DE102016212850A1 - Process for the biological production of methane - Google Patents

Abstract

A method for the biological production of methane (CH4) is given, which on the one hand is environmentally friendly and inexpensive and on the other hand dispenses with the use of fossil deposits. The process is divided into two steps, whereby first hydrogen (H2) and oxygen (O2) are produced from water (H2O) by peptides with hydrogenase activity. In the second step, methanogenesis bacteria produce methane (CH4) from the generated hydrogen (H2) and from carbon dioxide (CO2). To increase the yield of the process, interfering oxygen (O 2) can be bound in the hydrogenation reaction mixture and / or in the methanogenesis bacteria.

Description

The present invention relates to a process for the biological production of methane.

Methane is an important source of energy as well as an important source for the chemical industry. Methane is mainly produced from natural gas, the main component of which is. For this purpose, fossil deposits are exploited, in which mostly natural gas and oil are present together. Such deposits are found, for example, in Russia, Saudi Arabia, North America, China and Norway, often under the seabed, for example in the North Sea.

However, the availability of such fossil natural gas resources is limited. Furthermore, economic dependence on natural gas suppliers may occur. In addition, the methane is present in natural gas in addition to a variety of other components and must be separated by complex processes and thus with further energy consumption of the Beikomponenten to obtain pure methane.

However, the main problem with using methane from fossil sources is the strong global warming potential of methane. In the extraction and transport of fossil methane inevitably occurs a significant loss of the degraded gas, this share is released as a greenhouse gas into the atmosphere.

On the other hand, the use (ie the combustion) of fossil methane, as well as other fossil fuels, increases the greenhouse effect by releasing more carbon dioxide into the atmosphere and thus acting as a greenhouse gas. It is also known to obtain methane-containing gases from biomass (so-called biogas process). So is out of the DE 10 2004 035 997 A1 a biogas plant for the provision of methane-containing gases known. However, the biogas production has the disadvantage that only very impure methane is recovered, which is contaminated by carbon dioxide, water vapor, ammonia, hydrogen sulfide and other components. Furthermore, not enough biomass is available to ultimately replace the fossil deposits.

Finally, it is known that there are several trillion tons of methane in the form of methane hydrate on the earth's seabed. The promotion of these deposits is not yet commercially possible and seems to be feasible only with considerable costs.

With the aim of limiting the greenhouse effect promoted by the carbon dioxide-generating combustion of energy sources, for example fossil fuels, different methods and technologies for alternative energy generation have been developed.

For example, electricity obtained from wind or sunlight is now an important part of the general electricity supply. However, one problem with the generation of electricity by wind or photovoltaic systems is that the favorable weather conditions for electricity generation do not always match the prime demand of the end user. However, as there is still no adequate and / or cost-effective solution for long-term storage of such excessively produced electricity, alternative generation plants are regularly operated under capacity - especially at conditions that would be optimal for generating energy.

Another problem in this regard is the geographical distance between regions with favorable weather conditions for alternative energy production and the main sites of final energy consumers. For example, there are currently infrastructural bottlenecks in the electricity grid, which do not allow the electricity generated by wind turbines from the windy coastal regions of northern Germany in the densely populated metropolitan areas in the middle or even in southern Germany forward. This also means that wind turbines are purposefully operated under capacity.

There is therefore a need to provide energy carriers or storages which make it possible to store and / or redistribute excess energy that can be provided by alternative energy generation plants under optimal conditions.

In preferred embodiments of the present invention, therefore, an environmentally friendly and cost-effective method for producing methane is specified which, on the one hand, dispenses with the use of fossil deposits and, on the other hand, offers the possibility of making use of the excess energy arising in alternative energy generation.

• – Erzeugung von Wasserstoff (H₂) und Sauerstoff (O₂) aus Wasser (H₂O) durch isolierte, synthetische, und/oder rekombinante Peptide mit Hydrogenase-Aktivität in einem wässrigen Reaktionsgemisch (Hydrogenese),
• – Abtrennung des erzeugten Sauerstoffs vom erzeugten Wasserstoff,
• – Erzeugung von Methan aus dem erzeugten Wasserstoff und Kohlendioxid (CO₂) durch Methanogenese-Bakterien (Methanogenese),
• – Abtrennung und ggf. Verflüssigung des erzeugten Methans (CH₄).

According to the invention the above object is achieved with the features of claim 1. Thereafter, the method according to the invention for the biological production of methane comprises the following steps:

• Generation of hydrogen (H ₂ ) and oxygen (O ₂ ) from water (H ₂ O) by isolated, synthetic, and / or recombinant peptides having hydrogenase activity in an aqueous reaction mixture (hydrogenation),
• Separation of the generated oxygen from the generated hydrogen,
• - production of methane from the generated hydrogen and carbon dioxide (CO ₂ ) by methanogenesis bacteria (methanogenesis),
• - Separation and possibly liquefaction of the methane produced (CH ₄ ).

Advantageous embodiments of the method according to the invention can be taken from the subordinate claims.

According to the invention, it has been recognized that the production of methane can be divided into two steps, wherein in each case an environmentally neutral bioreaction can take place in both steps.

Thus, it is initially obtained by the use of suitable peptides with hydrogenase activity from water, hydrogen and oxygen.

The oxygen generated by the peptides with hydrogenase activity in the first step is separated and can be put to other uses. There remains the hydrogen generated in the first step.

In accordance with the invention, methane is now generated in a second step from the hydrogen produced and with the addition of carbon dioxide through the use of suitable methanogenesis bacteria.

The carbon dioxide used can be obtained from the ambient atmosphere, for example by an air liquefaction process with subsequent formation of dry ice. This reduces the proportion of greenhouse-active carbon dioxide in the atmosphere. Furthermore, carbon dioxide can be used from industrial or combustion processes, which directly reduces the carbon dioxide load on the atmosphere.

Finally, the methane produced can be separated from the remaining educts of hydrogen and carbon dioxide and possibly liquefied.

In an advantageous embodiment of the invention, isolated peptides having hydrogenase activity are selected from the group consisting of: hydrogenases; Hydrogenase-like peptides; Hydrogenasevarianten; and fragments or combinations thereof, used for hydrogen production.

In a further advantageous embodiment of the invention, synthetic or recombinant peptides having hydrogenase activity are selected from the group consisting of: hydrogenases; Hydrogenase-like peptides; Hydrogenasevarianten; Hydrogenase mimics; and fragments or combinations thereof, used for hydrogen production.

In the context of the present specification, the term "peptides with hydrogenase activity", hydrogenases, hydrogenase-like peptides; Hydrogenasevarianten; Hydrogenase mimics and fragments or combinations thereof, explicitly included.

Hydrogenases are enzymes that catalyze the reversible reduction of protons to hydrogen with a redox partner (hydrogenation). The process according to the invention makes it possible, in comparison to processes for the biological production of hydrogen by algae, to avoid to a large extent both the complex mechanical mixing and the expensive purification of the hydrogenereactor with partly highly polluting detergents.

The oxygen sensitivity of the hydrogenases may be an obstacle to their direct use for hydrogen production. However, not all hydrogenases are sensitive to oxygen and oxygen-insensitive (oxygen-resistant) hydrogenases are known. Oxygen-insensitive hydrogenases are "peptides with hydrogenase activity" within the meaning of the present invention.

The term "oxygen-resistant" with respect to the hydrogenase activity of the peptides of the invention includes both peptides which, at an oxygen content in the reaction mixture of ≤ 0.8% by volume, do not undergo any reduction in their hydrogenase activity, as well as peptides at an oxygen content in the reaction mixture of ≤ 0.8 vol .-% experience a reduction in their hydrogenase activity, but this reduction is less than 50% and the peptides remain usable in the process according to the invention

Due to the presence of various metals in the active site, four types of hydrogen are currently distinguished: iron [Fe] or [Fe Fe], nickel-iron [Ni Fe], nickel-iron-selenium [Ni Fe Se], and metal-free hydrogenases (the however, require a metal-containing cofactor for catalysis). Hydrogenases from algae are usually among the [Fe], [Fe Fe] or [Ni Fe] Hydrogenasetypen, which have particularly advantageous conversion rates. The active site of these hydrogenases is also referred to as H-cluster (H = hydrogen activating) and typically contains six iron atoms. In [Ni Fe] hydrogenases, in addition to iron, nickel also occurs in the active center. Peptides with hydrogenase activity in the context of the present invention include peptides that can be assigned to one of the known classes of hydrogenases due to the constellation of metal ions in the active center, but also peptides in which one or more of the metal ions are replaced by a replacement but continue to catalyze the reduction of protons to hydrogen with a redox partner.

In this context, the term "hydrogenase-like peptides" is to be understood, which inter alia includes the peptides with hydrogenase activity, which have one or more structures which are not considered as typical structural elements of a hydrogenase. For example, the term "hydrogenase-like peptides" includes peptides with hydrogenase activity which, because of their combinations of metal ions in the active site or by the presence or absence of certain protein domains, would not typically be considered to be hydrogenase, but which would still require the reduction of Catalyze protons to hydrogen with a redox partner.

The term "hydrogenase variants" is to be understood as meaning peptides with hydrogenase activity which represent truncated or, for example by mutation, altered variants of classified hydrogenases. For example, biotechnologically produced fusion proteins in general, and those suitable for affinity chromatography in particular, are also hydrogenase variants in the sense of the present invention. Fusion proteins suitable for affinity chromatography are, for example, those which attach an affinity tag to the peptide having hydrogenase activity, in particular a His, Myc, HA, FLAG, Avi, protein A, protein G, calmodulin , TAP, Strep, or GST tag.

The identification and structural biological characterization of hydrogenases allows the preparation of peptomimetic hydrogenase peptides, also known as hydrogenase mimics. "Hydrogenase mimics" in the context of the present invention are usually synthetically or recombinantly produced peptides, which may well contain artificial amino acids, such as proline and Brentztraubensäure derivatives, glycine derivatives or N-methyl amino acids. Hydrogenase mimics mimic the active site of hydrogenase. Although the biological activity of enzyme mimics generally does not correspond to the activity of the natural enzyme, enzyme mimics often represent a more robust, and possibly more cost effective, alternative to using the original enzyme. Hydrogenase mimics are advantageously oxygen insensitive and act as catalysts in the reduction of protons to hydrogen and therefore constitute peptides with hydrogenase activity in the context of the present invention.

The term "isolated" with respect to peptides having hydrogenase activity in the context of the present specification includes peptides taken out of one environment and thus separated from the other components of that environment. For example, a peptide naturally occurring in an organism with hydrogenase activity can be isolated from this organism. However, the term "isolated" in this context does not necessarily mean that the peptide is provided without other peptides or cellular constituents, that is, with a certain degree of purity.

The term "recombinant" with respect to peptides having hydrogenase activity in the context of the present specification includes peptides prepared using DNA technology known to those skilled in the art, ie, biotechnologically. Recombinant peptides with hydrogenase activity include, by truncation, mutation, or fusion, bioengineered hydrogenase variants. Hydrogenase mimics can also be made using recombinant DNA technology.

The term "synthetic" with respect to peptides having hydrogenase activity, in the context of the present specification, includes peptides made using techniques known to one of ordinary skill in the art of peptide synthesis, for example, by solid-phase peptide synthesis. In the formation of a synthetic peptide, the amino acids are typically added forward of the carboxylate groups, with ribosome-driven peptide synthesis in the cellular milieu linking amino acids beginning with the amino groups. In addition, the synthetic production of peptides with hydrogenase activity allows the incorporation of unusual amino acid building blocks, D-amino acids and linker molecules, as well as peptide cyclizations, which may have a positive effect on the hydrogenase activity per se, or their oxygen resistance.

In one or more advantageous embodiments of the present invention, peptides with hydrogenase activity are used which have [Fe], [Fe Fe], [Fe Ni] or [Ni Fe Se] complexes in their active center. Advantageously, the hydrogenase activity of the peptides of the invention is oxygen-resistant.

In the method of the invention, and as described in more detail below, it is advantageous that peptides with hydrogenase activity in their active sites can harbor carbon monoxide (CO) as the ligand of the metal ions, or that they are typically strong for a cell or organism toxic compound that is not detrimental to the activity of the hydrogenase. The use of such peptides, which may be hydrogenase variants, hydrogenase-like peptides or else hydrogenase mimics, is therefore likewise according to the invention.

• (a) aus Grünalgen und/oder Cyanobakterien isoliert sind, oder
• (b) rekombinant oder synthetisch hergestellt sind und eine Aminosäuresequenz beinhalten, die der Aminosäuresequenz von Peptiden mit Hydrogenase-Aktivität aus Grünalgen und/oder Cyanobakterien identisch oder ähnlich ist.

Typically, in the process according to the invention, peptides with hydrogenase activity are used which

• (a) are isolated from green algae and / or cyanobacteria, or
• (b) are produced recombinantly or synthetically and include an amino acid sequence identical or similar to the amino acid sequence of peptides having hydrogenase activity from green algae and / or cyanobacteria.

Methods for comparing amino acid sequences and for identifying homologous regions in the sequences to be compared are known to the person skilled in the art. A significant similarity of amino acid sequences is present when the sequences to more than 80%, in particular more than 85%, in particular more than 90%, in particular more than 95%, in particular more than 98%, in particular more than 99% are identical.

In an advantageous embodiment, the green algae and cyanobacteria are selected from the group consisting of: Chlamydomonas reinhardtii; Ralstonia eutropha; Paracoccus spp., Pyrococcus spp., Alcaligenes spp., Bradyrhizobium spp., Oligotropha spp., Acidovorax spp., Hydogenaphaga spp., Carboxydothermus spp., Desulfovibrio spp., Megasphaera elsdenii; Scenedesmus obliquus; Desulfomicrobium baculatum; Ralstonia metallidurans; Clostridium acetobutylicum; Clostridium pasteurianum; Aquifex aeolicus; Synechococcus BG04351; Synechococcus elongatus PCC 7942; Spirulina spp; Chroococcidiopsis spp .; Synechocystis PCC 6803; Anabaena PCC 7120; Thiocapsa roseopersicina; Allochromium vinosum; Desulfovibrio fructosovorans; Desulfovibrio gigas; Nostoc punctiforme; Fischerell muscicola; Anabaena siamensis; Cyanothece spp; Gloeocapsa alpicola; Aphanothece halophytica; Arthrospira maxima; Thiocapsa roseopersicina; Alteromonas macleodii; Aquifex aeolicus; Rubrivivax gelatinosus; and Shewanella oneidensis selected. Typically, the peptides used to generate hydrogen are provided with hydrogenase activity in an aqueous solution.

In some embodiments, the peptides having hydrogenase activity are attached to a support material, for example, by adsorption, by cross-linking, by ionic bonding, by covalent bonding, by immunoaffinity, by a spacer molecule (for example, by bi-reactive linker peptide, a bi reactive alkane or alkene, or by an alkyne). The binding to the carrier material allows the separation of the carrier material with the bound peptides from the reaction mixture, wherein the preferred carrier material according to the invention from the group consisting of: silica gel particles; Aluminum hydroxide (Al ₂ (OH) ₃ ), alumina hydroxide (AlO (OH)), alumina (Al ₂ O ₃ ); Materials useful for separation by ion exchange or affinity chromatography, for example, silica gel particles adapted to bind a peptide having hydrogenase activity, particularly by coating with antibodies specific for the peptides, or by coating with the respective affinity partner for His , Myc, HA, FLAG, Avi, protein A, protein G, calmodulin, TAP, Strep, or GST tags; titanium oxide; and silicides (for example, silicides with copper (Cu), titanium (Ti), platinum (Pt), palladium (Pd), iron (Fe), nickel (Ni), lead (Pb), gallium (Ga), indium (In ), Cadmium (Cd), ruthenium (Ru), cobalt (Co), silver (Ag), gold (Au), iridium (Ir), osmium (Os), mercury (Hg), zinc (Zn), hafnium (Hf ), Zirconium (Zr), vanadium (V), niobium (Nb), molybdenum (Mo), tungsten (W), chromium (Cr) or manganese (Mn)).

For example, the carrier with bound peptides having hydrogenase activity can be advantageously separated from the reaction mixture. As a result, for example, the amount of active peptides with hydrogenase activity in the reaction mixture can be easily regulated / scaled, thus also advantageously regulating / scaling the rate of production of the hydrogenase. In addition, so also advantageously a quick "shutdown" of the hydrogenatory reactor is possible. In addition, the uncomplicated and efficient separation of the peptides with hydrogenase activity allows the switching of the reaction mixture without leading to significant losses of the peptides. The separation further allows the regeneration of the hydrogenases outside the reaction mixture, for example under oxygen-free conditions.

• (a) durch das Anlegen einer elektrischen Spannung an Elektroden, die in das wässrige Reaktionsgemisch eingebracht sind, insbesondere durch Platin-, Kupfer-, Titan-, Silikon-, Palladium-, Eisen-, Nickel-, Blei-, Gallium-, Indium-, Kadmium-, Ruthenium-, Kobalt-, Silber-, Gold-, Iridium-, Osmium-, Quecksilber-, Zink-, Hafnium-, Zirkonium-, Vanadium-, Niob-, Molybdän-, Tungsten-, Chrom- oder Mangan-Elektroden; und/oder
• (b) durch eine Weitergabe von Elektronen über eine Elektronentransportkette im wässrigen Reaktionsgemisch, insbesondere eine Elektronentransportkette in der wenigstens ein Elektronendonator wie NADH mitwirkt, oder in der wenigstens ein Photosensibilisator aus der Gruppe der Porphyrine, zum Beispiel Zink-Tetraphenylprophyrin-Tetrasulfonat (ZnTPPS) oder Chlorophyll, oder mitwirkt, oder in der wenigstens ein Elektronen-Träger wie Methylviologen mitwirkt,

bereitgestellt. In preferred embodiments of the method of the present invention, the peptides with hydrogenase activity are activated by the uptake of electrons, the electrons being provided to the peptides by an electron donor. In particular, the electrons are preferred:

• (A) by the application of an electrical voltage to electrodes, which are introduced into the aqueous reaction mixture, in particular by platinum, copper, titanium, silicon, palladium, iron, nickel, lead, gallium, indium , Cadmium, ruthenium, cobalt, silver, gold, iridium, osmium, mercury, zinc, hafnium, zirconium, vanadium, niobium, molybdenum, tungsten, chromium or manganese electrodes; and or
• (B) by a transfer of electrons via an electron transport chain in the aqueous reaction mixture, in particular an electron transport chain in which at least one electron donor such as NADH participates, or in the at least one photosensitizer from the group of Porphyrins, for example zinc tetraphenylprophyrin tetrasulphonate (ZnTPPS) or chlorophyll, or in which at least one electron carrier such as methylviologen co-operates,

provided.

In a preferred embodiment, the electrical voltage is provided by excess energy from alternative energy production facilities. For example, for activation, the peptides of the invention having hydrogenase activity can be covalently linked to one of the electrodes - the anode - and thus directly utilize the electrons delivered by the electrode for activation, in particular by the direct transfer of electrons at the binding interface of peptide and electrode ,

Alternatively, a photosensitizer may be excited by light from at least one light source to provide the electrons required for activation. One possible photosensitizer that can independently contribute to hydrogenation is titanium or cobalt silicide. In particular, titanium silicide is furthermore advantageously able to bind oxygen and thus can act multimodally in the reaction mixture.

In a further advantageous electron transport chain, for example, the photosensitizer ZnTPPS may be present in the reaction mixture together with NADH. The photosensitizer can then be brought into its excited state by light taking up electrons from NADH and in turn pass electrons on to the electron carrier methylviologen. Methyl viologen may then function as an electron donor and pass electrons to the peptides with hydrogenase activity for activation. In particularly advantageous embodiments, the photosensitizer can be excited by light from multiple light sources, which are supplied with power wirelessly, and which are freely suspended in the aqueous reaction mixture during hydrogenation. Advantageously, by introducing the light sources into the reaction mixture, light can be distributed more homogeneously in the reactor space, and the reactor diameter no longer has to be limited by the limited light penetration depth of external illuminations. Through resonant inductive near-field coupling, the light sources, which are usually highly energy-efficient LED light sources, can be powered wirelessly. For example, a transmit coil may generate an alternating magnetic field in the reaction mixture that can inductively energize the light sources. In particular embodiments, the peptides to be activated with hydrogenase activity, as well as the photosensitizers, can be bound directly to the surfaces of the light sources suspended in the reaction mixture.

The aqueous reaction mixture is usually anaerobic and according to the invention has an oxygen content of up to 0.8% by volume, in particular up to 0.6% by volume, in particular up to 0.4% by volume, in particular up to 0.2% by volume. , in particular up to 0.1% by volume, in particular 0.05% by volume, or is oxygen-free.

In order to regulate the oxygen content, oxygen can be bound in the aqueous reaction mixture during or after the hydrogenation, in particular by adding at least one binder. In alternative embodiments, the oxygen content of the reaction mixture may be regulated by exposure to an oxygen-free atmosphere of, for example, carbon monoxide, carbon dioxide or an inert gas such as argon or nitrogen. In such embodiments, it is particularly advantageous to keep the volume of the gas phase as low as possible.

In other words, in the process according to the invention, the oxygen content of the reaction mixture can be continuously regulated already during the oxygen evolution. This has advantages in terms of constant reaction conditions, so that no disruption or interruption of hydrogen production occurs. It should be emphasized that - also in the following - the term "oxygen" explicitly includes oxygen radicals, which also inhibit the hydrogenase.

In addition, it has been recognized that even in the methanogenesis bacteria intracellular oxygen hinders the methanogenesis. Therefore, a preferred embodiment of the method is proposed in the following, in which the intracellular oxygen of the methanogenesis bacteria is also bound. The advantageous embodiments for binding the oxygen which are subsequently proposed by way of hydrogenation are therefore expressly not restricted to the hydrogenation step. Rather, these developments are also advantageously for binding the intracellular oxygen of the methanogenesis bacteria and are also proposed in this regard as embodiments of the method according to the invention.

An embodiment is preferred in which the oxygen is bound by adding at least one binder.

It is preferred that the or the binder is regenerated or after the absorption of oxygen. As a result, in the best case, the binder in the reaction mixture or remain within the bacterial cells and become effective again there. Furthermore, a cost-effective and environmentally friendly process is realized.

Preferably, the oxygen is bound biochemically or chemically and the binder (s) are regenerated after the uptake of oxygen. Binders which are added according to the invention for the biochemical binding of the oxygen to the reaction mixture are hemoglobin, myoglobin or porphyrin.

In this connection, a method is first proposed in which hemoglobin or myoglobin is added as a binding agent. In this case, biochemically produced hemoglobin or myoglobin can be introduced into the hydrogenation bioreactor. With respect to the introduction of hemoglobin or myoglobin during methanogenesis, this may be facilitated by opening the membranes of the bacteria by electroporation. During electroporation, the cells are briefly exposed to strong electric fields, whereby the plasma membranes are temporarily permeable. This makes it particularly easy for hemoglobin or myoglobin to enter cells and bind intracellular oxygen. In a further embodiment, the hemoglobin or the myoglobin is introduced by genetic engineering into the genome of the bacteria by means of biotechnological DNA transcription technology so that the hemoglobin or myoglobin can be produced by the cell itself in the future.

In a further development of the method, porphyrin is added as binder, which is preferably introduced in the form of porphyrin-iron complexes in the hydrogenase bioreactor. The porphyrins can be hindered sterically, so that the oxygen uptake prevents a reduction of the iron. Also, the penetration of porphyrin into the bacterial cell can be promoted by electroporation.

With regard to the abovementioned developments of the method, an embodiment is preferred in which the hemoglobin or myoglobin and / or the porphyrin is / are regenerated electrochemically and / or biochemically and / or physically after the absorption of oxygen. First, the hemoglobin or myoglobin or the porphyrin iron complexes can be reduced by electrochemical reduction to iron ion (Fe ²⁺ ) complexes. Furthermore, hemoglobin or myoglobin and porphyrin-iron complexes can be reduced by biochemical reduction with the enzyme NADPH to the already mentioned iron ion complexes.

Finally, hemoglobin or myoglobin may preferably be physically regenerated during a phase in which oxygen production is reduced by decreased hydrogenase activity by being exposed to carbon dioxide. The carbon dioxide molecules displace the oxygen molecules from the hemoglobin or myoglobin and take their places in the presence of a sufficiently high partial pressure. In embodiments in which hydrogenase activation is ensured by means of an electron transport chain which includes a photosensitizer, this can be done, for example, during a dark phase.

In a further preferred embodiment of the method according to the invention, the oxygen is chemically bound.

It is initially preferred that hydrazine and / or a hydrazine salt, in particular iron hydrazine, is added as the binder. The hydrazine is introduced directly into the hydrogenation bioreactor. The hydrazine or its salts exhibit a reducing effect, so that oxygen or oxygen radicals are bound.

Another possibility for the chemical bonding of oxygen is that a binder, in particular α-terpene and / or isoprene, and / or a derivative thereof is added as a binder. Terpenes generally have a reducing effect so that oxygen or oxygen radicals present in the reaction mixture are bound. In a particularly preferred manner, the triphenylmethane dye eosin and / or a heme protein of the cytochrome P450 family are added to the terpene. These substances, as so-called photosensitizers, enhance the reducing action of the terpene and, by the above-mentioned activation by light, can also provide electrons to the process for the activation of the peptides with hydrogenase activity.

In further embodiments of the method according to the invention, or the binders in the reaction mixture may be bound to a support material.

The support material may be selected from the group consisting of: silica gel particles; Aluminum hydroxide (Al ₂ (OH) ₃ ), alumina hydroxide (AlO (OH)), alumina (Al ₂ O ₃ ); Materials useful for separation by ion exchange or affinity chromatography, for example, silica gel particles adapted to bind the binder, particularly by coating with antibodies specific for the binder, or ions specific for the binding of the binder; Platinum; Copper; Titanium; Silicone; Palladium; Iron; Nickel; Lead; Gallium; indium; Cadmium; ruthenium; Cobalt; Silver; Gold; Iridium; Osmium; Mercury; Zinc; Hafnium; Zirconium; vanadium; Niobium; Molybdenum; Tungsten; Chromium and manganese, or alloy thereof, to be selected. Binding to the support material allows the continuous removal of oxygenated binder from the reaction mixture, regeneration of the binder outside the reaction mixture, for example, by the above-mentioned carbon dioxide impingement and the recycling of the thus regenerated binder into the reaction mixture.

In embodiments in which both the peptides with hydrogenase activity and the binder (s) are bound to a carrier material, the carrier material or carrier materials are selected such that, if required, a differentiated separation of the peptides with hydrogenase activity and the Binder is achieved. For example, the binder of a magnetic carrier material, such as Dynabeads ^® (Thermo Fischer) is attached, and is periodically removed by magnetic separation from the reaction mixture, and replaced with fresh / regenerated binder. In these embodiments, for example, the peptides having hydrogenase activity may be covalently attached to a non-magnetic, porous support material, such as silica gel particles.

• (a) die Abtrennung der gasförmigen Zwischenprodukte Wasserstoff und Sauerstoff von dem wässrigen Reaktionsgemisch, als auch
• (b) die Abtrennung des Methans von dem Bakterienmedium, über eine Membran, insbesondere über eine poröse Membran aus CLPE (cross-linked polyethylene), insbesondere über eine CLPE-Membran mit einer inneren Schicht aus PATBS (Poly(acrylamid-tert-butyl-sulfonsäure)).

Furthermore, both are carried out in the process according to the invention

• (a) the separation of the gaseous intermediates hydrogen and oxygen from the aqueous reaction mixture, as well
• (b) the separation of the methane from the bacterial medium, via a membrane, in particular via a porous membrane of CLPE (cross-linked polyethylene), in particular via a CLPE membrane with an inner layer of PATBS (poly (acrylamide-tert-butyl) sulfonic acid)).

With regard to the provision of an environmentally friendly process for producing methane, it should be noted that the consumption of carbon dioxide during the methane recovery according to the invention as a whole indicates a carbon dioxide-neutral process. This means that the methane produced does not produce any additional greenhouse-damaging carbon dioxide during a thermal conversion, since the corresponding amount of carbon dioxide has already been removed from the atmosphere during production.

In one or more advantageous embodiments of the present invention, sunlight is used as the energy source in the method according to the invention, which directly improves the energy balance of the method. The proposed method can therefore be used as an alternative to the environmentally friendly energy sources wind energy and solar energy for energy.

In further advantageous embodiments, however, the method according to the invention can also be used in particular for increasing the efficiency of existing wind power or photovoltaic systems. In particular, since the process makes available the excessively produced in favorable weather conditions electricity for methane production and thus, at least partially, converted into a conventional energy source. Through the regular use of natural gas as an energy source, Germany has a nationwide infrastructure of gas storage facilities, gas networks and other transport routes, which enables the distribution of the energy source methane (and thus indirectly the surplus electricity) without major infrastructural investments.

As a result, an environmentally friendly and cost-effective method of extracting methane is provided, which not only avoids the use of fossil resources, but also enables the more efficient use of alternative energy sources.

In a development of the method, the reaction mixture used to generate hydrogen contains a photosensitizer and is separated from the light source, in particular sunlight, by a substantially transparent pane. As a result, the peptides used with hydrogenase activity can utilize sunlight as an energy carrier.

If the hydrogenation takes place in an aqueous reaction mixture, an embodiment is preferred in which the gaseous intermediates hydrogen and oxygen are first separated before the gas mixture hydrogen and oxygen is separated. In a preferred embodiment, the gaseous intermediates hydrogen and oxygen are separated from the reaction mixture by a membrane, in particular a porous membrane made of CLPE (cross-linked polyethylene). In this case, the gaseous intermediates can diffuse through the membrane, while the generally aqueous reaction mixture is prevented by the membrane at the passage. It has been found that said membrane made of CLPE is particularly suitable for the separation of the present mixing partners.

In a development of this embodiment, the membrane is fixed on both sides in a hexagonal close-packed spherical packing. As a result, a particularly secure fixation of the membrane is achieved.

In a further embodiment, a CLPE membrane with an inner layer of PATBS (poly (acrylamide-tert-butyl-sulfonic acid)) is used. The inner layer of PATBS advantageously further enhances the performance of the proposed CLPE membrane for the separation of the present mixing partners.

In this context, a further development of the method is preferred, in which a multilayer membrane is used whose layers are at least partially verschraess each other, in particular in a circular or honeycomb pattern. By welding the various layers of the membrane, an increased pressure resistance is provided, so that the gas pressure can be increased in an advantageous manner.

In particularly advantageous developments of the process according to the invention, the aqueous reaction mixture completely fills the hydrogenereactor space, so that there is no or only a minimal gas phase between the reaction mixture and the membrane. Furthermore, the reactor space can be designed so that any gas phase can be removed via a valve.

In a further embodiment, a black membrane is used in the hydrogenation step. The black membrane absorbs much of the incident light so that heating of the membrane and the surrounding reaction mixture is achieved. The evolution of heat promotes the diffusion of both oxygen and hydrogen. In addition, the heat energy can be dissipated and used elsewhere, for example by a heat exchanger. Furthermore, it is possible to connect the black membrane directly to a heat transfer medium, so that the radiated heat energy is directly dissipated.

The gaseous mixing partners oxygen and hydrogen obtained in the first step are preferably separated from one another by a gas liquefaction, in particular by the Linde method. The Linde process is known per se. In this case, a gas or a gas mixture is cooled until the individual mixture partners reach their boiling point and accumulate as a liquid. In this case, the boiling point of oxygen is about 70 Kelvin above the boiling point of hydrogen, so that the oxygen first becomes liquid. The use of a gas liquefaction process is particularly advantageous when very pure components are to be recovered.

Pure pure water can thus be obtained from pure oxygen and pure hydrogen as a byproduct of the process, in particular using waste heat from the hydrogenation step.

Overall, an embodiment of the invention is preferred in which the methanogenesis bacteria used to produce methane are also provided in an aqueous solution. This solution can also be supplied periodically or continuously appropriate nutrients. This provides an optimal environment for the methanogenesis bacteria used. Furthermore, an uninterrupted implementation of the method according to the invention is made possible.

In a further embodiment, the methanogenesis bacteria used to produce methane have one or a mixture of the species Methanobacterium thermoautotropicum, Methanobacillus spp., Methanobacterium spp., Methanococcus spp., Methanosarcina spp. and Methanothrix spp. on. It has been found that the mentioned bacterial species harmonize particularly well with the other steps of the method according to the invention.

In the second step of the process, the hydrogen and carbon dioxide may be supplied to the methanogenesis bacteria under anaerobic conditions and / or at a temperature of about 60 ° C. The conditions mentioned have proven to be optimal in order to provide the highest possible methane yield in conjunction with a long life and a particularly uniform activity of the bacteria used.

It has also been recognized that oxygen or oxygen radicals present not only inhibit the hydrogen production of the peptides with hydrogenase activity, but also reduce the productivity of the methanogenesis bacteria. Therefore, a particularly preferred development of the method according to the invention is also to reduce the content of intracellular oxygen within the methanogenesis bacteria. To this end, all measures are proposed that have been addressed as preferred embodiments for reducing the oxygen within the hydrogenation. In other words, all of these measures are also advantageously for use on the methanogenesis bacteria, but are here to avoid repetition no further detailed mention.

An embodiment is preferred in which the separation of the methane from the bacterial medium takes place via a membrane, in particular via a porous membrane made of CLPE (cross-linked polyethylene).

Also, the membrane of the second step can be set in a hexagonal closest packing.

The use of a membrane having an inner layer of PATBS is preferred.

Again, a multilayer membrane can be used in an advantageous manner, the layers of which are at least partially welded together, in particular in a circular or honeycomb pattern.

With regard to the last-mentioned embodiments, reference is made to the statements relating to advantageous features of the membrane used in the first step in order to avoid repetition.

With regard to the second reaction step, a development of the method according to the invention is proposed in which the osmotic pressure of the carbon dioxide on the gas side of the membrane is increased. This ensures a consistently high concentration of carbon dioxide in the bacterial medium. This is advantageous because the carbon dioxide is ultimately converted by the bacteria to methane.

It is further proposed an embodiment of the method according to the invention, in which the separation of the methane produced in the second step of the hydrogen by a gas liquefaction, in particular according to the Linde method, takes place. With regard to the advantages of this embodiment, reference may be made to the explanations regarding the separation of oxygen and hydrogen.

With regard to the methanogenesis step of the process according to the invention, it is proposed that the carbon dioxide supplied to the methanogenesis bacteria is obtained from dry ice, the dry ice optionally being obtained from an air liquefaction, in particular by the Linde process. This can provide carbon dioxide or dry ice, which contains virtually no impurities. Furthermore, carbon dioxide is taken from the ambient air, which has a beneficial effect on the greenhouse effect.

Alternatively or additionally, the carbon dioxide supplied to the methanogenesis bacteria can be provided from carbon dioxide-rich gas streams, in particular from industrial and combustion processes. This development of the method is particularly advantageous in terms of improving the carbon dioxide balance of waste incineration and power plants. Thus, methane, which can be used as starting material for the chemical industry or for carbon dioxide-neutral combustion, can be provided from actually environmentally harmful exhaust gas streams with the aid of the present method.

With a view to improving the energy balance and reducing the emission of greenhouse-active carbon dioxide, a development of the method is proposed in which unused or unreacted carbon dioxide - in particular by means of a cold trap - recovered and returned to the process.

Finally, in view of an additionally improved methane yield of the process, an embodiment is preferred in which resulting excess bacterial material is taken periodically or continuously from the process and fed to a biogas process for additional methane production. In carrying out the method according to the invention continuously produces biomass, which must be removed from time to time or continuously due to the specified plant size. In this development of the method, this biomass, which would otherwise - possibly with further follow-up costs - should be disposed of, a further useful use will be supplied.

In terms of an optimized energy balance of the method according to the invention, heat can be removed from the hydrogenation step, in particular by a heat exchanger or a heat pump. To obtain a particularly large amount of heat, as already mentioned, a black membrane can be used in the hydrogenation step. The excess heat can be meaningfully reused or transferred within the process.

Heat may be transferred between the hydrogenase step and the methanogenesis step. In other words, waste heat from the hydrogenation step can be used advantageously for methanogenesis. However, since methanogenesis generally takes place at a significantly higher temperature than hydrogenation, the use of a heat pump is suitable.

There are now various possibilities for designing and developing the teaching of the present invention in an advantageous manner. For this purpose, on the one hand to the subordinate claims, on the other hand, to refer to the following explanation of a preferred embodiment of the method according to the invention with reference to the drawing. In connection with the explanation of the preferred embodiment with reference to the drawing are also generally preferred Embodiments and developments of the teaching explained. In the drawing show

1 1 is a schematic representation of the partial step of the embodiment of the method according to the invention, in which hydrogen and oxygen are produced,

2 1 is a schematic representation of the partial step of the embodiment of the method according to the invention, in which methane is recovered,

3 a schematic representation of the entire method according to the embodiment shown and

4 a schematic representation of a preferred membrane assembly for the separation of gaseous (intermediate) products of the reaction mixture or the bacterial medium, and

5 a schematic representation of two embodiments of multilayer membranes, which are preferably used for gas separation in the process according to the invention.

1 shows a schematic representation of a part of the proposed embodiment of the overall process. This first part relates to the generation of hydrogen and oxygen by peptides with hydrogenase activity and the separation of the generated oxygen from the hydrogen.

At the beginning of the process, an aqueous reaction mixture 1 with the peptides with hydrogenase activity in a hydrogenase bioreactor 2 provided. The aqueous reaction mixture 1 also contains a buffer solution, for example, a 20 mM phosphate buffer solution pH 7.0, and NADH, ZnTPPS and Methylviologen and is from the gas space of the bioreactor 2 through a membrane 3 separated from CLPE. The provided reaction mixture is steady with a pump 4 mixed, and, advantageously, recycled.

The reaction mixture 1 gets from the environment through a substantially transparent pane 5 separated. Due to the irradiation of light (h ט), especially sunlight, the peptides used produce hydrogen and oxygen. These intermediates can pass through the membrane 3 into the gas space of the bioreactor 2 to be crossed and deducted.

Alternatively, and also at unfavorable lighting conditions for the process, the hydrogenation reactor by means of a via the electrodes 6 applied voltage directly and optionally additionally electrons are supplied, which ultimately lead to the activation of the peptides.

To increase the osmotic pressure of oxygen on the reaction mixture side of the reactor 2 , is drawn off on the gas side of the reactor, the resulting oxygen directly with the hydrogen. In addition, the gas phase is steadily added another inert gas, in this case nitrogen, which is the passage of the resulting oxygen from the reaction mixture side to the gas side of the membrane 3 , ie in the gas space of the bioreactor 2 , supported.

In a further advantageous manner, oxygen and oxygen radicals can be bound simultaneously in the reaction mixture. This can be done by the bioreactor 2 a binder may be added. Suitable binders are, for example, hemoglobins, myoglobins, porphyrin, hydrazine or terpenes. The binders used can be regenerated in various ways, to which reference is made to the preceding statements. If the binder or binders are regenerated electrochemically, are within the bioreactor 2 Attached electrodes, which are not shown here.

The separation of the intermediates oxygen and hydrogen via a gas liquefaction 7 and about a fractionation 8th ,

The bioreactor 2 it is possible to continuously remove excess heat. For this purpose, a heat exchanger is preferably used (not shown). Furthermore, heat can be removed from the bioreactor for improved energy balance 2 branched off by means of a heat pump (not shown) and the bioreactor 10 for methanogenesis (see 2 ) are supplied at a higher temperature level.

2 shows a schematic representation of the second step of the illustrated embodiment of the method according to the invention, which relates to the production of methane from the hydrogen produced in the first step and the separation of the recovered methane.

The hydrogen obtained in the first step becomes another bioreactor 10 fed. In this bioreactor 10 is an aqueous solution of methanogenesis bacteria 9 in front of the gas space of the bioreactor 10 through a membrane 11 is separated. The membrane 11 exists as for the first step according to 1 from CLPE. The present in aqueous solution methanogenesis bacteria is fed both hydrogen and carbon dioxide. For this purpose - for example, originating from a gas liquefaction process - carbon dioxide in the dry ice production 12 converted into dry ice.

The educts carbon dioxide and hydrogen become anaerobic for the bacterial solution Conditions supplied at a temperature of about 60 ° C. In addition, the bacteria solution is continuously added appropriate nutrients. Both the bacterial solution and the gases in the gas space of the bioreactor 10 are continuously pumped 13 circulated.

In accordance with the invention, the methanogenesis bacteria produce from the supplied hydrogen and carbon dioxide by the adjustment of suitable environmental conditions and by the supply of nutrients 14 Methane (CH ₄ ). The resulting methane can pass through the membrane 11 from CLPE into the gas space of the bioreactor 10 diffuse. To prevent excessive diffusion of hydrogen and carbon dioxide into the gas space, the osmotic pressure of the two educts on the gas side of the membrane 11 elevated.

Although methanogenesis is carried out in principle under anaerobic conditions, the intracellular occurrence or even accumulation of oxygen and / or oxygen radicals in the methanogenesis bacteria can occur. However, this intracellular oxygen hinders methane production. Therefore, as an advantageous embodiment, it is proposed to bind the intracellular oxygen of the methanogenesis bacteria. For this purpose - as with the peptides with hydrogenase activity - a suitable binder can be added. For example, such a binder may act biochemically or chemically to bind the intracellular oxygen. Suitable binders include myoglobin, porphyrin, hydrazine or terpenes.

Preferably, the binder is regenerated after uptake of intracellular oxygen. It is possible to proceed as described with respect to the peptides with hydrogenase activity.

The gas mixture of carbon dioxide, hydrogen and methane 15 may be from the gas space of the bioreactor 10 be removed. After that, first in a cold trap 16 the carbon dioxide is separated and recycled. The separation of the remaining components hydrogen and methane via a gas liquefaction 17 and a downstream fractionation 18 , The separated hydrogen is also returned to the process.

What remains is the product methane (CH ₄ ) in high purity.

3 shows a schematic representation of the entire process. It becomes clear how the hydrogen obtained in the first step is transferred and used as starting material for the second step.

In the second step, the continuous growth of the methanogenesis bacteria used constantly results in excess biomaterial, which is discharged for further methane production in the biogas process.

To the other individual steps is to avoid repetition on the comments 1 and 2 directed.

• 1. Schritt (1): 2H₂O → 2H₂ + O₂

In the method according to the invention, the following gross reactions take place:

• Step 1 ( 1 ): 2H ₂ O → 2H ₂ + O ₂

Here, by the method according to the invention in an application of 1mg peptides with hydrogenase activity more than 0.5 liters of hydrogen, especially more than 1 liter of hydrogen, especially more than 1.5 liters of hydrogen, especially more than 2 liters of hydrogen, in particular more than 2.5 liters of hydrogen, in particular more than 3 liters of hydrogen, in particular more than 3.5 liters of hydrogen, in particular more than 4 liters of hydrogen, in particular more than 4.5 liters of hydrogen, in particular up to about 4.67 liters of hydrogen per liter Reaction mixture and hour are generated, which corresponds to a significantly increased hydrogen production compared to the production of hydrogen by means of algae reactors. In contrast, much lower hydrogen production rates are common for algae reactors. In particular, for algae reactors, for example of hydrogen production rates, 8 ml of hydrogen are known per liter per hour.

• 2. Schritt (2): CO₂ + 4H₂ → CH₄ + 2H₂O Gesamt (3): CO₂ + 2H₂O → 2O₂ + CH₄

Furthermore, more than 500 .mu.mol hydrogen, in particular more than 1500 .mu.mol hydrogen, especially more than 2000 .mu.mol hydrogen, especially more than 2500 .mu.mol hydrogen, in particular more than 3000 .mu.mol hydrogen, in particular up to 3500 .mu.mol hydrogen per minute are obtained.

• 2nd step ( 2 ): CO ₂ + 4H ₂ → CH ₄ + 2H ₂ O total ( 3 ): CO ₂ + 2H ₂ O → 2O ₂ + CH ₄

In the approach of the process of the invention shown here by way of example, 0.124 liters, in particular more than 0.248 liters, in particular more than 0.372 liters, in particular more than 0.496 liters, in particular more than 0.62 liters, in particular more than 0.744 liters, in particular more than 0.868 liters , in particular more than 0.992 liters, in particular more than 1.16 liters, in particular up to about 1.16 liters of pure, carbon dioxide-neutral methane are produced.

4 shows a schematic representation of a preferred definition of the membrane used in the invention 3 . 11 , In this case, in the method both in the hydrogen production by algae and in the methane production by methanogenesis bacteria preferably a membrane 3 . 11 from CLPE used to allow the passage of gaseous products from an aqueous medium.

This figure shows in a side view and in a plan view how the membrane 3 . 11 is anchored from CLPE in a hexagonal close-packed spherical packing in order to secure the membrane as securely as possible 3 . 11 to reach.

This type of fixing of the membrane is preferred in the process according to the invention, but not absolutely necessary for carrying out the process.

5 shows a schematic representation of two embodiments of multilayer membranes, which are preferably used in the method according to the invention. The membranes 3 . 11 consist of at least two layers, for example, can have an inner layer of PATBS. To increase the compressive strength are the layers of the membranes 3 . 11 at least partially verschscheißt each other. In 5 on the left a circular pattern is shown. The circles may touch or be slightly spaced apart. In 5 on the right are the different layers of the membrane 3 . 11 welded together in a honeycomb pattern, whereby an increased pressure stability is achieved.

When using the preferred membranes both in the bioreactor 2 as well as in the bioreactor 10 For example, the pressure on the gas side of the membrane can be increased or decreased without it failing. Accordingly, in particular, the osmotic pressure of oxygen is on the gas side of the reactor 2 further reducible and the pressure of carbon dioxide on the gas side of the reactor 10 further increase.

The membrane 3 in the bioreactor 2 can be made of black material. As a result, falls in the bioreactor 2 a larger excess of heat, which is advantageously used in the manner already described.

Finally, it should be emphasized that the above-described embodiment of the method according to the invention discusses the claimed teaching, but does not restrict it to the exemplary embodiment.

LIST OF REFERENCE NUMBERS

1

Hydro Genesis reaction mixture

2

Hydro Genesis bioreactor

3, 11

membrane

4, 13

pump

5

disc

6

electrodes

7, 17

Gas / air liquefaction

8, 18

fractionation

9

Methanogenesis reaction mixture

10

Methanogenic bioreactor

12

dry ice production

14

nutrient

15

Methanogenic gas mixture

16

cold trap

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102004035997 A1 [0005]

Claims (30)

A process for the biological production of methane (CH ₄ ), the process comprising the steps of: - generating hydrogen (H ₂ ) and oxygen (O ₂ ) from water (H ₂ O) by isolated, synthetic, and / or recombinant peptides Hydrogenase activity in an aqueous reaction mixture (Hydrogenese), - Separation of the generated oxygen from the generated hydrogen, - Production of methane from the generated hydrogen and carbon dioxide (CO ₂ ) by methanogenesis bacteria (methanogenesis), - Separation and possibly liquefaction of the generated methane (CH _4). A method according to claim 1, characterized in that the isolated peptides with hydrogenase activity from the group consisting of: hydrogenases; Hydrogenase-like peptides; Hydrogenasevarianten; and fragments or combinations thereof are selected. A method according to claim 1, characterized in that the synthetic or recombinant peptides with hydrogenase activity from the group consisting of: hydrogenases; Hydrogenase-like peptides; Hydrogenasevarianten; Hydrogenase mimics; and fragments or combinations thereof are selected. Method according to one of claims 1 to 3, characterized in that the peptides with hydrogenase activity [Fe], [Fe Fe], [Fe Ni] or [Ni Fe Se] have complexes in their active center. Method according to one of claims 1 to 4, characterized in that the hydrogenase activity of the peptides is oxygen-resistant. Method according to one of claims 1 to 5, characterized in that the peptides with hydrogenase activity (c) are isolated from green algae and / or cyanobacteria, or (d) are produced recombinantly or synthetically and contain an amino acid sequence corresponding to the amino acid sequence of peptides with hydrogenase activity from green algae and / or cyanobacteria is identical or similar. A method according to claim 6, characterized in that the green algae and cyanobacteria are selected from the group consisting of: Chlamydomonas reinhardtii; Ralstonia eutropha; Paracoccus spp., Pyrococcus spp., Alcaligenes spp., Bradyrhizobium spp., Oligotropha spp., Acidovorax spp., Hydogenaphaga spp., Carboxydothermus spp., Desulfovibrio spp., Megasphaera elsdenii; Scenedesmus obliquus; Desulfomicrobium baculatum; Ralstonia metallidurans; Clostridium acetobutylicum; Clostridium pasteurianum; Aquifex aeolicus; Synechococcus BG04351; Synechococcus elongatus PCC 7942; Spirulina spp; Chroococcidiopsis spp .; Synechocystis PCC 6803; Anabaena PCC 7120; Thiocapsa roseopersicina; Allochromium vinosum; Desulfovibrio fructosovorans; Desulfovibrio gigas; Nostoc punctiforme; Fischerell muscicola; Anabaena siamensis; Cyanothece spp; Gloeocapsa alpicola; Aphanothece halophytica; Arthrospira maxima; Thiocapsa roseopersicina; Alteromonas macleodii; Aquifex aeolicus; Rubrivivax gelatinosus; and Shewanella oneidensis are selected. Method according to one of claims 1 to 7, characterized in that the peptides used for the production of hydrogen are provided with hydrogenase activity in an aqueous solution. Method according to one of claims 1 to 8, characterized in that the peptides are provided with hydrogenase activity bound to a substrate. A method according to claim 9, characterized in that the peptides with hydrogenase activity are bound to the substrate by adsorption, or by cross-linking, or by ionic bonding, or by covalent bonding. A method according to claim 10, characterized in that the peptides are bound with hydrogenase activity by a bi-reactive linker peptide, a bi-reactive alkane or alkene, or by an alkyne to the substrate. Method according to one of claims 9 to 11, characterized in that the substrate is separated with the bound peptides from the reaction mixture. Method according to one of claims 9 to 12, characterized in that the substrate from the group consisting of: silica gel particles; Aluminum hydroxide (Al ₂ (OH) ₃ ); Aluminum oxide hydroxide (AlO (OH)); Alumina (Al ₂ O ₃ ); Materials useful for separation by ion exchange or affinity chromatography, for example, silica gel particles adapted to bind a peptide having hydrogenase activity, particularly by coating with antibodies specific for the peptides, or by coating with the respective affinity partner for His , Myc, HA, FLAG, Avi, protein A, protein G, calmodulin, TAP, Strep, or GST tags; titanium oxide; and silicides. A method according to claim 13, characterized in that the silicides of silicides with copper (Cu), titanium (Ti), platinum (Pt), palladium (Pd), iron (Fe), nickel (Ni), lead (Pb), gallium (Ga), indium (In), cadmium (Cd), ruthenium (Ru), cobalt (Co), silver (Ag), gold (Au), iridium (Ir), osmium (Os), mercury (Hg), zinc (Zn), hafnium (Hf), zirconium (Zr), vanadium (V), niobium (Nb), molybdenum (Mo), tungsten (W), chromium (Cr) or manganese (Mn) are selected. Method according to one of claims 1 to 14, characterized in that the peptides are activated with hydrogenase activity by the uptake of electrons, wherein the electrons are provided to the peptides by an electron donor, in particular: (a) by the application of an electrical voltage Electrodes introduced into the aqueous reaction mixture, in particular by platinum, copper, titanium, silicon, palladium, iron, nickel, lead, gallium, indium, cadmium, ruthenium, cobalt Silver, gold, iridium, osmium, mercury, zinc, hafnium, zirconium, vanadium, niobium, molybdenum, tungsten, chromium or manganese electrodes; and / or (b) by transfer of electrons via an electron transport chain in the aqueous reaction mixture, in particular an electron transport chain in which at least one electron donor such as NADH participates, or in which at least one photosensitizer from the group of porphyrins, for example zinc tetraphenylprophyrin tetrasulfonate ( ZnTPPS) or chlorophyll, or in which at least one electron carrier such as methyl viologen co-operates. A method according to claim 15, characterized in that the peptides with hydrogenase activity are covalently linked to an electrode. A method according to claim 15, characterized in that the photosensitizer is excited by light of at least one light source suspended in the aqueous hydrogenation reaction mixture. A method according to any one of claims 15 to 17, characterized in that the photosensitizer is excited by light from a plurality of light sources, which are supplied with power wirelessly, and which are suspended freely in the aqueous reaction mixture during the hydrogenation. Method according to one of claims 1 to 18, characterized in that the aqueous reaction mixture has an oxygen content of up to 0.8% by volume, in particular up to 0.6% by volume, in particular up to 0.4% by volume, in particular up to 0.2 vol .-%, in particular up to 0.1 vol .-%, in particular 0.05% by volume, or is oxygen-free. Method according to one of claims 1 to 8, characterized in that oxygen is bound in the aqueous reaction mixture during or after the hydrogenation, in particular by adding at least one binder. A method according to claim 20, characterized in that the or the binder is regenerated or after the absorption of oxygen. Method according to one of claims 1 to 121, characterized in that the oxygen is bound biochemically or chemically. A method according to claim 22, characterized in that (a) for the biochemical binding of oxygen hemoglobin, myoglobin or porphyrins is added as a binder, and (b) for the chemical bonding of the oxygen hydrazine and / or a, in particular iron hydrazine, or a terpene , in particular α-terpene and / or isoprene, and / or a derivative thereof is added as a binder. A method according to claim 23, characterized in that the myoglobin and / or the porphyrins is regenerated after the absorption of oxygen electrochemically and / or biochemically and / or physically. Method according to one of claims 1 to 24, characterized in that both (a) the separation of the gaseous intermediates hydrogen and oxygen from the aqueous reaction mixture, as well as (b) the separation of the methane from the bacterial medium, via a membrane, in particular via a porous membrane of CLPE (cross-linked polyethylene), in particular via a CLPE membrane with an inner layer of PATBS (poly (acrylamide tert-butyl-sulfonic acid)) takes place. Method according to one of claims 13 to 25, characterized in that the electrical voltage of alternative power generation systems, in particular of wind power or photovoltaic systems, is provided. A method according to claim 26, characterized in that the carbon dioxide is atmospheric carbon dioxide or carbon dioxide from industrial or combustion processes. Method according to one of claims 1 to 27, characterized in that when using 1 mg of peptides with hydrogenase activity more than 0.5 liters of hydrogen, in particular more than 1 liter of hydrogen, in particular more than 1.5 liters of hydrogen, in particular more as 2 liters Hydrogen, in particular more than 2.5 liters of hydrogen, in particular more than 3 liters of hydrogen, in particular more than 3.5 liters of hydrogen, in particular more than 4 liters of hydrogen, in particular more than 4.5 liters of hydrogen, in particular up to approximately 4, 67 liters of hydrogen per liter of reaction mixture and hour are generated. Process according to Claim 28, characterized in that 0.124 liters, in particular more than 0.248 liters, in particular more than 0.372 liters, in particular more than 0.496 liters, in particular more than 0.62 liters, in particular more than 0.744 liters, in particular more than 0.868 liters, in particular more than 0.992 liters, in particular more than 1.16 liters, in particular up to about 1.16 liters of pure, carbon dioxide-neutral methane can be produced. Method according to one of claims 1 to 28, characterized in that when using 1mg peptides with hydrogenase activity more than 500 .mu.mol hydrogen, especially more than 1000 .mu.mol hydrogen, especially more than 1500 .mu.mol hydrogen, especially more than 2000 .mu.mol hydrogen, in particular more than 2500 .mu.mol hydrogen, in particular more than 3000 .mu.mol hydrogen, in particular up to 3500 .mu.mol hydrogen per minute are obtained.

Images