater than this critical distance attenuates the overall reaction, which is determined by the electronic conduction rate; whereas at a closer distance, electronic conduction becomes so efficient that the kinetics of the enzymatic reaction become a fundamental measure. A similar phenomenon refers to radish peroxidase, where the critical distance is 18Â ° (Kulys and Smalius, 1984).

However, in some other cases, DET is determined by thickness rather than by the conductivity of the protein membrane where the active sites of an enzyme are located. To overcome this barrier, enzymes can be transformed to make them conductive through chemical modification (Willner et al., 1996; Guiseppi-Elie et al., 2002; Zhao etal., 2002; Cai and Chen, 2004). Another known strategy is the use of reduction reaction mediators, which facilitates shuttling of electrons between the enzyme's active sites and the electrode surface. This approach has been reported for both enzymatic microbial and biocell (Lewis 1966; Govil and Saran, 1982; Palmore and Whititesides 1994; Katz et al., 2003). Although mediators introduce one more step in the "fuel" reduction reaction for electron generation, a better biofuel efficiency can usually be observed. One of the challenges of using a mediator, which is often small and diffuse, is how to store the fuel cell where continuous system power is required.

The low charge density and short life span are additional problems for the actual application of fuel cells. To address these problems, much effort and significant improvements have been made over the past decade. For example, glucose oxidase (GOx) and amroperoxidase-11 were monolayered on a gold electrode on 0.4 cm diameter discs applied to a glucose / cumene peroxide fuel cell (Katz et al., 1999). A power of 520 μΐΐ was produced, which corresponds to 4.1 W / cm2, based on the projected area of the electrode. An improvement in charge density by volume or weight was obtained by miniaturization of the Glucose / 02 biocell (Heller, 2004). In this work, enzymes such as GOx ebilirubin oxidase (BOd) were introduced into the os (Os) - containing reducing polymers on the surface of two 7μπι carbon fibers. This generated a power of 4.3μ! / \ / In a volume of fibers of 0.0026 mm3, representing 1.65 μΐΛ // ιηιη3. In another study, a 45-day lifetime increase with enzymes introduced into a modified Nafion® membrane was found (Minteer et al., 2004; Moore et al., 2004).

Notably, improvements have occurred, mainly through proper choice of electrode material and enzymatic immobilization media to promote communication between enzymes and electrodes. Studies continue to be carried out to develop more fuel efficient biocells. It is estimated that the catalytic performance of biocatalysts may become the most important issue for the successful application of fuel cells.

Enzyme immobilization can be chemical or physical. Most enzyme biocells are constructed with physical enzyme mobilization, which is a common method for absorbing the enzyme into conductive particles such as carbon black or graphite powder. Hydrogenases and laccases have been physically adsorbed onto carbon black particles to build the composite electrode (Tarasevich et al., 2002). Pizzarielo et al. (2002) demonstrated a Glucose / H202 biofuel biocell using electrodecoded ferrocyanide. GOx and radish peroxidase (horseradishperoxidase, HRP) were first adsorbed onto synthetic graphite particles, and these adsorbed enzymes were suspended in 2-hexadecane and ferrocyanide in a chloroform solution. The electrode was prepared by suspension spraying on the polyester substrate. The cell was continuously working for 30 days with a negligible drop in voltage even though the power was low.

Another approach to physical immobilization is the entrapment of enzymes in polymeric matrices, which generally retain the enzyme better than electrode surface adsorption. US2004 / 0101741, published May 27, 2004, to Minteer et al., "Enzymeimobilization for use in biofuel cells and sensors, discloses a method for entrapping the enzyme in the Nafion® membrane. According to this method, dehydrogenase-dependent dehydrogenase NAD + (such as alcohol dehydrogenase, aldehyde dehydrogenase, formaldehyde dehydrogenase, glucose dehydrogenase and lactodehydrogenase) was physically mixed with the modified Nafion® bromide - solution, to which methylene green was then added and placed over the vitreous carbon electrode. Enzyme immobilization was performed at 140 ° C for 25 min The NAD + cofactor was co-immobilized by ion-tapping the Nafion® membrane, modifying the vitreous carbon electrode, Interestingly, there was no difference in enzymatic activity before and after heat treatment. Et0H / 02 fuel cell was constructed using this method generating a charge density as high as 2.04 pW.c m-2. Cyclic voltammetry measurements indicated that mass transference, not reaction kinetics, is the limiting factor in such a fuel cell.

US 2005/00954 66, published May 5, 2005, to Minter et al. , "Immobilized enzymes inbiocathodes", discloses the enhancement of a cathode - containing biofuel containing cell having a dual - function membrane, which comprises a membrane - buffered - compartment immobilized oxygen oxidoreductase enzyme and an electron transport mediator from a conducting electrode. electrons for a redoxcatalyzed reaction by an oxygen oxidoreductase enzyme. This enhanced fuel cell also has an anode which contains an oxidoreductase enzyme that uses an organic fuel such as alcohol as a substrate.

Several factors regulate the life of a fuel cell, which has always been a concern for its practical application. Reduction mediators and reduction in life span are also other concerns (Allen and Bennetto, 1993; Barton et al., 2004). In many cases, the stability of biocatalysts determines the life span of the cell. Generally enzymatic stability decreases after a few days (Willner et al., 1998; Katz et al., 1999; Kang et al., 2004). Immobilization may help extend the life span of enzymes. A miniature osmotic (Os) immobilized GOx eBOd fuel-cell miniature containing reducing polymers had a 20-day shelf life at 37 ° C (Mano et al., 2002). Moore et al. (2004) demonstrated promising results using tetrabutylammonium bromide, modifying Nafion® membrane to trap dehydrogenase. The half-life of pure enzymes is only 7 ~ 8 h in solution, but after immobilizing the enzyme, the lifespan increases by more than 45 days. In addition, immobilization-based fuel biocells showed no significant reduction in energy over several weeks of continuous operation (Winder, 2003).

One of the biggest problems in developing biocells is charge density, which is usually measured by the generation of power relative to the electrode surface area, or cell weight or volume. The high concentration of the enzyme is critical for high current density production. For example, when GOx was randomly placed as a monolayer on a flat surface, the enzyme charge was only 1.7x10-12 mol.cm-2 (0.27pg.cm-2), which was determined by the physical number of enzymes. Assuming that all enzymes are as active as in typical aqueous solutions number around 600s -1, the maximum current density limit was calculated to be only around 0.2 mA.cm-2 (Willner et al. 1996). GOx is known as one of the most efficient enzyme reducers. For other enzymes with low specific activity, the theoretical resulting density may be even lower. Since fuel cells typically work at voltages below IV, theoretically this current density may only provide less than 0.2mW.cm-2.

OBJECT OF THE INVENTION

Therefore, it is an object of the present invention to perform the treatment of previously carboxylated carbon tissues, deposition and immobilization of enzyme to obtain the bioacode or bioanode. It is also the object of this invention to use enzymatic extracts of Shiitake mushroom (Lentinula edodes), peroxidase ( Curcubita pepo) and yeast alcoholshydrogenase (Saccharomices cerevisae) in order to reduce the cost of biocell production.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 represents an enzyme fuel cell.

Figure 2 is a schematic representation of the components of a solid polymeric electrolyte unit fuel cell.

Figure 3 is a schematic representation of carbon tissue etching in order to obtain a biocode or bio anode of a fuel cell.

Figure 4 represents the results of the polarization (a), potential vs. curves. current, and power output vs. current (b) of the laccase-immobilized biocathode using Pt bioanode obtained from the fuel cell described in Figure 2;

Figure 5 represents the bias (a), potential vs current, and power vs. power curves. stream (b) of the immobilized peroxidase-containing biocathode using Pt bio anode obtained from the fuel cell described in Figure 2;

Figure 6 · represents the bias curves (a), potential vs. current, and power output vs. current (b) of the linked alcohol dehydrogenase-containing bioanode using Pt biocathode obtained in the fuel cell described in Figure 2.

DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to the accompanying drawings.

Figure 1 represents an enzyme fuel cell. In this Figure 1 there is on the right side the oxygen reducing enzyme adsorbed or immobilized on the CER reducing enzyme cathode and on the left side the oxidanteadsorbed or immobilized on the anode with oxidizing enzyme AEO, isolating the two electrodes CER and AEO has the conductive polymeric membrane of protons MP. In the anode with oxidising enzyme AEO the fuel C becomes residue R. Nocatode with reducing enzyme CER oxygen 02 becomes water H20.

The present invention relates to the treatment of previously carboxylated carbon tissues (TCs) in nitric acid between 4 and 10 mol.L-1 under reflux for 0 - 140h. After carbon is washed with water 3 times, the TCs are frozen and lyophilized to dryness or just dried in a greenhouse at 90 ° C. These fabrics are added in a chloroform solution (CHCl3) containing methylene blue or its derivative (1.0-2.0% (w / v) azure A, B or C) and 2.0 - N, N-dicyclohexylcarbodiimide. 4 mmol.L-1 for 12 - 24h under agitation at 25 ° C. After this period, the CTs are removed and air-dried to remove CHC13 and washed 3 times with water for 30min. Thereafter the TCs are dipped in phosphate buffer solution at pH 7.0 and OfOlmol.L-I for 12h and then 10.0-100 laccase, peroxidase, or alcohol-hydrogenase extracts added in Omg.mL-1 in this solution. After desertion to small bubbles and stirring for 2h, ethylene glycol is added to samples dipped in 3: 1 laccase or peroxidase solution (extract or enzyme: organic solvent) under stirring for 30min and left at 4 ° C without stirring for 12-24h. In the case of samples dipped in alcohol dehydrogenase solution, acetone is added in the same proportion. The TCs are then removed, frozen in liquid nitrogen and lyophilized until dry. The dried TCs are immediately immersed in 0.5% glutaraldehyde (GA) acetone solution for 30 min, washed 3 times with water and again frozen in lyophilized nitrogen to dryness. When assembling the fuel cell, the biocathodes are sprayed with 0-30% (w / v) Nafion emisopropanol solution.

Figure 2 is a schematic representation of the components of a solid polymeric electrolyte unit fuel cell. The fuel cell shown in this Figure 2 comprises aluminum plate 1; screw inlet 2; heater inlet 3; heaters 4; graphite board5; gas flow distributor 6; adjusting guide 7, spacers 8; membrane 9; gas diffusion electrode 10, thermocouple 11; O-ring 12; teflon tube 13; connectors 14.For electrochemical measurements the electrochemical fuel cell of Figure 2 is assembled, obtaining power values between 0.1 and 5.0mW.cm-2 with current densities between 0.1 and 20.0mA.cm-2. .

These biocathodes and bioanodes can be used for heart rate sensors and pacemakers.

Figure 3 is a schematic representation of the carbon tissue degradation steps to obtain a biocode or bioanode of a fuel cell, where in A pre-treatment of refluxing carbon dioxide (HN03) carbon tissue occurs; in B a methylene deazul monolayer or its derivative (A, B or C) is adsorbed or carbodiimide linked; C is deposited on methylene blue ammonium layer and D is immobilized on glutaraldehyde in the presence of polar solvent.

For electrochemical measurements the unit fuel cell is assembled, obtaining values of energy densities and electric current densities (see Figures 4, 5 and 6).

Figure 4 represents the results of the polarization (a), potential vs. curves. current, and power output vs. current (b) of the laccase-immobilized biocathode using Pt bioanode obtained from the fuel cell described in Figure 2;

Figure 5 represents the bias curves (a), potential vs. current, and power output vs. current (b) of the immobilized peroxidase-containing biocathode using Pt bioanode obtained in the fuel cell described in Figure 2. Figure 6 represents the bias (a), potential vs. curves. current, and power output vs. current (b) of the linked alcohol dehydrogenase-containing bioanode using Pt biocathode obtained in the fuel cell described in Figure 2.

All preparations are performed at temperatures below 30 ° C to avoid denaturing proteins (enzymes).

Example 1: Tissues of carbon (TCs) carboxylated previously in nitric acid between 4.0 and 8., -Omol. L-I overflows for 0 - 140h. After washing with water 3 times, the TCs are frozen and lyophilized to dryness or oven drying at 90 ° C. These tissues are added in chloroform solution (CHCl3) containing 1.0% (w / v) methylene blue and N, N-dicyclohexylcarbodiimide at 2.0 mmol.L-1 for 12h while stirring at 25 ° C. After this time, the CTs are removed and air-dried to remove CHC13 and washed 3 times with water for 30min. Thereafter the CTs are dipped in phosphate buffer solution at pH 7.0 and 0.01mol.L-1 for 12h and then the laccase extract at 10.0mg.mL-1 is added. After deaeration for bubble removal and stirring for 30 min, ethylene glycol is added to the laccase and acetone-treated samples for the 3: 1 alcohol dehydrogenase samples (extract: organic solvent) under stirring for 30 min, then left at 4 ° C without stirring. by 12pm. Immediately thereafter, the TCs are withdrawn, frozen in liquid nitrogen and lyophilized. The dried TCs are immediately dipped in 0.5% GA deacetone solution for 30min, washed 3 times with freshly frozen nitrogen water and lyophilized.

Example 2: The procedure of Example 1 is repeated, with laccase extract between 20.0 and 100, Omg.mL-1;

Example 3: Procedures 1 and 2 are repeated, and the concentrations of N, N-dicyclohexylcarbodiimide are between 0.04 and 4.0 mmol.L-I;

Example 4: Repeat procedures 1, 2 and 3, where GA concentrations are between 0.1 and 1.0% (v / v) Example 5: Repeat procedure 4 above, using crude extract zucchini peroxidase (Curcubita pepo) in place of the laccase extract;

Example 6: Procedure 4 above is repeated using crude alcohol dehydrogenase extract (Saccharomices cerevisae) and precipitating solvent, acetone;

Example 7: 0.1 to 0.5 ml of a solution of Nafion® in 1-30% (w / v) isopropanol by mass (g) of carbon tissue is sprayed together with the enzyme immobilized on all of them;

Example 8: All of the above methods are repeated using methylene blue, Azure A, B or C derivatives in place thereof.

The materials obtained through the proposed methods are highly satisfactory for the production of electrical energy in cardiac pacemakers or as embiosensor bioelectrodes, and in micrometer dimensions.

The advantages introduced by the present invention relate mainly to the use of plant or microorganism enzyme extracts or pure enzymes, which can reduce the cost of producing Nafion® membrane-free biocells as a polyelectrolyte membrane.

Claims (9)

1. Process for the treatment of carbon tissues to obtain biocathode or bioanode, characterized in that it comprises the following steps: - treating previously carboxylated carbon tissues in nitric acid (8, Omol. L-1) at reflux - preparing a monolayer of methylene blue or its derivative (azure A, B or C) adsorbed or bound with NfN-dicyclohexylcarbodiimide, - deposit enzyme obtained from vegetable enzymatic extracts or microorganisms onto the methylene blue monolayer; e- immobilizing the enzyme in the presence of polar solvent, in this case acetone containing glutaraldehyde. Process according to Claim 1, characterized in that chloroform (CHCl3) containing W, N-dicyclohexylcarbodiimide containing preferably between 0.5 and 4 mmol.L "1 is used to make the amide covalent bond between the carboxylic groups of the tissues. of carboxylated carbon and the methylene blue amino group or derivatives thereof (azure A, B or C) or their adsorption with concentration between 0.5 and 2mg.mL_1 porcarg (g) of the carboxylated TC. Process according to Claim 1, characterized in that the enzymes obtained from enzymatic vegetable extracts comprise laccase and peroxidase. Process according to Claim 1, characterized in that the enzymes obtained from microorganism enzymatic extracts comprise alcohol-hydrogenase. Process according to Claim 1, characterized in that it comprises the deposition of dalacase or peroxidase in an ethylene glycol organic solvent medium in the ratio of 1: 1 to 3: 1 (daenzyme: ethylene glycol solution). Process according to Claim 4, characterized in that the alcohol dehydrogenase is deposited in acetone in the ratio of 1: 1 to 3: 1 (dazyme: acetone solution). Process according to Claim 1, characterized in that the 0.1-1.0% (v / v) emacetone glutaraldehyde is used for periods of between 10 and 60 minutes for immobilization of the precipitated enzyme. Process according to Claim 1, characterized in that it uses glassy or pyrolytic carbon, carbon nanoparticles, activated carbon and other carbonaceous materials in place of carbon tissue. Process according to Claim 1, characterized in that Nafion® is sprayed between 1.0 and 30.0% (w / v) in isopropanol on the final obtained biocathode or bioanode followed by drying before use in the fuel cell to generate energy.