EP2176405A1

EP2176405A1 - Method for obtaining a cathodophilic, hydrogen-producing.microbial culture, microbial culture obtained with this method and use of this microbial culture

Info

Publication number: EP2176405A1
Application number: EP08778996A
Authority: EP
Inventors: René Alexander ROZENDAL; Hubertus Victor Marie Hamelers; Cees Jan Nico Buisman; Adriaan Willem Jeremiasse
Original assignee: Stichting Wetsus Centre of Excellence for Sustainable Water Technology
Current assignee: Stichting Wetsus Centre of Excellence for Sustainable Water Technology
Priority date: 2007-07-12
Filing date: 2008-07-08
Publication date: 2010-04-21
Also published as: WO2009008709A1; CN101784661A; NL1034123C2

Abstract

According to a first aspect, the present invention relates 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 bifunctional bioelectrode in which the cathodophilic, hydrogen-producing microbial culture is applied, and the bifunctional bioelectrode obtained with this method. Other aspects of the invention relate to a device for producing hydrogen in which the electrochemically active, hydrogen-producing microbial culture is applied.

Description

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

According to a first aspect, the present invention relates 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 bifunctional bioelectrode in which the cathodophilic, hydrogen-producing microbial culture is applied, and the bifunctional bioelectrode obtained with this method.

Other aspects of the invention relate to a device for producing hydrogen in which the electrochemically active, hydrogen-producing microbial culture is applied. Hydrogen-producing cathodes are typically used in water electrolysis processes. In water electrolysis water is split into hydrogen and oxygen under the influence of a potential difference applied between anode and cathode in accordance with the reaction:

H₂O → H₂ + 0.5 O₂ (1)

In water electrolysis the following electrode reactions take place:

An oxygen-producing oxidation reaction at the anode:

H₂O → 0.5 O₂ + 2 H⁺ + 2 e (2a) or 2 OH^" → 0.5 O₂ + H₂O + 2 e^" (2b)

A hydrogen-producing reduction reaction at the cathode:

2 H⁺ + 2 e^" → H₂ (3a) or

2 H₂O + 2 e → H₂ + 2 OH^" (3b)

Hydrogen-producing cathodes have recently also found application in a new type of electrolysis process, i.e. biocatalyzed electrolysis of dissolved bio-oxidizable material (e.g. in wastewater), hi 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 schematically represented as: [CH₂O] + H₂O → 2 H₂ + CO₂ (4)

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

The term biocatalyzed electrolysis is derived from the fact that the oxidation of bio- oxidizable material at the anode is catalyzed by electrochemically active micro-organisms. Conversely, the hydrogen-producing reduction reaction at the cathode is catalyzed chemically (e.g. with platinum) in its standard embodiment, just as in water electrolysis. In biocatalyzed electrolysis of dissolved bio-oxidizable material (e.g. in wastewater) the following electrode reactions take place:

The oxidation of bio-oxidizable material at the anode, schematically represented by:

[CH₂O] + H₂O → CO₂ + 4 H⁺ + 4 e- (5)

A hydrogen-producing reduction reaction at the cathode:

4 H⁺ + 4 e^' → 2 H₂ (6a) or

4 H₂O + 4 e- → 2 H₂ + 4 OH^" (6b)

Because hydrogen-producing electrolysis processes take place under the influence of a potential difference between anode and cathode, energy is consumed. This energy can be introduced by a power source. It is in principle the case that the higher the voltage required for electrolysis, the higher the consumption of electrical energy per quantity of produced hydrogen will be. Energy losses in the electrolysis process increase the required voltage, and therefore also the amount of electrical energy required per quantity of produced hydrogen. Such energy losses can also occur in the electrode reactions. Energy loss at a cathode is also referred to as cathode overpotential and is expressed in volts (V).

Overpotentials generally have the property of increasing (i.e. energy loss increases) as current density increases (i.e. as reaction speed increases). This is also the case for cathode overpotentials. The relation 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 assembled from two elements, i.e. a charge distributor of an electrically conductive material and a catalyst for accelerating the cathode reaction. Carbon is a frequently used charge distributor, since it has a low cost price, a high electrical conductivity and a high chemical resistance. Using only a charge distributor without catalyst the electrochemical production of hydrogen at a cathode is however very slow from a kinetic viewpoint. Large overpotentials (i.e. large energy losses) can hereby already occur at relatively low current densities. Use is often made of platinum as catalyst for electrochemical hydrogen production at a cathode.

For water electrolysis processes platinum is indeed a very effective cathode catalyst which can limit the overpotential to a minimum at very high current densities (e.g. 0.025 V overpotential at 1.08 A/cm as described in the book ''''Electrochemical oxygen technology'^'' written by Kinoshita, K; John Wiley & Sons Inc.: New York, 1992). Platinum has unfortunately been found for a number of reasons to be a much less suitable cathode catalyst for biocatalyzed electrolysis. Firstly, platinum is a very expensive material, hi addition, the current densities of biocatalyzed electrolysis are typically three to five orders of magnitude lower than those in water electrolysis (i.e. order of magnitude ~0.00001-0.001 A/cm²). The quantity of produced hydrogen per quantity of platinum is hereby much lower in biocatalyzed electrolysis than in water electrolysis. This makes the use of platinum in biocatalyzed electrolysis much too expensive to achieve a commercially attractive process.

It has further been found that platinum works nowhere near as effectively in biocatalyzed electrolysis as in water electrolysis, as described in the publication "Principle and perspectives of hydrogen production through biocatalyzed electrolysis" {International Journal of Hydrogen Energy 2006, 31, 1 632-1640) in the name of Rozendal. R.A. et al. At a current density of only 0.00005 A/cm² the cathode overpotential was already more than 0.28 V. The relatively mild conditions typically occurring in biocatalyzed electrolysis processes may be a possible reason for this low effectiveness of the platinum catalysis in biocatalyzed electrolysis. Relatively mild conditions are understood to mean, among others, 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 biocatalyzed electrolysis, and the relatively low effectiveness of platinum catalysis under the typical conditions of biocatalyzed electrolysis processes make it interesting to seek alternative methods of catalyzing electrochemical production of hydrogen at a cathode.

Prior art Attention has already been focused in the past on the problems of the use of platinum as cathode catalyst in the context of (bio)fuel cells and electrolysis cells. Biological catalysts, among others, have been investigated in this context. A (bio)fuel cell is understood to mean "an electrochemical 'device' that continuously converts chemical energy into electrical energy (and some heat) for as long as fuel and oxidant are supplied" (Hoogers, G, "Fuel Cell Technology Handbook", CRC Press 2003). The reverse takes place in an electrolysis cell: electrical energy is invested in order to cause desired chemical reactions (Bard, AJ., Faulkner, L.R., "Electrochemical Methods, Fundamentals and Applications", Wiley 2001), or electrical energy is converted into chemical energy (and some heat). Examples of electrolysis processes are water electrolysis and biocatalyzed electrolysis. Described in the international patent application WO 2004/015806 is a stainless steel electrode, the surface of which is covered with a biofilm (defined in WO 2004/015806 as "a film consisting of micro-organisms from biological water such as sea water, river water etc., which have been deposited spontaneously onto a surface"), which is intended to catalyze the reaction at the electrode of a fuel cell. The biofilm is formed by immersing the electrode in a medium which enhances the growth of biofilms, and by simultaneously applying a polarization potential to the electrode (value between -0.5 and 0.0 V relative to a standard calomel electrode). The electrode of a fuel cell covered with biofilm can be a cathode as well as 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. The patent application does not however relate to 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 the patent application JP 11057782 and by Sakakibara & Kuroda (Biotechnology and Bioengineering, vol. 42, p. 535- 537, 1993). However, these references describe the abiotic production of hydrogen at the cathode which is subsequently used by the biofilm to reduce nitrate to nitrogen and water. There is thus no mention in this case of a microbial culture catalyzing the production of hydrogen at the cathode, but of a biofilm which consumes electrochemically formed hydrogen.

Tatsumi et al. (Analytical Chemistry, vol. 71. p. 1753-1759, 1999), Tsujimura et al. (Phys. Chem. Chem. Phys., vol. 3, p. 1331-1335, 2001), Lojou et al. (Electroanalysis, vol. 14, p. 913-922, 2002) describe the use of electrodes with immobilized Desulfovϊbrio vulgaris Hildenborough (DvH) cells for the production and/or oxidation of hydrogen. The cells were immobilized by enclosing a suspension with the relevant cells between a membrane and an electrically conductive carrier material. For their catalytic effect on the electrode however, these DvH cells require externally supplied redox mediators. In addition to biofilm applications, systems have also been described in the literature and in patent applications in which isolated enzymes (and not whole micro-organisms) have been applied as biocatalyst. Guiral-Brugna et al. (Journal of Electroanalytical Chemistry, vol. 510, p. 136-143, 2001) and Morozov et al. (International Journal of Hydrogen Energy, vol. 27, p. 1501-1505, 2002) describe mediator-less hydrogen production at electrodes made from carbon material with immobilized hydrogenases. Morozov et al. (Russian Journal of

Electrochemistry, vol. 38, p. 97-102, 2002) moreover showed that, of one of such electrodes, 50% of the original activity was retained after conserving for six months in buffer solution at 4°C. Described in the patent application WO 2004/114494 is a fuel cell which makes use of immobilized hydrogenases at the anode for the purpose of catalyzing hydrogen oxidation, and immobilized oxidases at the cathode for the purpose of catalyzing oxygen reduction. The immobilization can be brought about by means of sorption from an aqueous solution or by means of chemical bonding. Described in US 2006/0159981 is 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 catalyzes the oxidation of a reductant (not further specified), and the enzyme on the cathode catalyzes the reduction of an oxidant (not further specified).

It will be apparent from the foregoing that, in the case isolated hydrogenases were used as catalyst for the electrochemical hydrogen production at a cathode, no mediators were necessary to achieve a catalytic effect. The use of isolated hydrogenases does however have the drawback that these isolated hydrogenases can only be obtained after a time-consuming purification. Isolated hydrogenases also lack the required stability for prolonged application, and they have no self-regenerative capacity. When whole micro-organisms were used as catalyst for the electrochemical hydrogen production at a cathode, externally added mediators (for instance methyl viologen or cytochrome c₃) have heretofore always been found necessary to bring about a catalytic effect. The use of mediators has the drawback that these are costly compounds having in a number of cases a high toxicity.

Specification of the invention The present invention has for its object to provide an alternative to the cathode systems described up to this point for electrochemical hydrogen production. According to a first aspect, the invention provides for this purpose 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 electric conductor, this microbial culture being capable of hydrogen oxidation; (ii) placing the bioelectrode in a medium, the culture medium, suitable for supporting the physiology of the microbial culture;

(iii) applying a potential to the bioelectrode which is lower than the equilibrium potential of the Ef¹VH₂ redox couple in the culture medium.

The bioelectrode provided in the method comprises an electrochemically active microbial culture on an electric conductor. This microbial culture is capable of hydrogen oxidation. The term electrochemically active microbial culture is understood in the context of the present invention to mean a culture of micro-organisms which can use an electrode directly, i.e. without the use of externally supplied redox mediators, as electron donor

(cathodophilic organisms) or as electron acceptor (anodophilic organisms). The existence of such anodophilic organisms is known in the field from, inter alia, "Principle 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. and from "Electricity production by Geobacter sulfurreducens attached to electrodes" Applied and Environmental Microbiology 2003, 69, 1548-1555 in the name of Bond, D.R. and Lovley, D.R.).

The microbial culture is present on an electric conductor, i.e. a material which can conduct an electric current. Carbon, for instance in the form of carbon felt or carbon paper or graphite felt or graphite paper, is particularly suitable as electric conductor. The skilled person may however select other suitable materials. An electric 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 H₂ is converted to protons and electrons in accordance with the reverse reaction of the above reaction equation (3 a and/or 3b). The skilled person will appreciate that the reactions in accordance with reaction equation 3 a and 3b are similar reactions, and that reaction 3 a is obtained by deleting 2 OH^" on the left and right from reaction 3b. The organisms of an electrochemically active microbial culture capable of hydrogen oxidation are herein able to relinquish the formed electrons directly, i.e. without an externally supplied redox mediator, to an anode as electron acceptor.

In the method according to the invention the bioelectrode is placed in a medium, the culture medium, suitable for supporting the physiology of at least a part of the organisms in the microbial culture. Supporting the physiology means here that the organisms can be metabolically active. Metabolic activity of the organisms in the microbial culture in respect of hydrogen formation via for instance one or more of the above reactions 3 a and 3b is of particular importance in the context of the present invention. Media suitable as culture medium are known to the skilled person. Such a suitable medium is for instance Postgate's medium. Other suitable media are described in examples 1 and 2. It is of further importance in hydrogen production that the culture medium has a low oxygen tension. The medium is therefore preferably microaerophilic, and more preferably substantially anaerobic.

Generally suitable is an aqueous solution of trace elements further comprising a carbon source, for instance carbon dioxide, with a pH of 2-10, such as pH 3-9, preferably pH 5-7. In order to limit growth of methano genie organisms, which consume hydrogen, it is recommended to use a low pH of below pH 5.0, such as below pH 4.0. It is known that methanogenic organisms are inhibited by such a low pH value. In addition, growth of methanogenic bacteria can be inhibited by minimizing the concentration of carbon dioxide once the microbial culture has grown sufficiently. A PCO₂ of below 0.0003 atm, such as below 0.0002 or below 0.0001 atm, can be used for this purpose.

In the method according to the invention a potential is applied to the bioelectrode which is lower than the equilibrium potential in the culture medium of the H⁺/H₂ redox couple. The equilibrium potential of the H⁺/H₂ redox couple in the culture medium can be determined by the skilled person on theoretical basis (on the basis of knowledge of the composition of the culture medium and the other reaction conditions). The equilibrium potential can further be determined in a number of cases by determining the open-circuit voltage of a cell. The potential can be applied using a potentiostat or other suitable electrical power source.

For electrochemically active micro-organisms adapted to hydrogen oxidation, wherein electrons are relinquished to the charge distributor of the electrode functioning as anode, the changeover to a potential which is lower than the equilibrium potential of the H⁺/H₂ redox couple in the culture medium means a reversal of their electrochemical reaction. Because this potential lies below the equilibrium potential of the H⁺ZH₂ couple, the cells will be forced to catalyze the reaction in the direction of proton reduction. They will thus produce hydrogen in electroactive manner.

The potential applied in step (iii) of the method is for instance 5, 10, 15, 20, 25, 40, 50, 60 mV lower than the equilibrium potential of the H⁺ZH₂ redox couple in the culture 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 H⁴VH₂ redox couple in the culture medium.

Small differences in the applied potential relative to the equilibrium potential of the H⁴VH₂ redox couple are advantageous here for determined applications, since organisms are hereby selected which can produce H₂ with a low energy investment.

The applied potential can be applied using a power source. A potentiostat is for instance suitable as power source. Such a potentiostat can be coupled to a reference electrode, for instance a standard hydrogen electrode, a calomel electrode or an Ag/ AgCl electrode, for the purpose of precisely regulating the potential of the bioelectrode relative to the equilibrium potential of the H⁴VH₂ couple in the culture medium. The use of a potentiostat is not however essential, and any other electric power source which the skilled person understands to be suitable for applying a potential to the bioelectrode can be used.

Alternatively, the potential can be regulated to below the equilibrium potential of the H⁴VH₂ redox couple by shifting, such as decreasing, this equilibrium potential of the H⁺ZH₂ redox couple, for instance by decreasing the pH of the culture medium and/or decreasing the hydrogen pressure or by other changes in the reaction conditions such as the temperature, as known to the skilled person. The equilibrium potential of the H⁺ZH₂ couple is for instance 0.0 V at pH 0.0 under standard conditions (T = 25°C and PH₂ = 1 atm). At pH 7.0 this equilibrium potential is -0.42 V under the same standard conditions. A potential rise of ± 60 mV per pH unit can be inferred herefrom. A pH reduction of the culture medium can thus also be used to give a potential present at a cathode a value below the equilibrium potential of the H⁺ZH₂ redox couple in the culture medium.

According to a preferred embodiment of the method, the bioelectrode is obtained by placing a bioanode, comprising an electrochemically active microbial culture on a charge distributor, this microbial culture being capable of oxidation of a biologically oxidizable carbon compound, in a culture medium which is able to support the physiology of at least a part of the microbial culture, at a potential higher than the equilibrium potential of the H-VH₂ redox couple in the culture medium. This takes place under conditions of limitation of the biologically oxidizable carbon compound in the presence of hydrogen. The applied potential can be for instance up to 1000 mV, up to 800 mV, such as up to 500 mV, higher than the equilibrium potential of the H¹VH₂ redox couple in the culture medium. The applied potential is preferably 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 instance short-chain (C₁-C₆) organic acids, such as lactic acid, ascetic acid and dissociated forms thereof, short-chain (C₁-C₆) alcohols, such as ethanol and propanol or carbohydrates such as glucose, fructose, lactose or saccharose.

Owing to this step an electrochemically active microbial culture capable of hydrogen oxidation is selected at the bioanode, for instance via a reaction which is the reverse of the reaction shown in the above stated 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 hydrogen electrochemically at a potential lower than the equilibrium potential of the H⁴VH₂ redox couple in the culture medium in which they are present.

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 instance carbon dioxide or a solution containing H₂CO₃ and/or HCO₃ ^" and/or CO₃ ^2'.

A further preferred embodiment comprises of inoculating the cathodophilic, hydro gen- producing microbial culture to a charge distributor. Inoculation of the cathodophilic, hydrogen-producing microbial culture makes it possible to multiply this culture in simple manner and/or to obtain a plurality of electrodes with the cathodophilic microbial culture. Inoculation can take place in any suitable manner known to the skilled person for the purpose of inoculating micro-organisms. A further aspect of the invention relates to a cathodophilic, hydrogen-producing microbial culture. Such a microbial culture able to produce hydrogen in electrochemical manner without use of external redox mediators has not been described earlier. The microbial culture comprises micro-organisms able to produce hydrogen by means of proton reduction and/or water reduction, as described in for instance reaction equations 3 a 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 producing hydrogen. As described above, the microbial culture is suitable for the production of hydrogen.

Other further aspects of the present invention relate to a method for manufacturing a bioelectrode which is for instance suitable as biocathode, for instance for use in biocatalyzed electrolysis of water. The method comprises of providing a body of an electrically conductive material and arranging a cathodophilic, hydrogen-producing microbial culture on 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) the cathodophilic, hydrogen-producing microbial culture is arranged on a first surface, the cathode surface; (iii) a catalyst for an electrochemical oxidation reaction is arranged on a second surface, the anode surface.

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

A body of an electrically conductive material, or a charge distributor, is provided in the method, hi the context of this invention the term charge distributor is understood to mean a material which can conduct electrical charge. Suitable charge distributors are for instance carbon, preferably carbon paper, a carbon plate, graphite paper, a graphite plate or an electrically conductive material such as copper or titanium.

In the method a cathodophilic, hydrogen-producing microbial culture according to the invention is arranged on the surface of the body of the electric conductor. The arranging can take place in any manner which the skilled person understands to be suitable for arranging a microbial culture. According to a preferred embodiment, the body comprises two separate surfaces. This is understood to mean that it must be possible to separate the two surfaces from each other such that one surface can serve as cathode and the other can serve as anode. This is possible for instance by selecting the body as a substantially two-dimensional body such as a plate-like body, preferably a flat plate. In this preferred embodiment a cathodophilic, hydrogen-producing microbial culture according to the invention is arranged on the first surface, the cathode surface. A catalyst for an electrochemical oxidation reaction, for instance an anaerobic oxidation reaction, is arranged on a second surface, the anode surface. The catalyst can be any suitable catalyst and the arranging can take place in any manner which the skilled person understands to be suitable for arranging the type of catalyst used. The catalyst can for instance be selected from the group of platinum and/or an electrochemically active microbial culture capable of electrochemical oxidation, for instance an anaerobic oxidation, of a biologically oxidizable substrate, such as a biologically oxidizable carbon compound. This latter microbial culture can comprise organisms from the group of Geobacter sulfurreducens, Shewanella putrefaciens, Geobacter metallireducens and Rhodoferax ferrireducens or other organisms from the genera to which these stated species belong, or a consortium of these organisms.

The bifunctional 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 a cathodophilic, hydrogen-producing microbial culture on the first surface, the cathode surface, and a catalyst for an electrochemical oxidation reaction on the second surface, the anode surface.

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 and those of the preferred embodiments stated in the claims will be apparent to the skilled person from the description relating to 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 electrolysis device. The device comprises a number of compartments, with an anode and a cathode placed at a mutual distance in each of the compartments. On the anode there is a catalyst for the electrochemical oxidation of an oxidizable substrate, and on the cathode there is a cathodophilic, hydrogen-producing microbial culture. Further present between the anode and cathode is an ion-conducting separation 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. Present in the cathode sub-compartments is a culture medium suitable for supporting the physiology of the cathodophilic, hydrogen-producing microbial culture. The device further comprises means for supplying to the anode sub- compartments a substrate medium comprising the oxidizable substrate and means for discharging hydrogen from the cathode sub-compartments. There is also 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.

A device for electrolysis of the above stated type is per se known in the field. The electrolysis device according to the invention is however distinguished from those known in the prior 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 lying therebetween. The number of anodes and cathodes is here present in alternating manner in the device. In this embodiment the electrical connection between the number of anodes and the number of cathodes is further adapted 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 adjacent to 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. These electrical connections are such that each anode is electrically connected to one cathode.

The electrolysis device according to this embodiment can be designed 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 the purpose of adjusting the potential of the cathode. If a power source is present, it will be accommodated 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 micro-organism selected from the group of Geobacter sulfurreducens, Shewanella putrefaciens, Geobacter metallkeducens and Rhodoferaxferrireducens or other organisms from the stated genera or a consortium of one or more organisms herefrom. It is known of these organisms that they can have an anodophilic action.

According to a further preferred embodiment of the electrolysis device, the anodes and cathodes of the intermediate compartments are adapted as bifunctional electrodes according to the invention. Owing to the low internal resistance of the bifunctional electrode, the internal resistance of the electrolysis device is also reduced compared to a prior art electrolysis device with a similar electrode surface area.

According to a further aspect, the invention relates to a method for producing hydrogen. The method comprises of: (i) providing a device for electrolysis according to the invention;

(ii) supplying a substrate medium comprising an oxidizable substrate to the anode sub-compartments; (iii) applying a potential to the number of cathodes, which potential lies below the equilibrium potential of the H⁴VH₂ redox couple in the culture medium; (iv) discharging the hydrogen produced at the cathode.

Supplying the substrate medium to the anode sub-compartments can take place 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 wastewater flow with a high content of organic compounds. Applying the potential to the number of cathodes, which potential lies below the equilibrium potential of the H⁴VH₂ redox couple, is possible using an optional power source to which an anode and cathode are connected in the electrolysis device according to the invention. As described above, the potential can alternatively be regulated to below the equilibrium potential of the H⁴VH₂ redox couple, by shifting, such as decreasing, this equilibrium potential of the H⁴VH₂ redox couple, for instance by reducing the pH of the culture medium and/or reducing the hydrogen pressure.

The potential applied to the cathode is preferably such that a desired quantity of H₂ is produced per unit of time. This desired quantity of H₂ can be predetermined. Another possible criterion for the production of H₂ is the energy invested in the form of the applied potential. A higher energy investment can cause an increase in the cost price of the produced H₂. The skilled person will be able to optimize the applied potential in respect of 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 will now be further elucidated on the basis of the following examples and the accompanying figures, which give non-limitative exemplary embodiments of the invention.

Figures IA- 1C show 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 makes use of 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 figures IA, IB, 1C. Such a cell consists for instance of two compartments 2, 3 separated by an ion-conducting separation 4 (for instance Nafion® 117). A compartment 3 comprises a carbon rod electrode 5. This electrode serves as the bioelectrode (working electrode) and is connected to a potentiostat 6 functioning as power source. The other compartment 2 comprises a platinum electrode 7 which is connected to the power source and which serves as counter-electrode. A reference electrode (not shown in figures IA, IB, 1C) can also be placed in the bioelectrode compartment. Both compartments 2, 3 are filled with a suitable medium (for instance a medium consisting of 0.74 g/L KCl, 1.36 g/L KH₂PO₄, 0.28 g/L NH₄Cl, 0.84 g/L NaHCO₃-, 0.1 g/L CaCl₂-2H₂O, 1 g/L MgSO₄-7H₂O and 1 mL/L trace elements). The bioelectrode compartment 3 of electrochemical cell 1 is inoculated with electrochemically active micro-organisms from the anode compartment of a biocatalyzed electrolysis cell. In the shown embodiment this culture is then fed with acetate and hydrogen and monitored at a potential of +0.1 V (vs. standard hydrogen electrode; NHE) and pH 7. Under these conditions electrochemically active micro-organisms form a biofilm 8 on the bioelectrode and produce an anodic current. The pH is then decreased to for instance pH 6 and the potential to -0.1 V (vs. NHE) and, after adapting biofilm 8 to these conditions, the bioelectrode is fed with hydrogen and medium in which only bicarbonate is dissolved as carbon source for the purpose of selecting hydrogen oxidizing micro-organisms 8a (Figure IB). Once a constant current is being generated under these conditions, the potential of the bioelectrode is decreased to -0.65 V (vs. NHE, at pH 6) so that the anodic current changes into a cathodic current (Figure 1C). After this decrease in potential, biofilm 8a will produce hydrogen. A cathodophilic, hydrogen-producing microbial culture 8a is thus obtained. Figure 2 shows an overview of a biocatalyzed electrolysis device. The biocatalyzed electrolysis cell comprises components similar to the electrochemical cell of figure 1C, i.e. two compartments 2, 3 separated by an ion-conducting separation 4. Both compartments 2, 3 comprise in this case a bioelectrode. Bioelectrode 5 in compartment 3 comprises a cathodophilic, hydrogen-producing microbial culture 8a according to the invention. Bioelectrode 7 in compartment 2 (the anode) comprises an anodophilic microbial culture 10 which is able to convert a biologically oxidizable carbon compound into CO₂, H⁺ and electrons. The electrons produced at anode 7 are conducted via an electrical circuit to cathode 5. Here the electrons are used by the cathodophilic microbial culture 8 to reduce protons to H₂. A power source 6 for supplying the required energy is incorporated in the electrical circuit. The protons are formed in anode compartment 2 and flow via the ion-conducting separation 4 (for instance a Nation membrane) to cathode compartment 3. The biologically oxidizable carbon compound OM comes from wastewater which enters anode compartment 2 via an inlet 11. The processed wastewater leaves the anode compartment as effluent via outlet 12. CO₂ and H₂ leave respectively the anode compartment and cathode compartment via outlets 13 and 14. The produced hydrogen can be further discharged 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 biofilm 8a on cathode 5. The schematic overview does not intend to present a stoichiometric representation of the reaction.

Figure 4 shows a bifunctional electrode 15 according to the invention. Bifunctional electrode 15 comprises an electric conductor 16, here a carbon plate. A film of a cathodophilic microbial culture 8a is arranged on one surface of carbon plate 16. A catalyst for an oxidation reaction is arranged on the other surface of carbon plate 16. In this case a biofilm of an anodophilic microbial culture. Figure 5 shows schematically the reactions which take place on both sides of bifunctional electrode 15. This schematic overview once again does not intend to show a stoichiometric representation of the reactions. It can be seen that on the anode side a biologically oxidizable carbon compound (OM) is converted by the anodophilic micro- organisms into electrons, CO₂ and H . The electrons flow through electric 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 applied 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.

Bifunctional electrodes 15 are placed between a terminal anode 18 and a terminal cathode 19. 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 sub-compartment 20 and a cathode sub-compartment 21. A feed flow 22 comprising wastewater is supplied to the anode sub-compartments. Biologically oxidizable carbon compounds in the wastewater are converted by the anodophile biofilm 10 into protons, CO₂ and electrons. The protons flow through the proton-conducting separation 4 to cathode sub- compartment 21. CO₂ leaves the anode sub-compartment via an outlet 22, for instance together with the effluent from the wastewater. The electrons generated on the anode side of a bifunctional electrode are conducted by the electrically conductive material of the bifunctional electrode directly 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 arranged in this electrical circuit. On the cathode side of bifunctional electrodes 15 the electrons are used by the cathodophilic hydrogen-producing microbial culture 8a to reduce protons. Hydrogen is herein produced which leaves cathode sub-compartments 21 via an outlet 23. Power source 6 supplies sufficient electrical energy for the production of hydrogen in the system. Owing to the low electrical resistance in the bifunctional electrodes the electrical resistance in this device is lower than in a prior art device with a similar electrode surface area. 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 cm². One compartment (volume: IL) comprised a carbon rod electrode with an area of 10 cm². This electrode served as the bioelectrode (working electrode) and was connected to a potentiostat 6 (μAutolablll, Eco Chemie B. V., the Netherlands). The other compartment (volume: 100 mL) comprised a platinum electrode 7 (1.25 cm²) which was comiected to the potentiostat and which was vertically oriented relative to the bioelectrode and served as counter-electrode. The distance of both electrodes to the membrane amounted to 1 cm. An Ag/ AgCl, 3 M KCl reference electrode was also placed in the bioelectrode compartment. Both compartments were filled with a medium consisting of 0.74 g/L KCl, 1.36 g/L KH₂PO₄, 0.28 g/L NH₄Cl,

0.84 g/L NaHCO₃ ^", 0.1 g/L CaCl₂-2H₂O, 1 g/L MgSO₄-7H₂O and 1 mL/L trace elements.

The bioelectrode compartment of the electrochemical cell was inoculated with electrochemically active micro-organisms from the anode compartment of a biocatalyzed electrolysis cell, subsequently fed with acetate and hydrogen and monitored at a potential of +0.1 V (vs. standard hydrogen electrode; NHE) and pH 7. Under these conditions electrochemically active micro-organisms formed a biofilm on the bioelectrode and produced anodic current. The pH was then decreased to pH 6 and the potential to -0.1 V (vs. NHE) and, after adapting the biofilm to these conditions, the bioelectrode was fed with hydrogen and medium in which only bicarbonate was dissolved as carbon source for the purpose of selecting hydrogen oxidizing micro-organisms. Once a constant current was being generated under these conditions, the potential of the bioelectrode was decreased to -0.65 V (vs. NHE, at pH 6) so that the anodic current changed into a cathodic current. During the following week the cathodic current increased to a value of 1 A/m² bioelectrode surface area, while the potential was held constant at -0.65 V (vs. NHE). The increase in the cathodic current density under unchanging conditions indicated a modification of the microbial community in the biofilm. The measured cathodic current density (1 A/m²) 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, p. 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 bioelectrode compartment (using a Shimadzu GC-2010 gas chromatograph). Example 2

The experiment described in example 1 was repeated in a somewhat modified setup. Use was now made of a cell design consisting of four plexiglass plates. The two inner plates formed the anode and cathode compartments, while the two outer plates served as heating jacket and reinforcement. The inner plates comprised channels (channel depth: 1 cm) for liquid transport (volume: 0.25 L) and a head-space for gas accumulation (volume 0.029 L). Two graphite felt electrodes (effective area: 250 cm²) separated by a cation-selective membrane (Fumasep® FKE, 20 x 30 cm) were placed between the inner plates. Both electrodes were connected to a potentiostat (Wenking Potentiostat/Galvanostat KP5V3A, Bank IC, Germany). The bioelectrode 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 a short distance from the electrodes in order to minimize the ohmic voltage loss between the reference electrode and working or counter-electrode. The bioelectrode compartment was filled with medium as specified in example 1. The counter-electrode compartment was filled with a solution of hexacyanoferrate(III) when the bioelectrode was operated anodically, and with a solution of hexacyanoferrate(II) when the bioelectrode was operated cathodically.

The bioelectrode compartment of the electrochemical cell was inoculated with electrochemically active micro-organisms from the anode compartment of a biocatalyzed electrolysis cell, subsequently fed with acetate and hydrogen and monitored at a potential of +0.1 V (vs. standard hydrogen electrode; NHE) and pH 7. Under these conditions electrochemically active micro-organisms formed a biofilm on the bioelectrode and produced anodic current. The bioelectrode was then fed with hydrogen and medium in which sodium bicarbonate was dissolved as only carbon source for the purpose of selecting hydrogen oxidizing micro-organisms. Once a constant current was being generated at a potential of -0.2 V (vs. NHE), the potential of the bioelectrode was decreased to -0.7 V (vs. NHE, at pH 7) so that the anodic current changed into a cathodic current. During the following period of time the cathodic current increased to a value of 1.1 A/m² bioelectrode surface area, while the potential was held constant at -0.7 V (vs. NHE). The increase in the cathodic current under unchanging conditions indicated a modification of the microbial community in the biofilm. The thus formed biocathode was then fed with medium which was prepared without carbon source.

At a cathode potential of -0.7 V (vs. NHE, at pH 7) it was found as shown in figure 7 that the cathodic current density of the biocathode (closed circles) was four times higher than the cathodic current of a control cathode (open circles) not inoculated with electrochemically active micro-organisms. The measured cathodic current (1.1 A/m²) of the biocathode was comparable to the current measured in example 1. The cathodic current density of a platinum catalyzed electrode from a previous study (Rozendal et al., International Journal of Hydrogen Energy, vol. 31, p. 1632-1640, 2006) under similar conditions (pH 7 and a cathode potential of -0.71 V) is shown in figure 7 as X. Analysis of the produced gas (using a Shimadzu GC- 2010 gas chromato graph) showed that hydrogen was produced with a measured efficiency of 50% (on the basis of electrons to H²). The quantity of hydrogen produced is shown in figure 8. This quantity of hydrogen was about 10 times higher for the biocathode (closed circles) than 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 hydro genases, the enzymes which catalyze hydrogen production in hydrogen-producing micro-organisms. The decrease in the cathodic current is an indication that electrochemically active micro-organisms catalyze the hydrogen production at the biocathode. After removal of carbon monoxide from the biocathode by means of flushing with nitrogen, the cathodic current recovered to 1.1 A/m². The control cathode was inoculated with electrochemically active micro-organisms by coupling the effluent from the biocathode to the influent of the control cathode. After eight days the control cathode was uncoupled from the biocathode and fed with the above specified 10 niM of 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 micro-organisms at the 'control' cathode. Electron microscopy also indicated that a microbial film was present on the biocathode and the later inoculated 'control' cathode. This demonstrates that a biocathode for hydrogen production can also be obtained by inoculating the electrochemically active micro- organisms from an already operative biocathode for hydrogen production.

Claims

1. Method for obtaining a cathodophilic, hydrogen-producing microbial culture, comprising of: (i) providing a bioelectrode comprising an electrochemically active microbial culture on an electric conductor, this microbial culture being capable of hydrogen oxidation; (ii) placing the bioelectrode in a medium, the culture medium, suitable for supporting the physiology of at least a part of the organisms in the microbial culture; (iii) applying a potential to the bioelectrode which is lower than the equilibrium potential of the H⁺ZH₂ redox couple in the culture medium.

2. Method as claimed in claim 1, wherein the bioelectrode is obtained by: a) providing a bioanode, comprising an anodophilic electrochemically active microbial culture on an electric conductor, this microbial culture being capable of hydrogen oxidation, placed in a culture medium suitable for supporting the physiology of at least a part of the microbial culture; b) applying a potential to the bioanode higher than the equilibrium potential of the H¹VH₂ redox couple in the culture medium in the presence of hydrogen and under conditions of limitation of a biologically oxidizable carbon compound.

3. Method as claimed in 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.

5. A microbial culture as claimed in claim 4 which is obtainable with the method according to any of the claims 1-3.

6. Use of a microbial culture as claimed in any of the claims 4-5 as electrochemical catalyst for producing hydrogen.

7. Method for manufacturing a bioelectrode, comprising of providing a body of an electrically conductive material and arranging a cathodophilic, hydrogen-producing microbial culture on the surface of the electrically conductive material.

8. Method as claimed in claim 7, wherein:

(iv) the provided body of the electrically conductive material comprises two separate surfaces; (v) the cathodophilic, hydrogen-producing microbial culture is arranged on a first surface, the cathode surface; (vi) a catalyst for an electrochemical oxidation reaction is arranged on a second surface, the anode surface.

9. Method as claimed in any of the claims 7-8, wherein the body is substantially two- dimensional, such as preferably a plate-like body.

10. Method as claimed in any of the 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 oxidation of a biologically oxidizable carbon compound.

11. A bifunctional bioelectrode, comprising a body of an electrically conductive material, which body comprises two separate surfaces, with a cathodophilic, hydrogen- producing microbial culture on the first surface, the cathode surface, and a catalyst for an electrochemical oxidation reaction on the second surface, the anode surface.

12. A bifunctional bioelectrode as claimed in claim 11, wherein the body is substantially two-dimensional, such as preferably a plate-like body, more preferably a flat plate.

13. A bifunctional bioelectrode as claimed in any of the claims 11-12, wherein the catalyst for the anaerobic oxidation reaction is selected from the group comprising platinum and/or an electrochemically active microbial culture capable of oxidation of a biologically oxidizable carbon compound.

14. Device comprising a number of compartments, with an anode and a cathode placed at a mutual distance in each of the compartments, with a catalyst on the anode for the electrochemical oxidation of an oxidizable substrate, and a cathodophilic, hydrogen- producing microbial culture on the cathode, wherein between the anode and cathode an ion- conducting separation is further present 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, with a culture medium in the cathode sub-compartment for supporting the physiology of the cathodophilic, hydrogen-producing microbial culture, and wherein the device further comprises means for supplying to the anode sub-compartments a substrate medium comprising the oxidizable substrate and means for discharging hydrogen from the cathode sub-compartments, and there is also an electrical connection between a number of anodes and a number of cathodes.

15. Device as claimed in claim 14, wherein the electrical connection between an anode and a cathode comprises a power source.

16. 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 lying therebetween, wherein the anodes and cathodes are present in alternating manner in the device and wherein the electrical connection between the number of anodes and the number of cathodes is adapted 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 adjacent to 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.

17. Device as claimed in claim 16, wherein the electrical connection between the anode and cathode of the terminal compartments comprises a power source.

18. Device as claimed in any of the 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 micro-organism selected from the group of Geobacter sulfurreducens, Shewanella putrefaciens, Geobacter metallireducens and Rhodoferax ferrireducens, other organisms from said genera or a consortium of one or more organisms therefrom.

19. Device as claimed in any of the claims 16-18, wherein the anodes and cathodes of the intermediate compartments are adapted as bifunctional electrodes according to claims 11- 13.

20. Method for producing hydrogen, comprising of:

(i) providing a device for electrolysis as claimed in any of the claims 14-19;

(ii) supplying a substrate medium comprising an oxidizable substrate to the anode sub-compartments;

(iii) applying a potential to the number of cathodes which is lower than the equilibrium potential of the H⁺/H₂ redox couple in the culture medium;

(iv) discharging the hydrogen produced at the cathode.