NL1034123C2 - Method for obtaining a cathodophilic, hydrogen-producing microbial culture, microbial culture obtained with this method and use of this microbial culture. - Google Patents

Description

Method for obtaining a cathodophilic, hydrogen-producing microbial culture, microbial culture obtained with this method and use of this microbial culture.

The present invention relates in a first aspect to a method for obtaining a cathodophilic, hydrogen-producing microbial culture.

According to further aspects, the invention relates to the cathodophilic, hydrogen-producing microbial culture and the use of this microbial culture for the production of hydrogen.

Further aspects of the invention relate to a method for manufacturing a bi-functional bioelectrode in which the cathodophilic, hydrogen-producing microbial culture is used and the bi-functional bioelectrode obtained with this method.

Other aspects of the invention relate to a device for the production of hydrogen in which the electrochemically active, hydrogen-producing microbial culture is used.

Hydrogen producing cathodes are typically used in water electrolysis processes. In water electrolysis, water is split into hydrogen and oxygen under the influence of an applied potential difference between anode and cathode according to the reaction: H2O2 H2 + 0.5O2 (1) In water electrolysis the following electrode reactions take place:

An oxygen-producing oxidation reaction at the anode: H 2 O - »0.5 02 + 2 H + + 2 e '(2a) or 2 OH' 0.5 02 + H 2 O + 2 e (2b) 30 A hydrogen-producing reduction reaction at the cathode: 034123 2 2 H + + 2 e '-> H 2 (3a) or 2 H 2 O + 2 e' - »H 2 + 2 OH '(3b)

Recently, hydrogen-producing cathodes have also found their application in a new type of electrolysis process, namely, bi-catalyzed electrolysis of dissolved bio-oxidisable material (e.g., in waste water). With biocatalyzed electrolysis, bio-oxidizable material is split into carbon dioxide and hydrogen under the influence of a potential difference between anode and cathode. This can be represented schematically as: [CH 2 O] + H 2 O -> 2 H 2 + CO 2 (4)

This process is described in the international patent application WO 2005/005981 and in the publication "Principles and perspectives of hydrogen production through biocatalyzed electrolysis" (International Journal of Hydrogen Energy 2006, 31, 1632-1640) in the name of Rozendal., R.A. et al.

The term bio-catalyzed electrolysis is derived from the fact that the oxidation of bio-oxidizable material at the anode is catalyzed by electrochemically active microorganisms. The hydrogen-producing reduction reaction at the cathode 20, on the other hand, is chemically catalyzed (e.g. with platinum) in its standard design, just like with water electrolysis. The following electrode reactions take place with biocatalyzed electrolysis of dissolved bio-oxidisable material (eg in waste water):

The oxidation of bio-oxidisable material at the anode, schematically represented by: [CH 2 O] + H 2 O 2 CO 2 + 4 H + + 4 e '(5)

A hydrogen-producing reduction reaction at the cathode: 4 H + + 4 e '2 H 2 (6a) or 4 H 2 O + 4 e' -> 2 H 2 + 4 OH '(6b) 3

Because hydrogen-producing electrolysis processes take place under the influence of a potential difference between anode and cathode, energy is used. This energy can be supplied by a power source. In principle, the higher the voltage required for electrolysis, the higher the consumption of electrical energy per amount of hydrogen produced. Energy losses in the electrolysis process increase the required amount of electricity and the required amount of electrical energy per amount of hydrogen produced. Such energy losses can also occur with the electrode reactions. Loss of energy at a cathode is also called 10 cathode over-potential and is expressed in Volts (V).

In general, overpotentials have the property of increasing (i.e., energy loss becomes greater) with increasing current density (i.e., increasing reaction rate). This is also the case for cathode over potentials. The relationship between the cathode potential and current density can be represented in a so-called E-j curve or polarization curve, which represents the cathode potential as a function of the current density (j).

A cathode is generally composed of two elements, namely a charge distributor of an electrically conductive material and a catalyst to accelerate the cathode reaction. Carbon is a widely used charge distributor, since it has a low cost, a high electrical conductivity and a high chemical resistance. However, with only a charge distributor without catalyst, the electrochemical production of hydrogen at a cathode is kinetically very slow. As a result, large over-potentials (i.e. large energy losses) can already arise at relatively low current densities. Platinum is often used as a catalyst for electrochemical hydrogen production at a cathode.

For water electrolysis processes, platinum is indeed a very effective cathode catalyst that can minimize over-potential at very high current densities (e.g., 0.025 V over-potential at 1.08 A / cm 2 as described in the book Electrochemical oxygen technology written by Kinoshita, K. John Wiley & 30 Sons, Inc .: New York, 1992).

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Unfortunately, platinum appears for many reasons to be a much less suitable cathode catalyst for biocatalyzed electrolysis. First, platinum is a very expensive material. In addition, the current densities of bio-catalyzed electrolysis are typically 3 to 5 orders of magnitude lower than that of water electrolysis 5 (i.e., order of magnitude -0.00001-0.001 A / cm 2). As a result, the amount of hydrogen produced per amount of platinum with biocatalyzed electrolysis is much lower than with water electrolysis. This makes the use of platinum in bi-catalyzed electrolysis far too expensive to be able to achieve a commercially interesting process.

Furthermore, it has been found that platinum is not nearly as effective in biocatalyzed electrolysis as in water electrolysis, as described in the publication "Principles and perspectives of hydrogen production through biocatalyzed electrolysis" (International Journal of Hydrogen Energy 2006, 31, 1632-1640) on name of Rozendal., RA et al. At a current density of only 0.00005 A / cm 2, the cathode was already over-potential over 0.28 V. The relatively mild conditions that typically occur in biocatalyzed electrolysis processes may be a possible reason for this low effectiveness of platinum catalysis in biocatalyzed electrolysis . Relatively mild conditions include, inter alia, the low temperature (e.g., room temperature), the low pressure (e.g., atmospheric pressure), and the mild pH (e.g., pH 7).

The high cost price of platinum in combination with the relatively low current densities in bio-catalyzed electrolysis and the relatively low effectiveness of platinum catalysis under the typical conditions of bio-catalyzed electrolysis processes make it interesting to look for alternative ways to electrochemically produce hydrogen to a cathode.

Prior art

In the past attention has already been paid to the problems of using platinum as a cathode catalyst in the context of (bio) fuel cells and electrolysis cells. In this context, biological catalysts have been examined, among other things. A (bio) fuel cell is understood to mean "an electrochemical" device "30 that continuously converts chemical energy into electrical energy (and some heat) as long as fuel and oxidizer are supplied" (Hoogers, G, "Fuel Cell Technology 5

Handbook, CRC Press 2003). In an electrolysis cell the opposite happens: electrical energy is invested to make desired chemical reactions proceed (Bard, AJ, Faulkner, AE, "Electrochemical Methods, Fundamentals and Applications", Wiley 2001), or electrical energy is converted into chemical energy ( and some 5 heat). Examples of electrolysis processes are water electrolysis and bio-catalyzed electrolysis.

International patent application WO2004 / 015806 describes a stainless steel electrode whose surface is covered with a biofilm (defined in WO2004 / 015806 a) is “a film consisting of microorganisms originating from biological water such as seawater, river water, etc., which spontaneously deposited on a surface ") intended to catalyze the reaction at the electrode of a fuel cell. The biofilm is formed by immersing the electrode in a medium that promotes the growth of biofilms and at the same time applying a polarization potential to the electrode (value between -0.5 and 0.0 V compared to a standard calomel electrode). The biofilm-covered electrode of a fuel cell can be either a cathode or an anode. In the case of a cathode, the biofilm catalyzes oxygen reduction. In the case of an anode, the biofilm catalyzes an anodic fuel cell reaction. However, the patent application does not focus on possible biofilm applications for catalysis of electrochemical hydrogen production at a cathode in an electrolysis cell, nor does this application describe how an electrochemically active microbial culture suitable for electrochemical hydrogen production could be obtained.

The use of a biofilm on a cathode is also described in patent application JP11057782 and by Sakakibara & Kuroda (Biotechnology and Bioengineering, vol. 42, pp. 535-537, 1993). However, these references describe the abiotic production of hydrogen at the cathode, which is then used by the biofilm to reduce nitrate to nitrogen and water. In this case, therefore, there is no question of a microbial culture that catalyzes the production of hydrogen at the cathode, but of a biofilm that consumes electrochemically formed hydrogen.

Tatsumi et al. (Analytical Chemistry, vol. 71, pp. 1753-1759, 1999), Tsujimura 30 et al. (Phys. Chem. Chem. Phys., Vol. 3, pp. 1331-1335, 2001) Lojou et al. al. (Electroanalysis, vol. 14, pages 913-922, 2002) describe the use of electrodes with 6 immobilized Desulfovibrio vulgaris Hildenborough (DvH) cells for the production and / or oxidation of hydrogen. The cells were immobilized by confining a suspension with affected cells between a membrane and an electrically conductive support material. However, for their catalytic action on the electrode, these DvH cells require externally supplied redox mediators.

In addition to biofilm applications, there are also systems described in the literature and in patent applications where isolated enzymes (and not whole microorganisms) were used as biocatalysts. Guiral-Brugna et al. (Journal of Electroanalytical Chemistry, vol. 510, pp. 136-143, 2001) and Morozov et al. (International Journal of 10 Hydrogen Energy, vol. 27, pp. 1501-1505, 2002) mediator-less hydrogen production on electrodes made of carbon material with immobilized hydrogenases. Moreover, Morozov et al. (Russian Journal of Electrochemistry, vol. 38, pp. 97-102, 2002) showed that of one of such electrodes, 50% of the original activity was retained after a half-year preservation in buffer 15 solution at 4 ° C. Patent application WO2004 / 114494 describes a fuel cell that uses immobilized hydrogenases at the anode to catalyze hydrogen oxidation and immobilized oxidases at the cathode for catalysing oxygen reduction. The immobilisation can be achieved by sorption from an aqueous solution or by chemical bonding. US 2006/0159981 20 describes a biological fuel cell consisting of an anode with an attached anode enzyme and a cathode with an attached cathode enzyme. The enzyme on the anode catalyses the oxidation of an undefined reducer and the enzyme on the cathode catalyses the reduction of an undefined oxidizer.

From the foregoing, it appears that, in the case that isolated hydrogenases were used as a catalyst for electrochemical hydrogen production at a cathode, no mediators were needed to effect a catalytic effect. The use of isolated hydrogenases, however, has the disadvantage that these isolated hydrogenases can only be obtained after a laborious purification. Furthermore, isolated hydrogenases lack the required stability for long-term use and have no self-regenerating capacity. When whole microorganisms were used as catalysts for electrochemical hydrogen production at a cathode, up to now 7 externally added mediators (for example methyl viologists or cytochrome C3) were always required to achieve a catalytic effect. The use of mediators has the disadvantage that these are expensive compounds with high toxicity in a number of cases.

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Description of the invention

It is an object of the present invention to provide an alternative to the electrochemical hydrogen production cathode systems described so far. In accordance with a first aspect, the invention provides a method for obtaining a cathodophilic, hydrogen-producing microbial culture. The method comprises the steps of: (i) providing a bioelectrode comprising an electrochemically active microbial culture on an electrical conductor, which microbial culture is capable of hydrogen oxidation; (Ii) placing the bioelectrode in a medium, the nutrient medium suitable to support the physiology of the microbial culture; (iii) applying to the bioelectrode a potential that is lower than the equilibrium potential of the HVH2 redox couple in the nutrient medium;

The bioelectrode provided in the method comprises an electrochemically active microbial culture on an electrical conductor. This microbial culture is capable of hydrogen oxidation. In the context of the present invention, the term electrochemically active microbial culture is understood to mean a culture of microorganisms that can use an electrode directly, i.e. without the use of externally supplied redox mediators, as an electron donor (cathodophilic organisms) or as an electron acceptor (anodophilic organisms). The existence of such anodophilic organisms is known in the art from, among others, "Principles and perspectives of hydrogen production through biocatalyzed electrolysis" (International Journal of Hydrogen Energy 2006, 31, 1632-1640) in the name of Rozendal., RA et al. And in “Electricity production by Geobacter sulfoaducens attached 30 to electrodes” Applied and Environmental Microbiology 2003, 69, 1548-1555 in the name of Bond, DR and Lovley, DR).

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The microbial culture is present on an electrical conductor, that is to say a material that can conduct electrical current. As an electrical conductor is particularly suitable, for example in the form of carbon felt or carbon paper or graphite felt or graphite paper. However, the skilled person can select suitable other materials.

An electrical conductor is also referred to in this description and the claims with the term charge distributor.

The electrochemically active microbial culture present on the bioelectrode is capable of hydrogen oxidation. Hydrogen oxidation is a process in which H 2 is converted to protons and electrons according to the reverse reaction of the above reaction equation (3a and / or 3b). The person skilled in the art will understand that the reactions according to reaction equation 3a and 3b are similar reactions, and that reaction 3a is obtained by stripping left and right 2 OH 'from reaction 3b. The organisms of an electrochemically active microbial culture capable of hydrogen oxidation are capable of directly delivering the electrons formed, i.e. without an externally supplied redox mediator, to an anode as an electron acceptor.

In the method according to the invention, the bioelectrode is placed in a medium, the nutrient medium, which is suitable to support the physiology of at least a part of the organisms in the microbial culture. Supporting physiology means that the organisms can be metabolically active. In the context of the present invention, in particular, metabolic activity of the organisms in the microbial culture with respect to hydrogen formation via, for example, one or more of the above reactions 3a and 3b is important. Media suitable as a nutrient medium are known to those skilled in the art. Such a suitable medium is, for example, Postgate's medium. Other suitable media are described in examples 1 and 2.

For hydrogen production, it is further important that the nutrient medium has a low oxygen voltage. The medium is therefore preferably microaerophilic, and more preferably substantially anaerobic.

In general, an aqueous solution of trace elements which further comprises a carbon source, preferably carbon dioxide, with a pH of 2-10, such as pH 3-9, preferably pH 5-7.

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To limit the growth of methanogenic organisms that consume water, it is preferable to use a low pH below pH 5.0, such as below pH 4.0. Methanoic organisms are known to be inhibited by such a low pH value. In addition, growth of methanogenic bacteria can be inhibited by minimizing the concentration of carbon dioxide after the microbial culture has grown sufficiently. A Pco2 of below 0.0003 atmosphere, such as below 0.0002 or below 0.0001 atmosphere can be used for this.

In the method according to the invention, a potential is applied to the bioelectrode which is lower than the equilibrium potential in the feed medium of the 10 H + / H 2 redox couple. The equilibrium potential of the H + / H2 redox couple in the nutrient medium can be determined by those skilled in the art on a theoretical basis (based on knowledge of the composition of the nutrient medium and the other reaction conditions). Further, in a number of cases, the equilibrium potential can be determined by determining the open circuit voltage of a cell. The application of the potential can be done with the aid of a potentiostat or another suitable electrical power source.

For electrochemically active microorganisms adapted to hydrogen oxidation, where electrons are delivered to the electrode's charge divider, the switch to a potential that is lower than the equilibrium potential of the H + / H2 redox couple in the nutrient medium reversal of their electro-chemical reaction. Because this potential is below the equilibrium potential of the H + / H2 couple, the cells will be forced to catalyze the reaction in the direction of proton reduction. Thus, they will produce hydrogen in an electro-active manner.

The potential applied in step (iii) of the method is, for example, at least 5, 10, 15, 20, 25, 40, 50, 60 mV lower than the equilibrium potential of the H + / H 2 redox couple in the nutrient medium, such as more than 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or more than 1000 mV lower than the equilibrium potential of the H + / H2 redox couple in the nutrient medium.

For certain applications, small differences of the imposed potential with respect to the equilibrium potential of the H + / H 2 redox torque are advantageous here, since this selects organisms that can produce H 2 with a low energy investment.

The imposed potential can be applied using a power source. A potentiostat, for example, is particularly suitable as a power source.

Such a potentiostat can be coupled to a reference electrode, for example a standard hydrogen electrode, a calomel electrode or an Ag / AgCl electrode, to accurately control the potential of the bioelectrode with respect to the equilibrium potential of the H + / H2 couple in the feed medium. However, the use of a potentiostat is not necessary and any other electrical power source, which the person skilled in the art understands to be suitable for applying a potential to the bioelectrode, can be used.

Alternatively, the potential can be controlled below the equilibrium potential of the H + / H2 redox couple, by shifting this equilibrium potential from the H + / H2 redox couple, such as lowering, for example, lowering the pH of the nutrient medium and / or lowering of the hydrogen tension or by other changes in the reaction conditions such as the temperature, as is known to those skilled in the art. The equilibrium potential of the H + / H2 couple is, for example, 0.0 V at pH 0.0 under standard conditions (T = 25 ° C and Ph, = 1 atm). At pH 7.0, this equilibrium potential is -0, under the same standard conditions. 42 V. From this a potential increase of ± 60 mV per pH unit can be deduced. A pH reduction of the nutrient medium can thus also be used to have a potential present on a cathode get a value that is below the equilibrium potential of the H + / H2 redox couple in the nutrient medium.

According to a preferred embodiment of the method, the bioelectrode is obtained by a bioanode comprising an electrochemically active microbial culture on a charge distributor, which microbial culture is able to place oxidation of a biologically oxidizable carbon compound in a nutrient medium capable of physiology of at least a portion of the microbial culture, at a potential higher than the equilibrium potential of the H + / H 2 redox couple in the nutrient medium. This is done under conditions of limitation of the biologically oxidizable carbon Compound in the presence of hydrogen. The applied potential can, for example, be up to 1000 mV, up to 800 mV, such as up to 500 mV higher than the equilibrium potential of the H + / H2 redox couple in the feed medium. Preferably, the applied potential is up to 200 mV higher, such as up to 150 mV, 100 mV, or 50 mV higher.

Suitable biologically oxidizable carbon compounds can be selected by the skilled person. Examples of suitable biologically oxidizable carbon compounds are, for example, short-chain (C 1 -C 6) organic acids, such as lactic acid, acetic acid and dissociated forms thereof, short-chain (C 1 -C 6) alcohols, such as ethanol and propanol or carbohydrates such as glucose, fructose, lactose or sucrose.

By this step, an electrochemically active microbial culture is selected on the bioanode that is capable of hydrogen oxidation, for example via a reaction that runs in reverse to the reaction shown in above reaction equation 3a or 3b. The inventors of the present invention have found that such an electrochemically active microbial culture capable of hydrogen oxidation can produce electrochemical hydrogen at a potential lower than the equilibrium potential of the HVH2 redox couple in the nutrient medium in which they are located.

According to a further preferred embodiment of the method, a biologically non-oxidizable carbon source, such as a carbon dioxide source, is present under conditions of limitation of the biologically oxidizable carbon compound. The presence of a carbon source makes microbial growth possible. A carbon dioxide source is, for example, carbon dioxide or a solution containing H2CO3 and / or HCO3 'and / or CO32'.

A further preferred embodiment comprises grafting the cathodophilic, hydrogen-producing microbial culture onto a charge distributor. By inoculating the cathodophilic, hydrogen-producing microbial culture, this culture can be multiplied in a simple manner and / or a plurality of electrodes can be obtained with the cathodophilic microbial culture. The inoculation can be carried out in any suitable manner known to the person skilled in the art for inoculating microorganisms.

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A further aspect of the invention relates to a cathodophilic, hydrogen-producing microbial culture. Such a microbial culture capable of producing hydrogen in an electrochemical manner without the use of external redox mediators has not been described previously. The microbial culture contains microorganisms that are capable of producing hydrogen by means of proton reduction and / or water reduction, as described in, for example, reaction equations 3a and 3b. The microbial culture can be a monoculture or a mixed culture. According to a preferred embodiment, the microbial culture is obtainable with the method according to the invention for obtaining said microbial culture.

Further aspects of the invention relate to the use of a microbial culture according to the invention for the production of hydrogen. As described above, the microbial culture is suitable for the production of hydrogen.

Still further aspects of the present invention relate to a method for manufacturing a bioelectrode which is suitable, for example, as a biocathode, for example for use in biocatalyzed electrolysis of water. The method comprises providing a body of an electrically conductive material and applying a cathodophilic, hydrogen-producing microbial culture to the surface of the electrically conductive material.

According to a preferred embodiment, this is a method wherein: (i) the provided body of the electrically conductive material comprises two separate surfaces; (ii) on a first surface, the cathode surface, the cathodophilic, hydrogen-producing microbial culture is applied; (iii) a catalyst for an electrochemical oxidation reaction is provided on a second surface, the anode surface.

Thus in this method use is made of the cathodophilic, hydrogen-producing microbial culture according to the invention.

In the method, a body of an electrically conductive material or a charge distributor is provided. In the context of this invention, the term charge distributor means a material that can conduct electrical charge. Suitable charge dividers are, for example, carbon, preferably carbon paper, a carbon plate, graphite paper, a graphite plate or an electrically conductive metal such as copper or titanium.

In the method, a cathodophilic, hydrogen-producing microbial culture according to the invention is applied to the surface of the body of the electrical conductor. The application can be made in any manner that the skilled person understands to be suitable for applying a microbial culture.

According to a preferred embodiment, the body comprises two separate surfaces. This means that it must be possible to separate the two surfaces from each other such that one surface can serve as a cathode and the other can serve as an anode. This is possible, for example, by selecting the body as a substantially two-dimensional body such as a plate-shaped body, preferably a flat plate.

In this preferred embodiment, a cathodophilic, hydrogen-producing microbial culture according to the invention is applied to the first surface, the cathode surface. A catalyst for an electrochemical oxidation reaction, for example an anaerobic oxidation reaction, is provided on a second surface, the anode surface. The catalyst can be any suitable catalyst and the application can be in any manner that is understood by those skilled in the art to be suitable for applying the type of catalyst used. The catalyst can for example be selected from the group of platinum and / or an electrochemically active microbial culture capable of electrochemical oxidation, for example an anaerobic oxidation, of a biologically oxidizable substrate, such as a biologically oxidizable carbon compound. The latter microbial culture may comprise organisms from the group of Geobacter sulferreducens, Shewanella putrefaciens, Geobacer metallireducens, and Rhodoferax ferricreducens or other organisms from the genera to which said species belong or a consortium of these organisms.

The bi-functional bioelectrode obtained with the above-described preferred embodiment of the method comprises a body of a charge distributor, which body comprises two separate surfaces, with on the first surface, the cathode surface, a cathodophilic, hydrogen-producing microbial culture and on the second surface, the anode surface, a catalyst for an electrochemical oxidation reaction.

The present invention also relates to the bioelectrode, in particular a bifunctional bioelectrode, obtained with this method. The characteristics of this bioelectrode and the bifunctional bioelectrode as well as those of the preferred embodiments mentioned in the claims will be clear to those skilled in the art from the description of the method for manufacturing this bifunctional bioelectrode.

According to a further aspect the invention relates to a device. This device is suitable for use as an electrolysis device. The device comprises a number of compartments, with an anode and a cathode spaced apart in each of the compartments. On the anode there is a catalyst for the electrochemical oxidation of an oxidizable substrate and on the cathode a cathodophilic, hydrogen-producing microbial culture. Furthermore, there is an ion-conducting separation between the anode and cathode, which divides the number of compartments into a cathode sub-compartment on the side of the cathode and an anode sub-compartment on the side of the anode. A nutrient medium is present in the cathode sub-compartments, which is suitable for supporting the physiology of the cathodophilic, hydrogen-producing microbial culture. The device further comprises means for supplying to the anode subcompartments a substrate medium comprising the oxidizable substrate and means for discharging hydrogen from the cathode subcompartments. Furthermore, there is an electrical connection between a number of anodes and a number of cathodes.

The ion-conducting separation can be a cation or anion-conducting material, such as a cation-selective membrane, an anion-selective membrane, or a bipolar membrane. (Micro) porous membranes such as microfiltration, ultrafiltration or nanofiltration membranes are also suitable. Such materials are known to the skilled person.

An electrolysis device of the above-mentioned type is known per se in the art. The electrolysis device according to the invention, however, differs from those known in the art by the presence of a cathodophilic, hydrogen-producing microbial culture on the cathode. In the context of this description, the term a number means one or more.

According to a preferred embodiment of the electrolysis device, the number of compartments is a plurality of compartments. The plurality of compartments is subdivided into a first and a second terminal compartment and a number of intermediate compartments between them. The number of anodes and cathodes is alternately present in the device. Furthermore, in this embodiment, the electrical connection between the number of anodes and the number of cathodes is arranged as an electrical connection between the anode and cathode of adjacent intermediate compartments, an electrical connection between the cathode of the first terminal compartment and the anode of the first terminal compartment adjacent intermediate compartment, an electrical connection between the anode of the second terminal compartment and the cathode of the intermediate compartment adjacent to the second terminal compartment and an electrical connection between the anode and cathode of the terminal compartments. These electrical connections are such that each anode is electrically connected to one cathode.

The electrolysis device according to this embodiment can be shaped as a stack of electrolysis cells.

According to a further preferred embodiment, the electrical connection between a number of anodes and cathodes comprises a power source for adjusting the potential of the cathode. If a power source is present, it will be included for a stack in the electrical connection between the anode and cathode of the terminal compartments.

According to another preferred embodiment of the electrolysis device, the catalyst for the electrochemical oxidation of an oxidizable substrate on the anodes comprises a catalyst from the group comprising platinum and / or a microorganism selected from the group of Geobacter sulferreducens, Shewanella putrefaciens, Geobacer metallireducens and Rhodoferax ferric reducts other organisms from said genera or a consortium of one or more organisms therefrom. These organisms are known to be anodophilic.

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According to a further preferred embodiment of the electrolysis device, the anodes and cathodes of the intermediate compartments are arranged as bifunctional electrodes according to the invention. Due to the low internal resistance of the bifunctional electrode, the internal resistance of the electrolysis direction is also reduced compared to an electrolysis device from the prior art with a comparable electrode surface.

In a further aspect, the invention relates to a method for producing hydrogen. The method comprises: (i) providing an electrolysis device according to the invention; (Ii) supplying a substrate medium comprising an oxidizable substrate to the anode sub-compartments; (iii) applying a potential to the plurality of cathodes which potential is below the equilibrium potential of the H + / H 2 redox couple in the nutrient medium; (Iv) draining the hydrogen produced at the cathode.

Supplying the substrate medium to the anode subcompartments can be done in any suitable manner such as (continuous) pumping. The oxidizable substrate can be any suitable substrate as known to the skilled person. The substrate medium can for instance be a waste water stream with a high content of organic compounds.

The application of the potential to the number of cathodes which potential is below the equilibrium potential of the H + / H2 redox couple can be made with the aid of any power source to which an anode and cathode are connected in the electrolysis device according to the invention. Alternatively, as described above, the potential can be controlled below the equilibrium potential of the H + / H2 redox couple, by shifting this equilibrium potential of the H + / H2 redox couple, such as lowering, for example, by lowering the pH of the nutrient medium and / or or reducing the hydrogen voltage.

The potential imposed on the cathode is preferably such that a desired amount of H2 is produced per unit of time. This desired amount of H2 can be determined in advance. Another possible criterion for the production of H2 is the 17 invested energy in the form of the imposed potential. A higher energy investment can increase the cost of the H2 produced. The person skilled in the art will be able to optimize the potential imposed with respect to the cost price of the hydrogen.

The hydrogen produced at the cathode sub-compartments can be discharged in any suitable manner for direct use and / or storage.

Figure description

The invention is now further elucidated with reference to the following examples and the enclosed figures, which give non-limitative exemplary embodiments of the invention.

1A-1C shows an overview of an embodiment of the method for obtaining the cathodophilic, hydrogen-producing microbial culture;

Figure 2 shows an embodiment of an electrolysis device according to the invention;

Figure 3 shows a detail of the biocathode of the electrolysis device of Figure 2;

Figure 4 shows a section through a bifunctional electrode according to the invention; Figure 5 shows a detail of the section through the bifunctional electrode of Figure 4;

Figure 6 shows another embodiment of an electrolysis device according to the invention, which uses the bifunctional electrode;

Figure 7 shows a polarization curve of the current density as a function of the cathode potential, as obtained using the cathodophilic, hydrogen-producing microbial culture and two reference experiments;

Figure 8 shows the volume of hydrogen as a function of time for a test in which the cathodophilic microbial culture is used, and a reference experiment;

In the method according to the invention use is made in an embodiment of an electrochemical cell 1 as shown in Figs. 1A, 1B, 1C. Such a cell consists, for example, of two compartments 2, 3 separated by an 18 ion-conducting separation 4 (for example Nafion® 117). A compartment 3 contains a carbon rod electrode 5. This electrode serves as the bio-electrode (working electrode) and is connected to a power source potentiostat 6. The other compartment 2 contains a platinum electrode 7 connected to the power source and serving as a against electrode. A reference electrode can also be placed in the bio-electrode compartment (not shown in Figs. 1A, 1B, 1C). Both compartments 2, 3 are filled with a suitable medium (for example a medium consisting of 0.74 g / L KCl, 1.36 g / L KH2 PO4) 0.28 g / L NH4 Cl, 0.84 g / L NaHCO3, 0.1 g / L CaCl2-2H20, 1 g / L MgSC> 4 · 7H 2 O and 1 mL / L trace elements).

The bioelectrode compartment 3 of the electrochemical cell 1 is inoculated with electrochemically active microorganisms from the anode compartment of a bio-catalyzed electrolysis cell. Subsequently, in the embodiment shown, this culture is fed with acetate and hydrogen and checked at a potential of +0.1 V (vs. standard hydrogen electrode; NHE) and pH 7. Electrochemically active microorganisms form a biofilm 8 on the bio-electrode and provide an anodic current. Subsequently, the pH is lowered to, for example, pH 6 and the potential to -0.1 V (vs. NHE) and after adaptation of the biofilm 8 under these conditions, the bio-electrode is fed with hydrogen and medium in which only bicarbonate as a carbon source is dissolved to hydrogen select oxidizing microorganisms 8a (Figure 1B). After a constant current is generated under these conditions, the potential of the bioelectrode is lowered to -0.65 V (vs. NHE, at pH 6) so that the anodic current changes to a cathodic current (Figure 1C). The biofilm 8a will produce hydrogen after this potential reduction. Thus, a cathodophilic, hydrogen-producing microbial culture 8a is obtained.

Figure 2 shows an overview of a biocatalyzed electrolysis device. The biocatalyzed electrolysis cell contains similar components to the electrochemical cell of Figure 1C, namely two compartments 2, 3, separated by an ion-conducting separation 4. Both compartments 2, 3 in this case contain a bioelectrode. The bioelectrode 5 in compartment 3 contains a cathodophilic hydrogen-producing microbial culture 8a according to the invention. The bioelectrode 7 in compartment 2 (the anode) contains an anodophilic microbial culture 10, which is capable of converting a biologically oxidizable carbon compound into CO2, H + and electrons. The electrons produced at the anode 7 are conducted via an electrical circuit to the cathode 5. Here, the electrons are used by the cathodophilic microbial culture 8 to reduce protons to H2. A power source 6 is included in the electrical circuit for supplying the required energy. The protons are formed in the anode compartment 2, and flow via the ion-conducting separation 4 (for example a Nafion membrane) to the cathode compartment 3. The biologically oxidizable carbon compound OM originates from wastewater that enters the anode compartment 2 via an inlet 11. The processed waste water leaves the anode compartment via outlet 12 as effluent 10. CO2 and H2 leave the anode compartment and cathode compartment, respectively, via the outlets 13 and 14. The hydrogen produced can be further removed for storage and / or use.

Figure 3 shows a schematic overview of the proton reduction reaction, which is catalyzed by the cathodophilic, hydrogen-producing microbial culture in the biofilm 8a on the cathode 5. The schematic overview is not intended to present a stoichiometric representation of the reaction.

Figure 4 shows a bifunctional electrode 15 according to the invention. The bi-directional electrode 15 comprises an electrical conductor 16, here a carbon plate. A film of a cathodophilic microbial culture 8a is provided on the carbon plate 16 on one surface. An oxidation reaction catalyst is provided on the other surface of the carbon plate 16. In this case a biofilm of an anodophilic microbial culture.

Figure 5 schematically shows the reactions that take place on both sides of the bifunctional electrode 15. Again, this schematic overview does not aim to show a stoichiometric representation of the reactions. It is visible that on the anode side a biologically oxidizable carbon compound (OM) is converted by the anodophilic microorganisms into electrons, CO2 and H +. The electrons flow through the electrical conductor 16 to the cathode side, where they are used by the cathodophilic, hydrogen-producing microbial culture 8a to reduce protons to H2.

The bifunctional electrode can be used in an electrolysis device according to the invention. An embodiment of an electrolysis device is shown in Figure 6. This electrolysis device 17 comprises a plurality of bifunctional electrodes 15 according to the invention. The bifunctional electrodes 15 are placed between a terminal anode 18 and a terminal cathode 19. The bifunctional electrodes 15 form compartments between the terminal anode and terminal cathode. These compartments are subdivided by an ion-conducting separation 4 into an anode part compartment 20 and a cathode part compartment 21. A feed stream 22 containing waste water is supplied to the anode part compartments. The anodophilic biofilm 10 converts biologically oxidizable carbon compounds in the waste water into protons, CO2 and electrons. The protons 10 flow through the proton-conducting separation 4 to the cathode part compartment 21.

CO2 leaves the anode part compartment via an outlet 22, for example together with the effluent from the waste water.

The electrons generated on the anode side of a bifunctional electrode are directly led by the electrically conductive material of the bifunctional electrode to the cathode side of the bifunctional electrode. The electrons generated at the monofunctional terminal anode are conducted via an electrical circuit to the monofunctional terminal cathode. A power source 6 is provided in this electrical circuit. On the cathode side of the bifunctional electrodes 15, the electrons are used by the cathodophilic hydrogen-producing microbial culture 8a for the reduction of protons. Hydrogen is thereby produced, which leaves the cathode part compartments 21 via an outlet 23. The power source 6 supplies sufficient electrical energy for the production of hydrogen in the system. Due to the low electrical resistance in the bifunctional electrodes, the electrical resistance in this device is lower than a device of the prior art with a comparable electrode surface.

Examples Example 1

An electrochemical cell was made from glass. The cell consisted of two compartments separated by a cation-selective membrane (Nafion® 117) with an area of 9.5 cm 2. A compartment (volume: 1L) contained a carbon rod 21 electrode with an area of 10 cm 2. This electrode served as the bio-electrode (working electrode) and w as connected to a potentiostat 6 (μAutolablII, E co C hemie B.V., the Netherlands). The other compartment (volume: 100 mL) contained a platinum electrode 7 (1.25 cm) connected to the potentiostat, which was oriented vertically 5 relative to the bio-electrode and which served as a counter-electrode. The distance of both electrodes from the membrane was 1 cm. An Ag / AgCl, 3 M KCl reference electrode was also placed in the bio-electrode compartment. Both compartments were filled with a medium consisting of 0.74 g / L KCl, 1.36 g / L ΙΟ ^ ΡΟ ^ 0.28 g / L NH4 Cl, 0.84 g / L NaHCCb ', 0.1 g / L CaCXrlYizQ, 1 g / L MgS (V7H20 and 1 mL / L trace elements.

The bioelectrode compartment of the electrochemical cell was inoculated with electrochemically active microorganisms from the anode compartment of a biocatalyzed electrolysis cell, then fed with acetate and hydrogen and checked at a potential of +0.1 V (vs. standard hydrogen electrode) (NHE) and pH 7. Electrochemically active microorganisms formed a biofilm on the bioelectrode under these conditions and provided anodic current. Subsequently, the pH was lowered to pH 6 and the potential to -0.1 V (vs. NHE) and after adjusting the biofilm under these conditions, the bioelectrode was fed with hydrogen and medium in which only bicarbonate as a carbon source was dissolved to hydrogen oxidizing micro select organisms. After a constant current was generated under these conditions, the potential of the bioelectrode was lowered to -0.65 V (vs. NHE, at pH 6) so that the necessary current changed and cathodic current. In the following week, the cathodic current increased to a value of 1 A / m2 bioelectrode surface while the potential was kept constant at -0.65 V (vs. NHE). The increase in cathodic current density at constant conditions indicated an adjustment of the microbial community in the biofilm. The measured cathodic current density (1 A / m) was more than twice as high as that measured with a platinum catalyzed electrode as used in a previous study (Rozendal et al., International Journal of Hydrogen Energy, vol. 31, page 1632 -1640, 2006) under similar conditions (pH 7 and a cathode potential of -0.71 V). Hydrogen was detected in the gas phase of the bio-electrode compartment (using a Shimadzu GC-2010 gas chromatograph).

22

Example 2

The experiment described in Example 1 was repeated in a slightly modified design. A cell design consisting of 4 plexiglass plates was now used. The 2 inner plates formed the anode and cathode compartments, while the 2 outer plates served as a heating jacket and reinforcement. The inner plates contain channels (channel depth: 1 cm) for liquid transport (volume: 0.25 L) and a headspace for gas accumulation (volume 0.029 L). Placed between the inner plates were 2 graphite felt electrodes (effective area: 250 cm 2), separated by a cation-selective membrane (Fumasep® FKE, 20 x 30 cm). Both electrodes were connected to a potentiostat (Wenking Potentiostat / Galvanostat KP5V3A, Bank IC, Germany). The bio-electrode was the working electrode. The Ag / AgCl, 3 M KCl reference electrodes (QM710X, ProSense BV, the Netherlands) were connected to Haber-Luggin capillaries. The Haber-Luggin capillaries were placed at a short distance from the electrodes to minimize the ohmic voltage drop between the reference electrode and work or against electrode. The bioelectrode compartment was filled with media as described in Example 1. The counter c ompartment was filled with a solution of hexacyanoferrate (III) when the bioelectrode was operated anodically and with a solution of hexacyanoferrate (II) when the bioelectrode 20 was operated cathodically.

The bioelectrode compartment of the electrochemical cell was inoculated with electrochemically active microorganisms from the anode compartment of a biocatalyzed electrolysis cell, then fed with acetate and hydrogen and checked at a potential of +0.1 V (vs. standard hydrogen electrode; NHE) and pH 7. Electrochemically active micro-organisms formed a biofilm on the bio-electrode under these conditions and provided anodic current. The bioelectrode was then fed with hydrogen and medium in which sodium bicarbonate was dissolved as the sole carbon source to select hydrogen-oxidizing microorganisms. After a constant current was generated at a potential of -0.2 V (vs NHE), the potential of the bioelectrode was lowered to -0.7 V (vs. NHE; pH 7) so that the anodic current changed to a cathodic current. The cathodic current increased in 23 the following time to a value of 1.1 A / m2 bio-electrode area while the potential was kept constant at -0.7 V (vs. NHE). The increase in cathodic current under constant conditions indicated an adjustment of the microbial community in the biofilm. The biocathode thus formed was then fed with medium prepared without a carbon source.

At a cathode potential of -0.7 V (vs NHE, at pH 7), as shown in Figure 7, it was found that the cathodic current density of the biocathode (closed circles) was 4 times as large as the cathodic current of a control cathode ( open circles) that was not inoculated with electrochemically active microorganisms. The measured cathodic current (1.1 A / m2) of the biocathode is comparable to the current measured in example 1. The cathodic current density of an average platinum catalyzed electrode from a previous study (Rozendal et al., International Journal of Hydrogen Energy, vol 31, pages 1632-1640, 2006) under comparable conditions (pH 7 and a cathode potential of -0.71 V) is shown in Figure 7 as X. Analysis of the gas produced (using a Shimadzu GC-2010 gas chromatograph ) showed that hydrogen was produced with a measured efficiency of 50% (based on electrons to H2). The amount of hydrogen produced is shown in Figure 8. This amount of hydrogen for the biocathode (closed circles) was approximately 10 times as high as that at the control cathode (open circles). In an experiment in which the biocathode was fed with carbon monoxide, the cathodic current of the biocathode decreased. Carbon monoxide is known as an inhibitor of hydrogenases, the enzymes that catalyze hydrogen production in hydrogen-producing microorganisms. The decrease in the cathodic current is an indication that electrochemically active microorganisms catalyze hydrogen production at the biocathode. After removal of carbon monoxide from the biocathode by flushing with nitrogen, the cathodic current recovered to 1.1 A / m2. The control cathode was inoculated with electrochemically active microorganisms by coupling the effluent from the biocathode to the influent from the control cathode. After 8 days, the control cathode was disconnected from the biocathode and fed with the previously described 10 mM bicarbonate medium. In the following 10 days, the current at the "control" cathode increased from -0.3 A / m2 to -1.0 A / m2. This indicates growth of electrochemically active microorganisms at the "control" cathode. Electron microscopy also revealed that a microbial film was present on the biocathode and the neighboring "control" cathode. Thus, it has been demonstrated that a biocathode for hydrogen production can also be obtained by inoculating the electrochemically active microorganisms from an already operating biocathode for hydrogen production.

1034123

Claims (20)

A method for obtaining a cathodophilic, hydrogen-producing microbial culture comprising: (i) providing a bioelectrode comprising an electrochemically active microbial culture on an electrical conductor, which microbial culture is capable of hydrogen oxidation; (ii) placing the bioelectrode in a medium, the nutrient medium, which is suitable to support the physiology of at least a portion of the organisms in the microbial culture; (iii) applying to the bioelectrode a potential that is lower than the equilibrium potential of the H + / H 2 redox couple in the nutrient medium. 2. A method according to claim 1, wherein the bioelectrode is obtained by: a) providing a bioanode comprising an anodophilic electrochemically active microbial culture on an electrical conductor, which microbial culture is capable of hydrogen oxidation, placed in a nutrient medium which is capable of supporting the physiology of at least a portion of the microbial culture; B) applying a potential to the bioanode in the presence of hydrogen under conditions of limitation of a biologically oxidizable carbon compound higher than the equilibrium potential of the H + / H 2 redox couple in the nutrient medium. The method of claim 2, wherein a biologically non-oxidizable carbon source, such as a carbon dioxide source, is present under conditions of limitation of the biologically oxidizable carbon compound. 4. A cathodophilic, hydrogen-producing microbial culture. 30 1034123 A microbial culture according to claim 4, which is available with the method according to any one of claims 1-3. 6. Use of a microbial culture according to any of claims 4-5, as an electrochemical catalyst for the production of hydrogen. 7. A method for manufacturing a bioelectrode, comprising providing a body of an electrically conductive material and applying a cathodophilic, hydrogen-producing microbial culture to the surface of the electrically conductive material. 8. Method according to claim 7, wherein: (iv) the provided body of the electrically conductive material comprises two separate surfaces; (v) a first surface, the cathode surface, the cathodophilic, hydrogen-producing microbial culture is applied; (vi) a second surface, the anode surface, is provided with a catalyst for an electrochemical oxidation reaction. 20 A method according to any of claims 7-8, wherein the body is substantially two-dimensional, such as preferably a plate-shaped body. 10. A method according to any one of claims 7-9, wherein the catalyst for the anaerobic oxidation reaction is selected from the group comprising platinum and / or an electrochemically active microbial culture capable of oxidizing a biologically oxidizable carbon Compound. 11. A bifunctional bioelectrode comprising a body of an electrically conductive material, which body comprises two separate surfaces, with on the first surface, the cathode surface, a cathodophilic, hydrogen-producing microbial culture and on the second surface, the anode surface, a catalyst for an electrochemical oxidation reaction. 12. A bi-functional bioelectrode according to claim 11, wherein the body is substantially two-dimensional, such as preferably a plate-shaped body, more preferably a flat plate. 13. A bifunctional bioelectrode according to any one of claims 11-12, wherein the anaerobic oxidation reaction catalyst is selected from the group comprising platinum and / or an electrochemically active microbial culture capable of oxidizing a biologically oxidizable carbon compound. 14. Device comprising a number of compartments, with in each of these compartments a spaced apart anode and a cathode with on the anode a catalyst for the electrochemical oxidation of an oxidizable substrate and on the cathode a cathodophilic, hydrogen-producing microbial culture, wherein furthermore an ion-conducting separation is present between the anode and cathode, which divides the number of compartments in a cathode sub compartment on the side of the cathode and an anode sub compartment on the side of the anode, with a nutrient medium for supporting the physiology in the kothode sub compartment of the cathodophilic, hydrogen-producing microbial culture and wherein the device further comprises means for supplying a substrate medium comprising the oxidizable substrate to the anode sub-compartments and means for discharging hydrogen from the cathode sub-compartments and furthermore there is an electrical connection between a number of anodes and a number of cathodes. The device of claim 14, wherein the electrical connection between an anode and a cathode comprises a power source. Device as claimed in claim 14, wherein the number of compartments is a plurality of compartments subdivided into a first and a second terminal compartment and a number of intermediate compartments between them, wherein the anodes and cathodes are alternately present in the device, and wherein the electrical connection between the the number of anodes and the number of cathodes is arranged as an electrical connection between the anode and cathode of adjacent intermediate compartments, an electrical connection between the cathode of the first terminal compartment and the anode of the intermediate compartment adjoining the first terminal compartment, an electrical connection between the anode of the second terminal compartment and the cathode of the intermediate compartment adjacent to the second terminal compartment and an electrical connection between the anode and cathode of the terminal compartments, such that each anode is electrically connected to one cathode. The device of claim 16, wherein the electrical connection between the anode and cathode of the terminal compartments comprises a power source. Device according to any of claims 14-17, wherein the catalyst for the electrochemical oxidation of an oxidizable substrate on the anodes comprises a catalyst from the group comprising platinum and / or a microorganism selected from the group of Geobacter sulferreducens, Shewanella putrefaciens , Geobacer metallireducens and Rhodoferax ferric reducts, other organisms from the aforementioned genera or a consortium of one or more organisms therefrom. Device as claimed in any of the claims 16-18, wherein the anodes and cathodes of the intermediate compartments are arranged as bifunctional electrodes according to claims 11-13. 25 A method for producing hydrogen comprising: (i) providing a device according to any of claims 14-19; (ii) supplying a substrate medium 30 comprising an oxidizable substrate to the anode sub-compartments; (iii) applying a potential to the number of cathodes that is lower than the equilibrium potential of the H + / H 2 redox couple in the nutrient medium; (iv) discharging the hydrogen produced at the cathode. 1 0 3 4 1 2 3