WO2006109057A1 - Pile a combustible - Google Patents

Pile a combustible Download PDF

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Publication number
WO2006109057A1
WO2006109057A1 PCT/GB2006/001333 GB2006001333W WO2006109057A1 WO 2006109057 A1 WO2006109057 A1 WO 2006109057A1 GB 2006001333 W GB2006001333 W GB 2006001333W WO 2006109057 A1 WO2006109057 A1 WO 2006109057A1
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WO
WIPO (PCT)
Prior art keywords
oxidant
fuel cell
oxygen
hydrogen
hydrogenase
Prior art date
Application number
PCT/GB2006/001333
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English (en)
Inventor
Fraser Andrew Armstrong
Kylie Alison Vincent
Oliver Lenz
Baerbel Friedrich
Original Assignee
Isis Innovation Limited
Humboldt-Universitat Zu Berlin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0507564A external-priority patent/GB0507564D0/en
Priority claimed from GB0518645A external-priority patent/GB0518645D0/en
Application filed by Isis Innovation Limited, Humboldt-Universitat Zu Berlin filed Critical Isis Innovation Limited
Publication of WO2006109057A1 publication Critical patent/WO2006109057A1/fr

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Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the invention relates to fuel cells and methods of operating fuel cells.
  • Fuel cells are electrochemical devices that convert the energy of a fuel directly into electrical and thermal energy.
  • a fuel cell consists of an anode and a cathode, which are electrically connected via an electrolyte.
  • a fuel which is usually hydrogen, is fed to the anode where it is oxidised with the help of an electrocatalyst.
  • an oxidant such as oxygen (or air) takes place.
  • the electrochemical reactions which occur at the electrodes produce a current and thereby electrical energy.
  • thermal energy is also produced which may be harnessed to provide additional electricity or for other purposes.
  • Fuel cells may also be adapted to utilise other hydrocarbon fuels such as methanol or natural gas.
  • Fuel cells have many advantages over traditional energy sources. The major attractions of these systems are their energy efficiency and their environmental benefits. Fuel cells can be operated at an efficiency which is higher than almost all other known energy conversion systems and this efficiency can be increased further by harnessing the thermal energy produced by the cell. Further, fuel cells are quiet and produce almost no harmful emissions, even when running on fuels such as natural gas, since the system does not rely on the combustion of the fuel. Particularly advantageous are cells which operate on hydrogen, as these systems produce no emissions other than water vapour and their fuel source is renewable.
  • Electrocatalysts are typically employed at both the cathode and the anode of the fuel cell in order to provide sufficiently rapid conversion of hydrogen and oxygen to water.
  • the most commonly used electrocatalyst is platinum.
  • Platinum is a very efficient catalyst and enables high currents to be produced in the fuel cell.
  • it is very costly and is of limited availability.
  • platinum since platinum is not a specific catalyst, it can only be used in a cell in which the anode and cathode are separated by a membrane that is essentially impermeable to fuel and oxidant. This provides a further disadvantage since membranes are expensive to produce and currently available materials do not provide a perfect barrier to fuel and oxidant.
  • Membranes can be used to reduce the presence of oxygen in the environment of the anode.
  • the use of membranes has a number of disadvantages. Membranes are expensive and the advantages of the reduced cost of the hydrogenase catalyst is therefore decreased by the need to use an expensive membrane material. Furthermore, the requirement for a membrane complicates the design of the fuel cell and increases its internal resistance. In addition to these difficulties, the membranes that are currently available are not able to completely prevent leakage of oxygen from the cathode to the anode side of the cell. Therefore, even when a membrane is used, oxygen presence around the anode cannot be completely avoided. Loss of catalytic activity of the hydrogenase therefore still occurs.
  • a new fuel cell is therefore required which addresses the above-described difficulties connected with the membranes currently in use in fuel cells. There is also a need to reduce the problem of oxygen inactivation of hydrogenase catalysts in fuel cells.
  • the present invention addresses these difficulties by providing a fuel cell which can be operated in the absence of a membrane separating the anode and the cathode. This is achieved, for example, by reducing an oxidant other than oxygen at the cathode.
  • the fuel cell can be operated in the complete absence of oxygen and oxygen-derived inactivation of the hydrogenase enzyme can be avoided. Since it is no longer necessary to prevent oxygen from passing from the environment of the cathode to that of the anode, the need for a membrane is obviated.
  • the membrane-less fuel cell employs a hydrogenase catalyst at the anode which is tolerant to the presence of oxygen, and a catalyst at the cathode which is tolerant to the presence of hydrogen. In this way, a membrane is not required since the presence of oxygen at the anode will no longer inactivate the fuel cell.
  • the membrane-less fuel cells of the present invention therefore operate at high efficiency due to the decreased rate of hydrogenase inactivation.
  • the efficiency is further increased since the ions in the electrolyte are free to move within the cell without having to pass through a membrane.
  • the internal resistance of the cell is thus reduced.
  • the present invention therefore provides a method of operating a fuel cell, which method comprises oxidising hydrogen at an anode having a first catalyst adsorbed thereon, the first catalyst comprising a hydrogenase, and reducing an oxidant at a cathode, wherein said fuel cell does not contain a membrane between the anode and the cathode, i.e. the anode and cathode are not separated by a membrane.
  • the cathode has a second catalyst adsorbed thereon, the second catalyst comprising an enzyme capable of catalysing the reduction of the oxidant, and the oxidant is substantially free from oxygen.
  • the first catalyst comprises an oxygen tolerant hydrogenase
  • the second catalyst comprises a hydrogen-tolerant substance, e.g. an oxidase enzyme.
  • oxygen may be employed as the oxidant.
  • the present invention also provides a fuel cell comprising - an anode having a first catalyst adsorbed thereon, said first catalyst comprising a hydrogenase;
  • the fuel cell does not contain a membrane between the anode and the cathode, i.e. the anode and cathode are not separated by a membrane.
  • the cathode has a second catalyst adsorbed thereon, the second catalyst comprising an enzyme capable of catalysing the reduction of an oxidant, which oxidant is substantially free from oxygen.
  • the first catalyst comprises an oxygen tolerant hydrogenase and a second catalyst absorbed on the cathode comprises a hydrogen- tolerant substance, e.g. an oxidase enzyme.
  • oxygen may be used as the oxidant.
  • the present invention also provides the use of an organic acid, a peroxide, or nitrate as an oxidant in a membrane-less fuel cell.
  • Figure 1 depicts a fuel cell according to the invention
  • FIG. 2 depicts an alternative fuel cell according to the invention.
  • Figures 3 a and 3b show the power output and voltage output of a fuel cell operating according to the method of the invention.
  • Figure 4 depicts the power output ( ⁇ W) vs load (k ⁇ , logarithmic scale) of a membraneless fuel cell according to the invention (solid squares) and a membraneless fuel cell employing a non-oxygen-tolerant hydrogenase (open circles).
  • Figure 5 depicts a further fuel cell according to the invention.
  • the fuel cells of the invention comprise:
  • an oxidant source which provides an oxidant to a cathode
  • a cathode which may be coated with a catalyst e.g. a catalyst comprising a reductase or an oxidase, at which the oxidant is reduced and which is electrically connected to the anode via an electrical conductor; and
  • an electrolyte which serves as a conductor for ions between the anode and the cathode.
  • the fuel cells of the invention do not have a membrane separating the anode and the cathode, i.e. anode and cathode are present in the same compartment of the fuel cell.
  • a membrane is herein defined as any solid porous material which hinders movement of oxygen molecules from one side of the membrane to the other.
  • the term membrane therefore includes solid electrolyte proton exchange membranes such as NafionTM.
  • the anode and cathode are therefore typically separated only by a liquid electrolyte and generally no solid material separates the electrodes.
  • the absence of a membrane separating the anode and the cathode means herein that ions and other materials including protons and oxygen molecules can move from anode to cathode and vice versa through the electrolyte without passing through a membrane.
  • the same meaning is ascribed to the term 'fuel cell does not contain a membrane between the anode and the cathode'.
  • the present invention may be used in combination with any fuel cell, as long as the operating conditions are sufficiently mild that the hydrogenase, and any reductive enzymes used, are not denatured. For example, fuel cells which operate at very high temperatures, or which require extreme pH conditions, may well cause the enzymes to denature.
  • FIG. 1 An example of a fuel cell according to the invention is depicted in Figure 1.
  • the anode 1 and cathode 2 are separated physically but are electrically connected via the external circuit 3 and the electrolyte 4. Electrons flow from the anode to the cathode via the external load 5. Ions flow between the electrodes through the electrolyte. No membrane is present between the anode and the cathode.
  • the fuel cells of the present invention utilise hydrogen as a fuel.
  • the reaction of hydrogen which occurs at the anode can be described according to the following equation (1):
  • the electrons produced are transferred via the conductor to the cathode and, similarly, the protons are transferred to the cathode via the electrolyte.
  • the source of hydrogen may be hydrogen gas itself. If desired, the hydrogen may be derived from a source such as an alcohol, including methanol and ethanol, or from fossil fuels such as natural gas.
  • the hydrogen may be in a crude form and thus may contain impurities, or purified hydrogen may be used.
  • the fuel source is typically a gas which comprises hydrogen and which is provided to the anode. It is also conceivable that the fuel may be provided in liquid form.
  • the fuel source also comprises an inert gas, although substantially pure hydrogen may also be used.
  • an inert gas for example, a mixture of hydrogen with one or more gases such as nitrogen, helium, neon or argon may be used as the fuel source.
  • the hydrogen fuel source may optionally comprise further components, for example other additives.
  • hydrogen is present in the fuel source in an amount of at least 0.5%, e.g. at least 2% by volume, preferably at least 5% and more preferably at least 10% by volume, for example 25%, 50%, 75% or 90% by volume.
  • the remainder of the fuel source is typically an inert gas, although it may be air.
  • Provision of hydrogen to the anode encompasses supplying hydrogen to the electrode directly, to the electrolyte and/or to a space in the fuel cell to which the electrolyte is exposed.
  • the fuel and oxidant are provided in mixed form, for example a mixture of hydrogen in air may be used.
  • a mixture may be provided in gaseous or liquid form and may be supplied directly to the electrolyte surrounding the electrodes, or to a space in the fuel cell to which the electrolyte is exposed. This latter embodiment is discussed further below.
  • the fuel source is supplied from an optionally pressurised container 6 of the fuel source in gaseous or liquid form.
  • the fuel source is supplied to the electrode via an inlet 7, which may optionally comprise a valve.
  • An outlet 8 is also provided which enables used or waste fuel source to leave the fuel cell.
  • the oxidant is a material which can be reduced at the cathode.
  • the oxidant is not oxygen.
  • the oxidant is, for example, substantially, or totally, free from oxygen.
  • substantially free from oxygen means containing no more than 1%, preferably no more than 0.5%, 0.2%, 0.1% or 0.05% oxygen.
  • the fuel cell may be operated in the substantial or total absence of oxygen.
  • the oxidant may be substantially or totally free from carbon monoxide or cyanide.
  • substantially free from carbon monoxide means containing no more than 1%, preferably no more than 0.5%, 0.2%, 0.1% or
  • Substantially free from cyanide means containing no more than 1%, preferably no more than 0.5%, 0.2%, 0.1% or 0.05% of cyanide. It is further preferred that the reduced product of the oxidant, as well as the oxidant itself, does not cause substantial inactivation of the hydrogenase.
  • a material which does not cause substantial inactivation of the hydrogenase means that hydrogenase activity is substantially or fully maintained in the presence of the material.
  • the hydrogenase maintains at least about 50%, 70%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of its activity when the material is present, compared to its normal activity, i.e. its activity when the material is absent.
  • Maintenance of activity may include include the situation in which the activity drops initially in the presence of the material, but is substantially or fully recovered after supply of the material is terminated.
  • Substantial recovery of activity means that at least about 50%, 70%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of its normal activity (i.e. its activity when the material is absent) is recovered after termination of supply of the material.
  • This assay can be carried out to determine whether a hydrogenase maintains its activity in the presence of a given material.
  • This assay comprises:
  • adsorbing the hydrogenase to be tested onto a measuring electrode typically a graphite electrode for example a pyrolytic graphite edge (PGE) electrode
  • a measuring electrode typically a graphite electrode for example a pyrolytic graphite edge (PGE) electrode
  • PGE pyrolytic graphite edge
  • a counter electrode e.g. a platinum wire
  • a reference electrode e.g. a saturated calomel electrode
  • step (a4) applying a potential of -40OmV or a more negative potential to activate the enzyme; (a5) changing the potential to a value in the range of from 0 to +30OmV and measuring the current generated by the cell over a period of time; and (bl) repeating the above steps, but following application of the potential in step (a5), supplying a quantity of the given material (e.g. by injection of a buffer containing the material) to the environment of the anode at a time T 0 .
  • Steps (al) to (a5) act as a control and indicate the current generated over time in the absence of the material.
  • Li step (bl) if the current does not decrease from the values measured in step (a5) on addition of the material to the anode environment (or decreases only to at least about 50%, 70%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of the values measured in step (a5)), the hydrogenase is deemed to maintain its activity in the presence of the material.
  • step (bl) if the activity measured in step (bl) is initially reduced compared with that measured in step (a5) but then after a time t recovers to substantially the values measured at the same time t in step (a5) (or recovers to at least about 50%, 70%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of the values measured in step (a5)), the hydrogenase is deemed to substantially fully recover its activity following termination of supply of the material. Typically, substantially full recovery of activity is achieved at no more than 100 seconds, e.g. 60, 30 or 10 seconds after time T 0 .
  • Preferred materials for use as the oxidant include organic acids (any reducible organic acid may be used), peroxides and nitrate.
  • Preferred organic acids include fumaric acid.
  • Preferred peroxides include hydrogen peroxide.
  • the most preferred oxidants are fumaric acid, hydrogen peroxide and nitrate.
  • the reduction of the oxidant preferably has the same stoichiometry as oxidation of hydrogen.
  • the electrons/protons produced at the anode and cathode should balance overall. This avoids, for example, a build up of protons and a pH change of the electrolyte.
  • the reaction which occurs at the anode can typically be described according to the following equation (2):
  • the electrolyte 4 comprises the oxidant.
  • the oxidant may be separately supplied in gaseous or liquid form, hi the depicted embodiment, a fixed amount of the oxidant is included in electrolyte 4 prior to use. Thus, a fixed amount of oxidant is present in the fuel cell. In this embodiment, the oxidant is gradually used up as the fuel cell is operated. Fuel cells of this type are therefore typically suitable for relatively short term use and may, for example, be disposable.
  • an inlet 10 is provided to supply oxidant to the fuel cell, for example as a substantially constant supply, so that the oxidant in the cell is constantly refreshed.
  • a source of oxidant 9 may also be provided.
  • the oxidant alone may be supplied, or alternatively a mixture of oxidant and electrolyte is supplied.
  • An outlet 11 is also provided to allow spent oxidant, or electrolyte containing spent oxidant, to exit the cell.
  • the anode maybe made of any conducting material, for example stainless steel, brass or carbon, e.g. graphite.
  • the surface of the anode 22 may, at least in part, be coated with a different material which facilitates adsorption of the first catalyst.
  • the surface onto which the first catalyst is adsorbed should be of a material which does not cause the hydrogenase to denature. Suitable surface materials include graphite, for example a polished graphite surface or a material having a high surface area such as carbon cloth, carbon sponge or porous carbon. Materials with a rough surface and/or with a high surface area are generally preferred.
  • the cathode may be made of any conducting material, for example stainless steel, brass or carbon, e.g. graphite.
  • the surface of the cathode 21 may, at least in part, be coated with a different material which facilitates adsorption of the second catalyst.
  • the surface onto which the second catalyst is adsorbed should be of a material which does not cause an enzyme to denature. Suitable surface materials include graphite, for example a polished graphite surface or a material having a high surface area such as carbon cloth, carbon sponge or porous carbon. Materials with a rough surface and/or with a high surface area are generally preferred.
  • the anode has a first catalyst adsorbed onto its surface 22.
  • the first catalyst comprises (or optionally consists of) one, or a mixture of, hydrogenase enzymes.
  • the hydrogenase(s) are capable of oxidising hydrogen but substantially do not react with the oxidant used.
  • the first catalyst may also comprise further additives if desired.
  • Suitable hydrogenases include those having a [Ni-Fe] and/or [Fe-Fe] and/or [Ni-Fe-Se] active site, preferably a [Ni-Fe] active site.
  • Hydrogenases having a [Ni-Fe] and/or [Fe-Fe] and/or [Ni-Fe-Se] active site are found in many microorganisms and enzymatically catalyse the oxidation of hydrogen and/or reduction of protons in those microorganisms.
  • the microorganisms containing hydrogenases include methanogenic, acetogenic, nitrogen-fixing, photosynthetic, such as purple photo synthetic, and sulfate- reducing bacteria and those from purple photosynthetic bacteria are preferred.
  • suitable hydrogenases include the hydrogenases from
  • Allochromatium vinosum Desulfovibrio gigas and Desulfomicrobium baculatum.
  • the Allochromatium vinosum hydrogenases i.e. the Allochromatium vinosum [Ni-Fe] hydrogenase or the Desulfomicrobium baculatum [Ni-Fe-Se] hydrogenase may be employed. Fragments or variants of these enzymes which retain hydrogenase activity may also be used. For example, variants which have been adapted for better attachment to an electrode surface are envisaged. Oxygen tolerant hydrogenases as described herein may also be employed.
  • the bacteria discussed above and other suitable microorganisms can generally be obtained commercially (for example from DSMZ in Germany).
  • the bacteria or other microorganism may be cultured to provide a sufficient quantity of enzyme for use in the fuel cell. This may be carried out, for example by culturing the enzyme in a suitable medium in accordance with known techniques. Cells may then be harvested, and the hydrogenase isolated and purified by any known technique.
  • the cathode of the fuel cell of the invention typically comprises a second catalyst.
  • the second catalyst may, for example, be coated or adsorbed onto the surface of the cathode.
  • the second catalyst is typically specific to reduction of the oxidant and does not react with hydrogen.
  • the second catalyst typically retains at least about 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% activity in the presence of hydrogen, compared to its normal activity, i.e. its activity in the absence of hydrogen.
  • the skilled person can easily determine the relative activity of a catalyst in the presence of hydrogen by carrying out the following simple assay.
  • the assay can be performed in a gas-tight standard electrochemical cell with an electrode coated with the chosen cathode catalyst as the working electrode.
  • the working electrode is polarised at an appropriate potential for reduction of the oxidant (e.g. fumaric acid, O 2 ) and the current response is monitored.
  • Oxidant is maintained at essentially constant concentration throughout the assay (e.g. ImM fumaric acid in the solution, 10% O 2 in the gas space).
  • the remaining gas mixture is comprised of inert gases (e.g. N 2 or Ar).
  • the experiment is repeated using a mixture of inert gases and H 2 in place of the inert gas in the gas space.
  • An H 2 -tolerant catalyst will show substantially no difference in current response in the presence or absence of H 2 .
  • the activity of the catalyst in the presence of H 2 can be determined as a percentage of the activity in the absence of hydrogen.
  • the second catalyst typically comprises or optionally consists of one or more enzymes, e.g. reductases, typically one reductase. Further additives may be present in the second catalyst if desired.
  • Suitable reductases are those which catalyse the reduction of the oxidant used in the cell.
  • fumarate reductase is typically used as the reductase.
  • Reductases such as fumarate reductases can be readily accessed by the skilled person. For example, fumarate reductase can be isolated from E. coli by standard techniques.
  • the first catalyst comprises an oxygen tolerant hydrogenase.
  • oxygen tolerant we mean that the hydrogenase activity is either maintained, or is decreased and then substantially fully recovered, following introduction of oxygen into the environment of the anode.
  • oxygen tolerant hydrogenase enzymes maintain at least about 50%, preferably 60%, 70%, 80%, 90% or 95%, preferably at least about 99%, 99.5%, 99.9% or 99.99%, of their activity when oxygen is introduced into the environment of the anode as compared to their activity when oxygen is absent.
  • substantially fully recovered means returning to substantially the same activity, i.e. at least about 50%, preferably 60%, 70%, 80%, 90% or 95% preferably at least about 99%, 99.5%, 99.9% or 99.99%, of the activity that is observed if oxygen is not introduced.
  • an oxygen tolerant hydrogenase will recover substantially full activity within 1000 seconds, preferably within 800 seconds, more preferably within 600 seconds, particuarly preferably within 100 seconds, 60 seconds, 30 seconds, 10 seconds or even 5 or 2 seconds, from termination of oxygen supply to the environment of the anode.
  • the rate constant for recovery of activity is at least 0.001s "1 or 0.005s "1 at an applied potential of from 0 to +10OmV.
  • the oxygen tolerant hydrogenase enzymes have the above-described activity when the amount of oxygen in the environment of the anode is up to 20 ⁇ M, 30 ⁇ M or up to 40 ⁇ M, or even up to 80 ⁇ M or 90 ⁇ M detectable oxygen (e.g. [O 2 ] in injection of buffer solution), and when the applied potential is in the range of from 0 to +10OmV or from 0 to +30OmV.
  • detectable oxygen e.g. [O 2 ] in injection of buffer solution
  • Preferred oxygen tolerant enzymes maintain or achieve substantially full recovery of activity at potentials of from 0 to +30OmV.
  • Non-oxygen tolerant enzymes may achieve partial recovery when the potential is reduced to between -10OmV and OV, but do not achieve significant recovery of activity at potentials of from 0 to +30OmV.
  • a simple assay can be carried out to determine whether a hydrogenase is oxygen tolerant.
  • This assay comprises carrying out steps (al) to (a5) set out above and then in step (bl), repeating steps (al) to (a5), but following application of the potential in step (a5), supplying a quantity of oxygen (e.g. 40 ⁇ M or 90 ⁇ M) to the anode at a time T 0 .
  • Oxygen is typically supplied by injection, at time To, of an oxygen-saturated buffer into the environment of the anode.
  • Steps (al) to (a5) act as a control and indicate the current generated over time in the absence of oxygen (anaerobic).
  • the current does not decrease from the values measured in step (a5) on addition of oxygen to the anode environment. That the current does not decrease means that the current is at least about 50%, preferably at least about 99.99% of the measured anaerobic current as discussed above.
  • the current will decrease and then increase again to the values obtained in step (a5).
  • a time t is designated as the time lapse after time T 0 at which hydrogenase activity is substantially recovered, and the measured current reaches substantially the same value as is obtained in step (a5) at the same time t.
  • Substantial recovery of the current means that the current returns to at least about 50% preferably at least about 99.99% of the measured anaerobic current as discussed above.
  • the time t at which the current will return to the values measured in step (a5) is no more than 1000 seconds, preferably no more than 800 seconds, more preferably no more than 600 seconds, e.g. no more than 100, 60, 30, 10, 5 or 2 seconds.
  • step (bl) the current measured in step (bl) will not return to the values measured in step (a5) after oxygen supply to the system.
  • a skilled person can determine whether any particular hydrogenase is an oxygen tolerant hydrogenase within the meaning of the present invention.
  • the oxygen tolerant hydrogenase enzyme used in the invention is typically also carbon monoxide tolerant.
  • carbon monoxide tolerant we mean that hydrogenase activity is substantially or fully maintained in the presence of carbon monoxide.
  • carbon monoxide tolerant enzymes maintain at least about 80%, 85%, 90%, 95%, 98%, 99% or 99.5% of their activity, preferably at least 99.9% or 99.99% of their activity, when carbon monoxide is introduced into the environment of the anode as compared to activity when carbon monoxide is totally absent.
  • the carbon monoxide tolerant enzymes have the above-described activity when the amount of carbon monoxide in the environment of the anode is up to 40 ⁇ M, 80 ⁇ M, 120 ⁇ M, 200 ⁇ M, 400 ⁇ M, 600 ⁇ M or up to 800 ⁇ M detectable carbon monoxide.
  • a simple assay can be carried out to determine whether a hydrogenase is carbon monoxide tolerant.
  • This assay comprises carrying out steps (al) to (a5) described above with reference to the oxygen tolerance assay, and then (cl) repeating steps (al) to (a5), but following application of the potential in step (a5), supplying a quantity of carbon monoxide (e.g. a 120 ⁇ M aliquot) to the anode.
  • a quantity of carbon monoxide e.g. a 120 ⁇ M aliquot
  • steps (al) to (a5) act as a control to indicate the current generated over time in the absence of carbon monoxide.
  • step (cl) where a carbon monoxide tolerant enzyme is used, at least 80%, 85%, 90%, 95%, 98%, 99% or 99.5%, preferably at least 99.9% or 99.99%, of hydrogenase activity is maintained.
  • the activity of the hydrogenase following introduction of carbon monoxide is at least 80%, 85%, 90%, 95%, 98%, 99% or 99.5%, preferably at least 99.9% or 99.99%, of that measured in steps (al) to (a5) at the same time t after application of the potential in the range 0 to +30OmV.
  • the oxygen tolerant hydrogenase enzyme used in the invention is typically also tolerant to the presence of sulfides such as H 2 S.
  • H 2 S tolerant we mean that hydrogenase activity is substantially or fully maintained in the presence OfH 2 S.
  • H 2 S tolerant enzymes maintain at least about 80%, 85%, 90%, 95%, 98%, 99% or 99.5% of their activity, preferably at least 99.9% or 99.99% of their activity, when H 2 S is introduced into the environment of the anode as compared to activity when H 2 S is totally absent.
  • the H 2 S tolerant enzymes have the above-described activity when the amount OfH 2 S in the environment of the anode is up to 40 ⁇ M, 80 ⁇ M, 120 ⁇ M, 200 ⁇ M, 400 ⁇ M, 600 ⁇ M or up to 800 ⁇ M detectable H 2 S.
  • An assay analogous to that described above with regard to oxygen and carbon monoxide tolerance may be used to determine the H 2 S tolerance of an enzyme.
  • Preferred oxygen tolerant hydrogenases for use in the present invention are those from bacteria of Has Ralstonia genus, or similar genera including Wautersia, Alcaligenes, and Hydrogenomonas (for example Wautersia or Hydrogen omonas) according to
  • Ralstonia metallidurans CH34 ATCC No. 43123, DSM No. 2839, e.g.
  • Membrane-bound, or membrane-associated oxygen tolerant hydrogenases are preferred.
  • a most preferred oxygen tolerant hydrogenase is the membrane-bound hydrogenase from Ralstonia eutropha.
  • alternative oxygen tolerant hydrogenases can also be used, in particular those from bacteria or other organisms which contain an ortholog of the hypX gene from Ralstonia eutropha Hl 6 and/or are found in oxygen-rich environments such as in the soil.
  • hydrogenases from aerobic Knallgas bacteria are advantageously used.
  • Examples for alternative genera whose hydrogenases would be suitable are Alcaligenes, Aquifex, Azotobacter, Bradyrhizobium, Burkholderia, Chromobactrium, Dechloromonas, Hydrogenovibrio, Magnetococcus, Magnetospirillum, Microbulbifer, Paracoccus, Pseudomonas, Rhizobium, Rhodobacter, Rubrivivax, and Streptomyces.
  • Aquifex, Azotobacter, Bradyrhizobium, Burkholderia, Chromobactrium, Dechloromonas, Hydrogenovibrio, Magnetococcus, Magnetospirillum, Microbulbifer, Paracoccus, Pseudomonas, Rhizobium, Rhodobacter, Rubrivivax, and Streptomyces are suitable.
  • the membrane-bound hydrogenase from Ralstonia eutropha comprises a small subunit HoxK and a large subunit HoxG.
  • the large subunit HoxG incorporates the [Ni- Fe] active site.
  • the amino acid sequence of the membrane-bound hydrogenase HoxK from Ralstonia eutropha is shown in SEQ ID NO: 2, with the corresponding DNA sequence pHGl shown in SEQ ID NO: 1.
  • the amino acid sequence of the hoxG protein is shown in SEQ ID NO:4, with the corresponding DNA sequence pHGl shown in SEQ ED NO: 3.
  • the oxygen tolerant hydrogenase used in the invention may comprise or consist of the hydrogenase from Re or may be a fragment or variant thereof having oxygen-tolerant hydrogenase activity.
  • the fragment is one which retains oxygen-tolerant hydrogenase activity.
  • the fragment is typically from about 50 to 750 amino acids in length, for example, the fragment may be at least about 100, 200, 300, 400, 500 or 600 amino acids in length.
  • the variant retains oxygen-tolerant hydrogenase activity.
  • the oxygen-tolerant hydrogenase activity of the variant may be enhanced or reduced compared to the hydrogenase from Re.
  • the variant typically shares at least about 40%, for example at least about 50%, 60%, 70%, 80%, 90% or 95% sequence identity with the Re hydrogenase.
  • the oxygen tolerant hydrogenase used in the invention may comprise or consist of the hydrogenase from Ralstonia metallidurans (Rm) or may be a fragment or variant thereof having oxygen-tolerant hydrogenase activity.
  • Rm Ralstonia metallidurans
  • the fragment is one which retains oxygen-tolerant hydrogenase activity.
  • the variant retains oxygen-tolerant hydrogenase activity.
  • the oxygen-tolerant hydrogenase activity of the variant may be enhanced or reduced compared to the hydrogenase from Rm.
  • the variant typically shares at least about 40%, for example at least about 50%, 60%, 70%, 80%, 90% or 95% sequence identity with the Rm hydrogenase.
  • Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
  • Variant polypeptides within the scope of the invention may be generated by any suitable method, for example by site-directed mutagenesis.
  • a functional mimetic or derivative of the hydrogenase from Re or Rm which has oxygen tolerant hydrogenase activity may also be used in the invention.
  • Such an active fragment may be included as part of a fusion protein.
  • the oxygen tolerant hydrogenase for use in the invention may be modified by the addition of suitable amino acid residues for the covalent attachment of linkers to the electrode surface.
  • the oxygen tolerant hydrogenase may also be chemically modified, e.g. post-translationally modified. For example, it may be glycosylated or comprise modified amino acid residues. It may also be modified by the addition of an affinity tag such as a histidine stretch, a strep-tag or a FLAG-tag to assist purification or by post translational modification including hydroxylation or phosphorylation.
  • oxygen tolerant hydrogenases used in the present invention can be obtained using standard techniques.
  • Oxygen tolerant hydrogenases can, for example, be isolated from a source of the bacterium to be used, and optionally cultured to provide a sufficient quantity of enzymes to use in the cell. Cells may then be harvested, isolated and purified by any known techniques.
  • the hydrogenase from Ralstonia eutropha is purified either by Strep-Tactin affinity chromatography using a C-terminally Strep- tagged derivative of the small subunit HoxK or by a procedure described by Podzuweit et ⁇ l in Biochim. Biophys. Acta 905, 435-446 (1987).
  • the hydrogenases may be produced synthetically, or may be genetically modified, for example to produce variants, using standard techniques.
  • an oxygen tolerant hydrogenase in this embodiment of the invention enables a membrane to be omitted from the fuel cell even when oxygen is employed as the oxidant.
  • the oxidant typically comprises oxygen, although any other suitable oxidant may be used.
  • the oxidant source typically provides the oxidant to the cathode in the form of a gas which comprises the oxidant. It is also envisaged, however, that the oxidant may be provided in liquid form. Generally, the oxidant source also comprises an inert gas, although the oxidant in its pure form may also be used.
  • a mixture of oxygen with one or more gases such as nitrogen, helium, neon or argon may be used.
  • the oxidant source may optionally comprise further components, for example alternative oxidants or other additatives.
  • An example of a suitable oxidant source is air.
  • the oxidant may be provided in the form of a mixture with the fuel, e.g. a mixture of hydrogen in air may be used.
  • oxygen is present in the oxidant source in an amount of at least 2% by volume, preferably at least 5% and more preferably at least 10% by volume.
  • Provision of oxidant to the cathode encompasses direct supply of oxidant to the electrode, supply to the electrolyte or supply to a space in the fuel cell to which the electrolyte is exposed.
  • the oxidant source is supplied from an optionally pressurised container 9 ( Figure 2) of the oxidant source in gaseous or liquid form.
  • the oxidant source is supplied to the electrode via an inlet 10, which may optionally comprise a valve.
  • An outlet 11 is also provided which enables used or waste oxidant source to leave the fuel cell.
  • the second catalyst typically comprises or optionally consists of one or more enzymes which do not react with hydrogen.
  • the second catalyst typically retains at least about 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% activity in the presence of hydrogen, compared to its normal activity, i.e. its activity in the absence of hydrogen.
  • Typical catalysts were used in this embodiment include the reductases described above and other oxidase catalysts known in the art.
  • alternative oxidase catalysts include those enzymes capable of reducing oxygen to water, typically those capable of reducing oxygen directly to water. This means that the oxidase enzyme is capable of reducing oxygen to form water, substantially without producing any intermediates as by-products. Some oxidase enzymes are known which do not fully reduce oxygen to water. Rather, such enzymes produce undesirable intermediate products such as hydrogen peroxide. The presence of such intermediate products may interfere with the functioning of the fuel cell and their presence should be minimised. Therefore, oxidase catalyts which are capable of directly and fully reducing oxygen to water are preferred.
  • the oxidase should have a potential of at least 0.5 V, when measured at pH 7 against a standard hydrogen electrode.
  • Preferred oxidase enzymes have a potential of at least 0.6V under the same conditions, more preferably at least 0.7V. This leads to a high voltage fuel cell. Lower potentials can, however, be tolerated where the enzyme has a very high turnover number. This provides a relatively efficient fuel cell, despite the low potential.
  • Preferred oxidase catalysts for use in the present invention include bilirubin oxidase and laccases. These enzymes are monomeric glycoproteins and have highly stable structures. They can therefore withstand a reasonable amount of variation in temperature and pH which allows more flexibility in the operation of the fuel cell.
  • Preferred laccase enzymes are fungal laccases, for example the blue copper oxidase enzymes.
  • the natural co-substrates for fungal laccases are phenolic products of lignin degradation that are oxidised to radicals. These enzymes therefore catalyse the reduction of oxygen to water with no intermediate. Further, these enzymes typically operate at potentials of 0.6V or greater.
  • Examples of useful blue copper oxidases are those from the white rot fungus Coriolus hirsutus, and the fungus Trametes versicolor.
  • Other useful fungi as the source of the laccase include Pycnoporus cinnabarinus.
  • Fragments, variants, functional mimetics and derivatives of the above described oxidases may also be used provided they maintain oxidase activity.
  • oxidase activity For example, glycosylated, hydroxylated or phosphorylated oxidases, or those modified by the addition of an affinity tag such as a histidine stretch, a strep-tag or a FLAG-tag.
  • the fungi discussed above, and other suitable fungi, yeast or bacteria can generally be obtained commercially.
  • the fungi, yeast or bacteria may be cultered to provide a sufficient quantity of enzymes to use in the fuel cell. This may be carried out, for example, by culturing the enzyme in a situable medium in accordance with known techniques. Cells may then be harvested, isolated and purified by any known technique.
  • FIG. 5 A further alternative embodiment of the invention, in which hydrogen and oxidant may be supplied in the form of a mixture, is depicted in Figure 5.
  • the electrolyte 4 is exposed to an environment or space 12 which contains hydrogen and may optionally contain an oxidant.
  • the catalyst used at the anode is an oxygen tolerant hydro genase as described above, the environment 12 may comprise hydrogen and oxygen, for example a mixture of hydrogen in air.
  • the environment 12 acts as the fuel source and optionally also as the oxidant source.
  • Hydrogen and optionally oxidant are supplied to the electrodes by diffusion of dissolved hydrogen/oxidant through the electrolyte and no separate supply of hydrogen or oxidant to the electrodes is required.
  • inlets 7 and 10, and containers 6 and 9, of Figures 1 and 2 are therefore not required, although direct supply of hydrogen and/or oxidant to the electrodes may also be provided via such inlets, if desired.
  • Electrodes typically only a thin layer of electrolyte separates the electrodes from the environment 12 (15 of Figure 5). This layer usually has a thickness of from 0.01 to 5 mm, for example from 0.1 to 1 mm. Hydrogen and oxygen (or other oxidant) will readily exchange between the thin layer of electrolyte and the environment 12, and quickly diffuse to the electrodes. Thus, a constant supply of hydrogen and oxygen (or other oxidant) is provided to the electrodes.
  • the oxidant may be supplied by alternative means, such as described above with reference to Figures 1 and 2.
  • hydrogen may be present in environment 12, whilst oxidant is present in the electrolyte 4 as described with reference to Figure 1, or oxidant may be supplied directly to the electrode via an inlet 10 as described with reference to Figure 2.
  • oxidant is present in environment 12, whilst hydrogen is supplied directly to the electrode via inlet 7 as described with reference to Figure 1.
  • the environment 12 to which the electrolyte is exposed may be any gas containing hydrogen and/or oxidant, preferably hydrogen and/or oxygen, where an oxygen tolerant hydrogenase enzyme is used.
  • the environment is air containing a trace amount of hydrogen.
  • Inert gases containing small amounts of hydrogen may also be used where the oxidant is supplied to the cathode separately.
  • no more than 4% hydrogen is present when combined with air or oxygen in order to reduce combustion risks. More preferably, up to 3%, for example up to 2% or 1% hydrogen may be used.
  • the minimum hydrogen present is that which provides sufficient fuel to operate the cell. For example, 0.01% or more, preferably 0.1% or 0.5% or more hydrogen may be present.
  • the required gases may be provided to the environment 12, which may be a sealed container, via inlet 13 with waste gas exiting via outlet 14.
  • a slow release cartridge 16 may be provided, for example a hydrogen-storage material which will slowly release hydrogen into the atmosphere. Such slow release materials are well known in the art. Examples include Pd, TiFeo. 8 Ni 2 alloy and AB 2 or AB 5 materials such as LaNi 5-y Al y compounds.
  • the catalysts are coated or adsorbed, preferably adsorbed, onto their respective electrodes so that they are immobilised on the electrode surface. This helps to prevent migration of the catalysts to the opposing electrode (i.e. migration of the hydrogenase to the cathode and of the reductase or oxidase to the anode).
  • the electrode surface is polished prior to attachment of the catalyst using any suitable polishing means, for example an aqueous alumina slurry or sandpaper.
  • the electrode surface may also be modified by other means, for example using oxygen plasma or other chemical modification.
  • Adsorption of the catalyst may then be carried out, for example by applying a concentrated solution of the catalyst, optionally mixed with a suitable attachment means, to the electrode surface, e.g. by pipette.
  • the catalyst, optionally together with attachment means may be made up into a dilute aqueous solution.
  • the electrode is then inserted into the solution and left to stand.
  • a potential may be applied to the electrode during this period if desired. The potential enables the degree of coating with the catalyst to be easily monitored.
  • the potential will be increased and then subsequently decreased within a range of from approximately -50OmV to +20OmV vs SHE and the potential cycled in this manner for up to 10 minutes at a rate of 10mV/s, typically for about 5 or 6 minutes.
  • the catalysts may be applied in a sub-monolayer, a monolayer, or as multiple layers, for example 2, 3, 4 or more layers.
  • the catalysts need not be applied to the entire surface of the electrodes.
  • at least 10% of the available surface of the anode is coated with the first catalyst.
  • at least 25%, 50%, 75% or 90% of the available surface is coated with the first catalyst.
  • at least 10% of the available surface of the cathode is typically coated with the second catalyst.
  • at least 25%, 50%, 75% or 90% of the available surface is coated with the second catalyst.
  • the "available surface" of the anode or cathode is the surface which is in contact with the fuel or with the oxidant respectively.
  • the 'available surface' is the surface which is in contact with the electrolyte.
  • the catalyst layers are, for example, adsorbed to the surface of the electrodes using an attachment means.
  • the attachment means is, for instance, a polycationic material.
  • suitable attachment means include large polycationic materials such as polyamines including polymyxin B sulfate and neomycin.
  • the catalyst may alternatively be covalently attached by means of a linker molecule.
  • Adsorption of the catalysts on to the electrode surfaces has a number of advantages.
  • the enzymes are typically in direct electronic contact with the electrodes and electron transfer from enzyme to electrode may occur rapidly. This means that the fuel cell of the invention can be operated without the need for a separate electron mediator to transfer charge at the electrodes. In one embodiment of the invention, the fuel cell is operated in the substantial or total absence of an electron mediator.
  • the fuel cells of the present invention comprise an electrolyte suitable for conducting ions between the two electrodes.
  • the electrolyte should preferably be one which does not require the fuel cell to be operated under extreme conditions which would cause the hydrogenase to denature. Thus, electrolytes which rely on high temperature or extreme pH should be avoided.
  • the electrolyte is typically an aqueous solution containing salts such as alkali metal halides, e.g. NaCl or KCl. Appropriate concentrations are in the range of 0.05 to 0.5 M, e.g. about 0.1 M.
  • a pH buffer may also be present in the electrolyte, e.g. a phosphate, citrate or acetate buffer.
  • Other additives may also be present as desired, including glycerol, polymyxin B sulphate or other attachment means which may help to stabilise the enzymes.
  • the electrolyte is typically a non- chloride containing electrolyte since chloride inhibits laccase. In such a case, citrate or phosphate may be used to act as both the electrolyte and buffer.
  • the conditions under which the fuel cell is operated must be controlled so that the enzymes do not denature. Furthermore, the conditions can be optimised to provide a maximum amount of the enzymes in the active state and thereby increase the efficiency of the system.
  • the fuel cell is operated at a temperature of from 10 to 65 0 C, preferably from 15 to 55°C, more preferably from 20 to 45 0 C.
  • the preferred pH of the cell is from 4 to 9, e.g. 5 to 9, preferably from 6 to 8.
  • hydrogen is supplied directly to the anode, it is typically supplied at such a rate as to provide a partial pressure of from 1x10 3 to 1x10 5 Pa. Hydrogenases have been found to show hydrogen oxidation activity within this pressure range.
  • the partial pressure may be at least IxIO 4 , 2xlO 4 or 5xlO 4 Pa.
  • the potential at the anode when working at pH7 is typically maintained at -40OmV or greater (i.e. at -40OmV or a less negative potential).
  • Preferred potentials at the anode are from -40OmV to + 40OmV, preferably from -20OmV to + 30OmV, for example from 0 to +30OmV. Each of these potentials is measured against a standard hydrogen electrode.
  • a fuel cell as described above, may be operated under the conditions described above, to produce a current in an electrical circuit.
  • the fuel cell is operated by supplying hydrogen to the anode and supplying oxidant to the cathode, for example by using an electrolyte which comprises the oxidant.
  • hydrogen and/or oxidant may be provided via diffusion through the electrolyte from a surrounding environment containing the hydrogen and/or oxidant.
  • two or more cells of the invention may be used either in series or in parallel. If the cells are connected in parallel, the same electrolyte may be employed for each cell. If series cells are employed, a separate electrolyte is required for each individual cell.
  • the fuel cell of the present invention is therefore envisaged as a source of electrical energy which might replace conventional platinum electrode-based fuel cells.
  • An anode coated with the Allochromatium vinosum hydrogenase was prepared by polishing a pyrolytic graphite edge plane electrode (lcm 2 ) with an aqueous slurry of l ⁇ m alumina, and inserting the electrode into a dilute (0.1 to l ⁇ M) solution of
  • Allochromatium vinosum hydrogenase was then applied to the electrode, the potential being increased and then subsequently decreased within a range of from approximately -50OmV to +20OmV vs SHE and the potential cycled in this manner for about 5 or 6 minutes at a rate of 10mV/s.
  • a cathode coated with the Escherichia coli fumarate reductase was prepared by polishing an electrode in the same manner as the anode, and running a dilute solution (0.1 to l ⁇ M) of Escherichia coli fumarate reductase over the surface of the electrode.
  • the hydrogenase coated anode and reductase coated cathode were employed in an electrochemical cell in accordance with Figure 1.
  • the electrolyte solution contained NaCl (100 mM), 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (HEPES) buffer (20 mM) and sodium fumarate (0.5 mM) and was adjusted to pH 7 with aqueous HCl or NaOH. No membrane was present between the anode and cathode.
  • FIG. 3a and 3b depict the variation in power output with applied load and the voltage/current relationship.
  • Reference Example 1 preparation of Ralstonia eutropha (Re) hydrogenase
  • MBH membrane bound hydrogenase
  • a Strep-tag II sequence was fused to the 3' end of the MBH small subunit gene, hoxK to facilitate purification.
  • Re cells containing the MBH overproduction plasmid were grown at 30 0 C in fructose- glycerol mineral medium in the presence of 80% H 2 , 10% CO 2 and 10% O 2 .
  • the cells were collected by centrifugation, resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 50 mM NaCl) and broken by passage three times through a French pressure cell.
  • the membranes were separated by ultra-centrifugation (1 hour, 90,00Og and 4 °C) and the MBH was solubilized by incubating the membranes at 4 0 C in 7.5 vol/g buffer A containing 2 % Triton-Xl 14.
  • the cleared solubilizate was applied to a Strep-Tact ⁇ n.
  • Superflow column which was then washed with 8 column volumes of buffer A using a BioCAD Sprint purification system.
  • the Strep-tagged MBH was eluted with 6 column volumes of buffer A containing 5 mM desthiobiotin. Fractions containing MBH were combined and concentrated.
  • Tr ⁇ metes versicolor (Tv) laccase Crude powdered extract of Tv laccase (Fluka) was suspended in sodium acetate buffer (50 mM, pH 5.5). The same buffer was used throughout the purification. Insoluble material was removed by centrifugation, and the extract was applied to a DEAE Toyopearl 650M column, washed with buffer and released from the resin with buffer containing ammonium sulphate (100 mM). Laccase-containing fractions were diluted ten-fold with buffer and applied to a Q-Sepharose column (Amersham Biosciences), washed with buffer and eluted with a 0-100 mM ammonium sulfate gradient in buffer.
  • Tv laccase Tr ⁇ metes versicolor
  • An electrode coated with Re MBH was prepared by soaking a pyrolytic graphite edge strip ('edge' area 0.7 cm 2 , freshly polished with 1 ⁇ m alumina) in a dilute solution of Re MBH for 5 minutes in a glove box. During removal from the box, the film was stored in anaerobic buffer solution.
  • An electrode coated with Tv laccase was prepared by soaking a pyrolytic graphite edge strip ('edge' area 0.7 cm 2 , freshly polished with coarse sandpaper) in a dilute solution of JV laccase for 20 minutes. Preparation was carried out in air.
  • H 2 and air inlet tubes were positioned close to the hydrogenase and laccase electrodes respectively. No membrane was used to separate the electrodes, but rather both were placed in the same compartment of the cell.
  • Example 2 was repeated but replacing the Re hydrogenase with Av hydrogenase. The results are depicted in Figure 4 (open circles). A maximum power output of 0.2 ⁇ W was recorded.

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Abstract

L’invention concerne une pile à combustible et un procédé de fonctionnement de la pile à combustible. La pile à combustible comprend une anode réalisant l’oxydation d’hydrogène et une cathode réalisant la réduction d’un oxydant. Un catalyseur à hydrogénase est absorbé par l’anode. Un catalyseur à réductase est typiquement absorbé par la cathode, la réductase étant capable de catalyser la réduction de l’oxydant. La pile à combustible peut fonctionner sans membrane de séparation de l’anode et de la cathode.
PCT/GB2006/001333 2005-04-14 2006-04-12 Pile a combustible WO2006109057A1 (fr)

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GB0507564A GB0507564D0 (en) 2005-04-14 2005-04-14 Fuel cell
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GB0518645.7 2005-09-13

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040101741A1 (en) * 2002-11-27 2004-05-27 St. Louis University Enzyme immobilization for use in biofuel cells and sensors
US20040214053A1 (en) * 2001-08-24 2004-10-28 Fraser Armstrong Fuel cell
WO2004114494A2 (fr) * 2003-05-06 2004-12-29 The Chemistry Faculty Of The Moscow State University Pile a combustible a hydrogene/oxygene
US20050095466A1 (en) * 2003-11-05 2005-05-05 St. Louis University Immobilized enzymes in biocathodes
WO2006030196A1 (fr) * 2004-09-13 2006-03-23 Isis Innovation Limited Pile a combustible biochimique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040214053A1 (en) * 2001-08-24 2004-10-28 Fraser Armstrong Fuel cell
US20040101741A1 (en) * 2002-11-27 2004-05-27 St. Louis University Enzyme immobilization for use in biofuel cells and sensors
WO2004114494A2 (fr) * 2003-05-06 2004-12-29 The Chemistry Faculty Of The Moscow State University Pile a combustible a hydrogene/oxygene
US20050095466A1 (en) * 2003-11-05 2005-05-05 St. Louis University Immobilized enzymes in biocathodes
WO2006030196A1 (fr) * 2004-09-13 2006-03-23 Isis Innovation Limited Pile a combustible biochimique

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Title
ARMSTRONG ET AL.: "Electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels", PNAS, vol. 102, no. 47, 22 November 2005 (2005-11-22), pages 16951 - 16954, XP002390503 *
ARMSTRONG ET AL.: "Hydrogen cycling by enzymes: eletrocatalysis and implication for future energy technology", JCS DALTON TRANS., 2005, pages 3397 - 3403, XP009069529 *
KARYAKIN A A ET AL: "HYDROGEN FUEL ELECTRODE BASED ON BIOELECTROCATALYSIS BY THE ENZYME HYDROGENASE", ELECTROCHEMISTRY COMMUNICATION, ELSEVIER, AMSTERDAM, NL, vol. 4, no. 5, 2002, pages 417 - 420, XP001174128, ISSN: 1388-2481 *
MOROZOV ET AL: "Tolerance to oxygen of hydrogen enzyme electrodes", ELECTROCHEMISTRY COMMUNICATION, ELSEVIER, AMSTERDAM, NL, vol. 8, no. 5, May 2006 (2006-05-01), pages 851 - 854, XP005424817, ISSN: 1388-2481 *

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