Ex-situ biological methanation method

Abstract

The present invention relates to an ex-situ biomethanation process and an apparatus for carrying out the process. The present invention relates to an ex-situ methane production process comprising the step of contacting at least one methanogen, a culture medium and optionally a first culture medium in a gas / liquid bioreactor, said gas / liquid bioreactor optionally comprising a second culture medium, and producing methane by reacting the influent gas with at least one methanogenic microorganism, characterized in that the culture medium is optionally immobilized in a gas / liquid bioreactor; and at least one methanogenic microorganism immobilized on the culture medium; the invention also relates to a device for carrying out the method.

Description

Technical Field

[0001] The present invention relates to a non-in-situ biomethanation method and an apparatus for implementing the method.

[0002] The present invention relates to a non-in-situ methane production method, which comprises the steps of: contacting at least one methanogenic microorganism, a culture substrate and optionally a first culture medium in a gas / liquid bioreactor, the gas / liquid bioreactor optionally containing a second culture medium, and producing methane by reacting an inflowing gas with at least one methanogenic microorganism, characterized in that the culture substrate is optionally immobilized in the gas / liquid bioreactor; and at least one methanogenic microorganism is immobilized on the culture substrate; and also relates to an apparatus for implementing the method. Prior Art

[0003] Methanation is the reaction of synthesizing methane from hydrogen molecules and carbon dioxide.

[0004] In recent years, methanation has been directly related to the development of wind energy and solar energy, and the development of wind energy and solar energy depends on the ability to store on a large scale the electric energy that has been produced but not consumed. Power-to-gas is a promising solution for converting this excess electric energy into hydrogen by electrolyzing water. However, since the hydrogen industry is still under construction, converting this hydrogen into methane by methanation makes it possible to store this energy on a large scale using the existing natural gas infrastructure, which is the concept of power-to-methane. Other advantages of methanation are that it captures and stores carbon dioxide during the conversion process, thereby reducing the environmental impact associated with carbon dioxide emissions (biogas produced by methanation, synthesis gas obtained by pyrolysis or gasification, combustion gas effluents or equivalent sources). This dual advantage of methanation makes it a technology for the future.

[0005] There are two competing pathways for methanation: the catalytic pathway, which has been striving for industrialization for many years, and the biological pathway, which is more robust (with respect to impurities such as CO, NH3, H2S, etc.) and has a smaller environmental footprint, especially due to reduced energy costs.

[0006] However, so far, neither in-situ nor non-in-situ biomethanation technologies have been mature or economically viable, and various dedicated methods still need further research.

[0007] The main disadvantage of the biological route is that the kinetics of methane production are slower than those of the catalytic route. This kinetic bottleneck is due in part to the limitations of the physico-chemical processes of converting gaseous hydrogen molecule feedstock into the liquid phase. Conventional solutions are to increase pressure and / or intensify agitation, which can be antagonistic to the biological process and may reduce their productivity due to the sensitivity of the microorganisms, and especially hydrogenotrophic methanogenic archaea, to mechanical stress. There is a need for the industrialization of biological methods with acceptable energy costs in order to reduce economic costs. Summary of the Invention

[0008] The present invention provides a non-in-situ biological methanation method and apparatus, which unexpectedly addresses the disadvantages of known biological methods and is an attractive alternative to catalytic methods that are difficult to industrialize. Thus, the present invention operates at lower pressures and temperatures compared to the catalytic route, improving the robustness and service life of the method, and also reducing the need for external energy supply compared to known prior art, increasing the methane content of the outlet gas, thereby improving the conversion efficiency, productivity, and robustness of the biological method.

[0009] A first object of the present invention is a non-in-situ methane production method, which comprises the following steps:

[0010] a) contacting at least one methanogenic microorganism, a culture substrate, and optionally a first culture medium;

[0011] b) introducing the mixture obtained in step a) into a gas / liquid bioreactor optionally containing a second culture medium;

[0012] c) contacting an inlet gas in the gas / liquid bioreactor obtained in step b);

[0013] d) reacting the inlet gas with at least one methanogenic microorganism;

[0014] e) recovering the outlet gas obtained in step d);

[0015] characterized in that

[0016] - the culture substrate is optionally immobilized in the gas / liquid bioreactor; and

[0017] - at least one methanogenic microorganism is immobilized on the culture substrate.

[0018] Advantageously, steps a) and b) can occur sequentially or simultaneously.

[0019] Advantageously, steps c), d), and e) can occur sequentially and / or simultaneously.

[0020] "At least one methanogenic microorganism" is defined herein as an aggregate of microorganisms comprising at least one methanogenic strain or a pure methanogenic strain.

[0021] Advantageously, the at least one methanogenic microorganism may comprise microorganisms belonging to the phylum Euryarchaeota and may be selected from the classes Methanobacteria, Methanococci, Methanopyri or Methanomicrobia. Preferably, the at least one methanogenic microorganism may comprise at least one strain of the genus Methanothermobacter.

[0022] Citation of deposited biological materials

[0023] In a particularly advantageous embodiment of the invention, the at least one methanogenic microorganism is the strain CLERMONT of Methanothermobacter marburgensis deposited on October 20, 2022 under the Budapest Treaty; its DSMZ (Leibniz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraβe 7B 38124 Braunschweig GERMANY) number is DSM34405.

[0024] The at least one methanogenic microorganism may be included in a pre-culture medium. The pre-culture medium may be the same as or different from the first medium.

[0025] "Culture substrate" means a medium that can be colonized by hydrogenotrophic microorganisms and / or methanogenic microorganisms. The substrate may be in the form of spheres, tablets, rollers, gels, foams, granules, rings, towers or biochips. Preferably, the culture substrate may be spherical or spheroid-like.

[0026] Advantageously, the culture substrate may be an organic or inorganic substrate of natural or synthetic origin.

[0027] Advantageously, the organic, natural-source culture substrates may be selected from: alginate, κ-carrageenan, chitosan, wood chips, straw, charcoal, plant fibers, corncobs, bagasse, rice, sunflower seed husks, diatomaceous earth, mycelium and mixtures thereof.

[0028] Advantageously, the organic culture matrix of synthetic origin can be a polymer. The polymer can be expanded or non-expanded. The polymer can be selected from: polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyurethane, polyacrylonitrile, polyvinyl alcohol, polyamide (PA) and polylactic acid (PLA) and mixtures thereof. Preferably, the organic culture matrix of synthetic origin is an expanded polymer. Preferably, the culture matrix can be selected from a porous cube made of polyurethane foam impregnated with powdered activated carbon and / or a rigid polyethylene biochip.

[0029] Advantageously, the inorganic culture matrix of natural origin or of synthetic origin may be selected from the group consisting of magnetite, volcanic rock, vermiculite, porous glass, silicon-based materials, ceramics, nanoparticles and mixtures thereof. Preferably, the culture matrix may be selected from the group consisting of sepiolite, pozzolan and / or porous glass (e.g., More preferably, the culture matrix may be selected from sepiolite, volcanic ash and / or porous glass, and may be spherical or quasi-spherical.

[0030] Advantageously, the culture matrix may be selected from porous cubes made of polyurethane foam impregnated with powdered activated carbon, rigid polyethylene biochips, sepiolite, volcanic ash and / or porous glass.

[0031] In this context, "optionally immobilized" means, in the context of "immobilization", that all elements of the solid matrix do not move relative to each other or relative to the bulk of the reactor (in the case of a downflow, a low-velocity upflow, which is used for solids denser than the liquid, or solid matrices whose movement is mechanically blocked, for example by means of a grid). In contrast, "non-immobilized" means that the solid matrices are suspended in an upflow in a "fluidized bed" mode, moving relative to each other and relative to the bulk of the reactor, but the net velocity of all solid matrices relative to the bulk of the reactor is zero.

[0032] In this article, "at least one methanogenic microorganism is fixed on the culture matrix" means that the microorganism is not free in its environment (culture medium or other environment), and when at least one methanogenic microorganism fixed on the culture matrix contacts the culture medium, a system with two different phases will appear. Usually, 0.01% to 100% (by weight) of at least one methanogenic microorganism is fixed on the culture matrix, preferably 60% to 100%, more preferably 80% to 100%.

[0033] "Culture medium" refers to a medium capable of sustaining at least one microorganism to produce a gas mixture and whether or not the culture medium is capable of producing biomass into which the influent gas is injected and dissolved.

[0034] Advantageously, the culture medium may comprise water, nutrients, trace elements or a mixture thereof. Preferably, the culture medium may comprise sources of nutrients (nitrogen, calcium, sodium, potassium, sulfur, phosphorus, magnesium) and trace elements (iron, zinc, copper, cobalt, nickel, molybdenum, iodine and boron) necessary for the maintenance of microorganisms or for the growth of microorganisms and for the activity of microorganisms.

[0035] Advantageously, the culture medium may be a continuous liquid phase. In the context of the present invention, "continuous liquid phase" means a volume of liquid having physical continuity and not a discontinuous liquid volume consisting of a set of liquid phases that do not contact each other, such as droplets percolating in a gas phase.

[0036] Advantageously, the pH of the culture medium may be from 7 to 9. Preferably, the pH of the culture medium may be 8.

[0037] Advantageously, at least one of step a) or step b) may use at least one culture medium. The contact in step a) may be carried out between at least one methanogenic microorganism and the culture substrate before introducing the obtained mixture into a gas / liquid bioreactor containing the culture medium in step b). The contact in step a) may be carried out between at least one methanogenic microorganism, the culture substrate and the culture medium before introducing the obtained mixture into a gas / liquid bioreactor that does not yet contain the culture medium in step b). The contact in step a) may be carried out between at least one methanogenic microorganism, the culture substrate and the first culture medium before introducing the obtained mixture into a gas / liquid bioreactor containing a second culture medium in step b). The first culture medium and the second culture medium may be the same or different.

[0038] Advantageously, the method according to the present invention may be continuous, semi - continuous or discontinuous (batch operation).

[0039] In a first variant, the non - in - situ methane production method according to the present invention comprises the following steps:

[0040] a) bringing at least one methanogenic microorganism into contact with a culture substrate;

[0041] b) introducing the mixture obtained in step a) into a gas / liquid bioreactor containing a culture medium;

[0042] c) contacting an inflowing gas in the gas / liquid bioreactor obtained in step b);

[0043] d) reacting the inflowing gas with at least one methanogenic microorganism;

[0044] e) recovering the outflowing gas obtained in step d);

[0045] characterized in that

[0046] - The culture medium is optionally immobilized in a gas / liquid bioreactor; and

[0047] - At least one methanogenic microorganism is immobilized on the culture medium.

[0048] In a second variant, the ex-situ methane production method according to the invention comprises the following steps:

[0049] a) bringing at least one methanogenic microorganism, a culture medium and a culture into contact;

[0050] b) introducing the mixture obtained in step a) into a gas / liquid bioreactor;

[0051] c) contacting an inflowing gas in the gas / liquid bioreactor obtained in step b);

[0052] d) reacting the inflowing gas with at least one methanogenic microorganism;

[0053] e) recovering the outflowing gas obtained in step d);

[0054] characterized in that

[0055] - The culture medium is optionally immobilized in a gas / liquid bioreactor; and

[0056] - At least one methanogenic microorganism is immobilized on the culture medium.

[0057] In a third variant, the ex-situ methane production method according to the invention comprises the following steps:

[0058] a) bringing at least one methanogenic microorganism, a culture medium and a first culture into contact;

[0059] b) introducing the mixture obtained in step a) into a gas / liquid bioreactor containing a second culture;

[0060] c) contacting an inflowing gas in the gas / liquid bioreactor obtained in step b);

[0061] d) reacting the inflowing gas with at least one methanogenic microorganism;

[0062] e) recovering the outflowing gas obtained in step d);

[0063] characterized in that

[0064] - The culture medium is optionally immobilized in a gas / liquid bioreactor; and

[0065] - At least one methanogenic microorganism is immobilized on the culture medium.

[0066] In the present text, "gas / liquid bioreactor" refers to a fermenter capable of carrying out a biological reaction that requires at least one reagent present in gaseous form to be dissolved in the culture medium.

[0067] Advantageously, the gas / liquid bioreactor may be selected from pneumatically stirred reactors such as bubble columns or airlift reactors with upward or downward liquid circulation, mechanically stirred columns with upward or downward liquid circulation, continuous stirred tank reactors (CSTR), submerged fixed bed reactors, fluidized bed reactors, and spray bed reactors. Preferably, the gas / liquid bioreactor may be selected from pneumatically stirred reactors, preferably pneumatically stirred reactors with downward liquid circulation.

[0068] Advantageously, steps a) and b) of the method according to the present invention may be carried out at a temperature comprised between 25 °C and 70 °C. Preferably, the temperature of steps a) and b) may be 55 °C.

[0069] Advantageously, steps a) and b) of the method according to the present invention may be carried out at a pressure comprised between 1 bar and 20 bar (absolute pressure). Preferably, the pressure of steps a) and b) may be comprised between 1 bar and 3 bar. More preferably, the pressure of steps a) and b) may be 2 bar.

[0070] Advantageously, the method according to the present invention may further include an intermediate step a') carried out between steps a) and b), which allows the at least one methanogenic microorganism to proliferate on a substrate before being introduced into the gas / liquid bioreactor. The proliferation step a') may last less than or equal to 6 months. Preferably, the duration of step a') may be 30 days.

[0071] Steps a) and a') allow the formation of a methanation catalyst (the substrate is colonized by at least one methanogenic microorganism). At the end of step a) or optional step a'), the methanation catalyst may or may not be separated from the first liquid culture medium before carrying out step b). Thus, "the mixture obtained in step a)" may denote a mixture comprising the catalyst and the first culture medium, or, when there is no culture medium in step a), or when the culture medium has been separated therefrom, it may denote only the catalyst.

[0072] Advantageously, the method according to the present invention may further include an intermediate step b') carried out between steps b) and c), which allows the at least one methanogenic microorganism to proliferate on a substrate in the gas / liquid bioreactor before introducing the influent gas. The proliferation step b') may last less than or equal to 3 months. Preferably, the duration of step b') may be 15 days.

[0073] Advantageously, the inflowing gas in step c) of the method according to the invention can be carbon dioxide (CO2) and hydrogen (H2). The inflowing gas is not limited to CO2 and H2. It can contain any other gases, such as methane, carbon monoxide, nitrogen, ammonia, and / or hydrogen sulfide. The H2 / CO2 volume ratio can be in the range of 2:1 to 6:1. Preferably, the H2 / CO2 volume ratio is 4:1.

[0074] Advantageously, the flow rate of the inflowing H2 in step c), step d) or step e) of the method according to the invention can be in the range of 0.1 NL / L 反应器 / hour to 10 NL / L 反应器 / hour, which is equivalent to 2.4 NL / L 反应器 / day to 240 NL / L 反应器 / day. Preferably, the flow rate of the inflowing H2 can be 10 NL / L 反应器 / hour, which is equivalent to 240 NL / L 反应器 / day.

[0075] Advantageously, the flow rate of the inflowing CO2 in step c), step d) or step e) of the method according to the invention can be in the range of 0.025 NL / L 反应器 / hour to 2.5 NL / L 反应器r / hour, which is equivalent to 0.6 NL / L 反应器 / day to 60 NL / L 反应器 / day. Preferably, the flow rate of the inflowing CO2 can be 2.5 NL / L 反应器 / hour, which is equivalent to 60 NL / L 反应器 / day.

[0076] Advantageously, the liquid is recycled in the reactor through a recirculation loop to homogenize the reaction medium. The recirculation rate of the liquid is in the range of 0.1 L / L 反应器 / minute to 1 L / L 反应器 / minute.

[0077] Advantageously, the reaction temperature in step d) of the method according to the invention can be in the range of 25 °C to 70 °C. Preferably, the temperature can be 55 °C.

[0078] Advantageously, during steps c), d) and e) of the method according to the invention, the pressure in the bioreactor can be in the range of 1 bar to 20 bar. Preferably, the pressure in steps c), d) and e) can be in the range of 2 bar to 6 bar or 1 bar to 3 bar. More preferably, the pressure in steps c), d) and e) can be 2 bar.

[0079] Advantageously, the outflowing gas can contain methane (CH4). The outflowing gas can also contain water vapor.

[0080] Advantageously, the volume ratio of methane (CH4) to the other off-gases (CO2 and H2) can be comprised between 2:3 and 9.9:10.

[0081] Advantageously, the flow rate of the off-gas can be comprised between 0.025 NL / L 反应器 / h and 2.5 NL / L 反应器 / h, which corresponds to 0.6 NL / L 反应器 / day and 60 NL / L 反应器 / day. Preferably, the flow rate of the off-gas is 2.5 NL / L 反应器 / h, which corresponds to 60 NL / L 反应器 / day.

[0082] Advantageously, the method according to the invention may further comprise step d’) of supplementing the culture medium with nutrients. This step makes it possible to compensate for the decrease in the concentration of nutrients in the culture medium, these nutrients being consumed by the microorganisms during the methane production in step d) of the method.

[0083] A second object of the invention is a device 1 for non-in-situ methane production, comprising:

[0084] - a gas / liquid bioreactor 11, which comprises a container 12, a gas inlet 13, a gas outlet 14, a liquid inlet 15 and a liquid outlet 16, at least one methanogenic microorganism 121 immobilized on an optionally immobilized culture substrate 122 and a culture medium 123;

[0085] - a gas injection device 17 for injecting the influent gas into the continuous liquid phase 123 comprised in the gas / liquid bioreactor 11;

[0086] - a liquid injection device 18 for injecting the continuous liquid phase 123 into the gas / liquid bioreactor 11;

[0087] - a recovery device 19 for recovering the off-gas from the gas / liquid bioreactor 11.

[0088] Where applicable, the above definitions also apply to the device.

[0089] Advantageously, the volume of the bioreactor container 12 can be comprised between 0.01 m 3 and 150 m 3 . Preferably, for an agricultural installation, the volume of the container 12 of the bioreactor 11 can be less than 50 m 3 , and for an industrial installation, the volume of the container 12 of the bioreactor 11 can be greater than 50 m 3 . More preferably, for an industrial installation, the volume of the container 12 of the bioreactor 11 can be 100 m 3The height / diameter ratio of the container 12 may be included in the range of 10:1 to 10:3.

[0090] The "gas injection device" refers to any device for injecting an inflowing gas into the continuous liquid phase contained in the gas / liquid bioreactor.

[0091] Advantageously, the gas injection device 17 may be selected from microbubble diffusers, such as porous bottom column diffusers, perforated pipes, porous membranes made of polymer or ceramic materials, baffle bubble columns, or may be selected from bubbleless membrane contactors, such as hollow fiber membranes, or may be selected from water injectors or static mixers. Preferably, the gas injection device 17 may be a porous bottom column diffuser. The gas / liquid bioreactor 11 may be configured to continuously supply the inflowing gas, particularly during the processes of step d) and step e). The gas injection device 17 is connected to the gas inlet 13 of the bioreactor 11.

[0092] In this text, "connected" means a direct connection or an indirect connection between two elements of a device.

[0093] The "liquid injection device" refers to any device for injecting the continuous liquid phase into the gas / liquid bioreactor. The liquid injection device 18 is connected to the liquid inlet 15 of the bioreactor 11.

[0094] Advantageously, the device according to the present invention may include a liquid recirculation loop 24 connected to the liquid outlet 16 and connected to the liquid inlet 15. The recirculation loop may be connected to a purging device 20. The liquid recirculation loop may include a pump 23 and the liquid injection device 18. The pump may be a peristaltic pump.

[0095] Advantageously, the liquid injection device 18 is selected from porous static dispersion systems (such as coronal or comb-shaped). Preferably, the liquid injection device 18 may be comb-shaped porous.

[0096] Advantageously, the device 1 may further include a purging device 20 for purging the continuous liquid phase included in the gas / liquid bioreactor 11. The purging device 20 is connected to the liquid outlet 16 of the bioreactor 11.

[0097] The gas / liquid bioreactor 11 may be configured such that the nutrient input and / or purging of the culture medium is carried out continuously or discontinuously.

[0098] The "recovery device" refers to any device for recovering the outflowing gas from the gas / liquid bioreactor.

[0099] Advantageously, the recovery device 19 can be selected from a simple gas outlet, a gas outlet with a condenser (especially to eliminate the residual water vapor contained in the effluent gas), and an outlet associated with a recirculation system for the effluent gas. Preferably, the recovery device 19 can be an outlet associated with a recirculation system for the effluent gas. The gas / liquid bioreactor 11 can be configured such that the effluent gas is recovered on a continuous basis. The recovery device 19 is connected to the gas outlet 14 of the bioreactor 11. The water vapor condensate can be reinjected into the bioreactor 11 or extracted from the device 1. Since the methanation reaction produces water, there is the advantage of being able to regulate the amount of water in the device 1.

[0100] Advantageously, the device 1 can include a gas outlet 14 equipped with a condenser.

[0101] Advantageously, the device 1 can include a gas outlet 14 equipped with instrumentation.

[0102] Advantageously, the device 1 can include a gas circuit connected to an analyzer, preferably an analyzer of chromatographic type.

[0103] Advantageously, the device 1 can include a gas recirculation circuit from the upper part to the lower part of the bioreactor 11.

[0104] Advantageously, the device 1 can include a mixer for the recirculated gas and the incoming gas.

[0105] Advantageously, the device 1 can include a set of detectors for verifying (directly in the bioreactor 11) the concentration of the incoming gas, the redox potential, or the pH in the culture medium 123. The pH detector is an advantageous way to measure the temperature.

[0106] Advantageously, the device 1 can include gas cylinders to allow the supply of the incoming gas. The gas cylinders can be connected to the gas injection device 17. The gas injection device 17 can also include a mixer to control the CO2 / H2 ratio of the incoming gas. The mixer can be the same as or different from the mixer for the recirculated gas and the incoming gas.

[0107] "Bottle" refers to a gas storage device; the storage device can be replaced by any other compatible storage device.

[0108] Advantageously, the device 1 can include a set of flow meters, preferably mass flow meters. These mass flow meters allow the regulation of the inlet flow rate of the incoming gas.

[0109] Advantageously, the device 1 can include a gas flow meter.

[0110] Advantageously, the device 1 can include a sampling device. For example, the sampling device can collect liquid samples for compound analysis and monitor the progress of the method.

[0111] Advantageously, the device 1 may include means for sampling the composition of the effluent gas mixture for analysis of compounds.

[0112] Advantageously, the device 1 may include a set of valves.

[0113] Advantageously, the device 1 may include a pH adjustment system.

[0114] Advantageously, the device 1 may include sub-devices housed in a container so that they can be separated by a grid. Preferably, the sub-devices are present in cases where several different types of culture substrates are used.

[0115] The present invention also relates to a kit which, once assembled, gives access to the device 1 according to the invention, and which comprises:

[0116] - a substrate 122;

[0117] - at least one methanogenic microorganism 121;

[0118] - an optional gas / liquid bioreactor 11;

[0119] - an optional gas injection device 17;

[0120] - an optional liquid injection device 18;

[0121] - an optional recovery device 19.

[0122] The present invention also relates to a methanation catalyst comprising the substrate 122 obtained in step a) of the method according to the invention. The catalyst comprises at least one methanogenic microorganism 121 immobilized on the culture substrate 122.

[0123] Another object of the present invention relates to the use of the strain Methanothermobacter marburgensis CLERMONT DSM 34405 for the production of methane.

[0124] Another object of the present invention relates to the use of the strain Methanothermobacter marburgensis CLERMONT DSM 34405 for the implementation of a biomethanation process.

[0125] With reference to the accompanying drawings, other advantages will become apparent from the following examples, which are given for illustrative purposes and not for the purpose of exhaustion. Description of the Drawings

[0126] Figure 1 Figure 1 There is shown a device 1 for the production of methane according to the invention, which comprises:

[0127] ​​- A gas / liquid bioreactor 11, which comprises a container 12, a gas inlet 13, a gas outlet 14, a liquid inlet 15 and a liquid outlet 16, at least one methanogenic microorganism 121 fixed on a culture medium substrate 122, a culture medium 123, a gas injection device 17, a gas recovery device 19 and a liquid recirculation loop 24 connected to the liquid inlet 15 and the liquid outlet 16, wherein the liquid recirculation loop 24 comprises a pump 23 and a liquid injection device 18 for injecting the continuous liquid phase 123 into the gas / liquid bioreactor 11;

[0128] - Two inflow gas sources, which are connected to the gas injection device 17 through two valves 21;

[0129] - A chromatograph 22, which is connected to the recovery device 19 through a valve 21;

[0130] - A purge device 20, which is connected to the recirculation loop 24 and comprises a valve 21.

[0131] Figure 2 Figure 2 Shows the variation of the growth of bacteria and archaea with time in a stirred bioreactor under the conditions of 100 RPM and 55 °C, supplemented with H2 and CO2, and in the presence of a modified basal BA medium, in the absence (■ without culture medium substrate) or in the presence of a polyethylene culture medium substrate with a low filling rate (● FR_low) or a high filling rate (▲ FR_high).

[0132] Figure 3 Figure 3 Shows the variation of the total number of archaea and bacteria under the conditions of absence (without culture medium substrate, left) or presence of a polyethylene culture medium substrate with a low filling rate (● FR_low, middle) or a high filling rate (▲ FR_high, right). Cultivation was carried out in a stirred bioreactor under the conditions of 100 RPM and 55 °C, supplemented with H2 and CO2, and in the presence of a modified basal BA medium.

[0133] Figure 4 Figure 4 Is an image of the methanation catalyst in a polyethylene biochip at day 0 of the growth of Methanothermobacter marburgensis Clermont DSM 34405 in a modified BA medium under the conditions of 55 °C and supplemented with H2 and CO2, taken by SEM.

[0134] Figure 5 Figure 5 Is an image of the methanation catalyst in a polyethylene biochip at day 14 of the growth of Methanothermobacter marburgensis Clermont DSM 34405 in a modified BA medium under the conditions of 55 °C and supplemented with H2 and CO2, taken by SEM.

[0135] ​​​​​​​​​Figure 6 Figure 6 It is an image of the methanation catalyst in a polyethylene biochip on the 34th day of the growth of Methanothermobacter marburgensis Clermont DSM 34405 in a modified BA medium under the conditions of 55 °C and supplemented with H2 and CO2, taken by SEM. Example

[0136] Other advantages, objectives and specific features of the present invention will become apparent from the following examples, which are for illustrative purposes and not for restrictive purposes.

[0137] In the following examples, different parameters were measured using the techniques described in detail below:

[0138] Measurement of the tolerance of at least one methanogenic microorganism to exposure to oxygen (O2)

[0139] Several exposures to open air were carried out in the enclosed space of a laminar flow hood (PSMIICytosafe, Faster) for 1 minute, 10 minutes, 30 minutes, 60 minutes and 180 minutes.

[0140] During each exposure, 3 mL of a culture medium containing at least one methanogenic microorganism in the exponential growth phase was inoculated into three vials containing 30 mL of a culture medium containing at least one methanogenic microorganism. To restore anaerobic life, the vials were purged with an H2 / CO2 mixture to remove O2, and then the pressure was adjusted to 1.5 bar. The cultures were incubated at 55 °C for 10 days. The ability to regrow after exposure to O2 was monitored by CH4 dosage and spectrophotometry (Cell Density Meter, Fisherbrand).

[0141] Measurement of the gas phase composition

[0142] The gas phase composition was continuously analyzed by a gas chromatograph (Agilent analyzer, Technologies) equipped with two separation modules and a thermal conductivity detector (TCD). Module A was equipped with molecular sieve to separate hydrogen, nitrogen, oxygen and methane under an argon flow. Module B was equipped with a PoraPlot U (Agilent) column to separate carbon dioxide and hydrogen sulfide under a hydrogen flow. A hydrophobic filter was placed at the inlet of the analyzer to protect it from humidity. The gas analysis of the bioreactor was carried out once an hour.

[0143] Measurement of metabolite content and organic acid content

[0144] ​The contents of metabolites and organic acids were analyzed by a high-performance liquid chromatograph (HPLC Agilent Technologies, 1260 Infinity). Separation was provided by two serially mounted ion exclusion columns (Rezex ROA 300x7, 8 nm, Phenomenex, USA) heated to 50 °C. The detector used for the identification of different compounds was a refractometer (HP 1100 series). The liquid phase consisted of a sulfuric acid solution (2 mM) and was circulated at a rate of 0.7 mL / min. Before injection, the samples were deproteinized. A 1 mL volume of the culture sample was taken, mixed with 125 μL of Ba(OH)2·8H2O (0.3 M) and 125 μL of ZnSO4·7H2O (5 wt / vol %) and centrifuged (5 min; 10,000 g). The supernatant was then filtered through a 0.22 μm membrane before injection and transferred to an HPLC vial for storage at 4 °C.

[0145] Measurement of the growth of microorganisms

[0146] Changes in the growth of microorganisms were monitored at 600 nm by a spectrophotometer (Uvisco, V-1800).

[0147] Microscopic observation

[0148] Collect 0.5 ml volume of the culture on the BA (basic anaerobic) medium described and modified by Bu et al. (Reference 1), and fix it with formaldehyde solution (2%, final concentration). Then load the cells onto a 400-mesh carbon-coated copper grid (Formvar, Pelanne Instruments, France) by centrifugation at 18000 g (20 minutes, 14 °C), and then stain with 20 μL of 2% uranyl acetate. After rinsing with distilled water and drying on absorbent paper, observe using a JEOL 2100plus transmission electron microscope (TEM) (Akishikma, Tokyo, Japan, UCA Partner CYSTEM platform) equipped with a Gatan CMOSRIO 9 camera (Gatan Ametek, Pleasanton, USA) at an accelerating voltage of 80 kV. Fix the samples in the culture matrix with 2.5% glutaraldehyde and 0.15% ruthenium red solution, and incubate overnight at 4 °C in 0.2 M sodium cacodylate buffer at pH 7.4. After rinsing in the same buffer (3 x 10 minutes), post-fix for 1 hour at room temperature with a solution of 1% osmium tetroxide in 0.2 M sodium cacodylate buffer at pH 7.4. Then rinse the samples with distilled water for 20 minutes, dehydrate in an ethanol bath with increasing degrees (25° to 100°), 10 minutes / bath. The final dehydration step is to dehydrate with a mixture of 100° ethanol V / V and hexamethyldisilazane for 10 minutes, followed by dehydration with pure hexamethyldisilazane (evaporate overnight under a fume hood). Then place the samples on a metal pad with a double-sided carbon tape. Perform chromium metallization (5 nm) using a Quorum Q150 TES Plus metallizer. Observe using a Hitachi Regulus 8230 scanning electron microscope at an accelerating voltage of 1 kV using a secondary electron detector.

[0149] Example 1: Evaluation of the tolerance of Methanothermobacter marburgensis strain Clermont DSM 34405 to exposure to oxygen (O2)

[0150] By screening hydrogenotrophic microorganisms, the consortium was simplified from the methanation unit (Ennezat, 63), enabling the isolation of new methane-overproducing methanogenic strains.

[0151] To isolate the archaeal strain with the highest abundance in the bioreactor, a 50 mL sample was extracted from the aggregates of the digestate from the methanation unit (Ennezat, 63) under sterile and anaerobic conditions. In some cases, the sample was cultured for 6 weeks at a temperature of 55 °C in the presence of a H2 / CO2 mixture at a ratio of 4:1 under the screening conditions for methanogenic hydrogenotrophic strains. After incubation at 55 °C (bioreactor temperature) for 72 hours, it was transplanted into a modified BA medium (10% inoculum: 0.5 mL inoculum was inoculated into 5 mL medium). Serial dilutions (from 10-1 to 10-10) were carried out according to the physico-chemical conditions of the bioreactor (55 °C, 1.5 bar, pH = 7.5). The last positive dilution tube (turbid and producing CH4) was used as the inoculum for the next series. Growth was monitored by spectrophotometry (Cell Density Meter, Fisherbrand) and gas measurement by microchromatography (Agilent analyzer, Technologies) equipped with two separation modules and a thermal conductivity detector (TCD). Module A has molecular sieve, which separates hydrogen, nitrogen, oxygen and methane under an argon flow. Module B is equipped with a PoraPlot U (Agilent) column, which can separate carbon dioxide and hydrogen sulfide in the gas phase under a hydrogen flow. Then the roll-tube method was used for separation on a solid medium (Hungate, 1969). For this purpose, 0.1 g of agar (BactoTM Agar) was added to 5 mL of the modified BA medium (Hungate tube) under a N2 flow, and then autoclaved. Then the agar medium was liquefied in a water bath at 100 °C and then placed at 45 °C to slow down its gelation. After inoculation with the last series of positive dilutions in the medium, the agar medium was evenly placed in a thin layer on the inner side of the test tube by the rotational movement of the Spinner tube and using ice cubes on the outer surface to solidify the medium. Then the test tube was incubated at 55 °C in a vertical position for 168 hours to avoid contamination of the colonies by condensation. The test tube showed the presence of macroscopic parental microbial entities (colonies) containing a large number of identical microorganisms and CH4 production. The test tube was opened in a controlled atmosphere (atmospheric pressure) anaerobic culture chamber, and the colonies were collected using a sterile Pasteur pipette. The colonies were resuspended in 5 mL of medium. The purity of the liquid culture was confirmed by multiple serial dilutions (10-1 to 10-5) and light microscopy (Labophot, Nikon). Analysis could be carried out on the isolated strains.

[0152] Table 1: Effects of oxygen exposure on the growth and metabolic activity of Methanothermobacter marburgensis isolate CLERMONT DSM 34405

[0153] [Table 1]

[0154]

[0155] Exposed to oxygen according to the above method, exposure for 0 minutes to 60 minutes does not seem to have a significant effect on the growth, hydrogenotrophy, and methanogenic activity of Methanothermobacter marburgensis strain CLERMONT DSM 34405. After 180 minutes of exposure to oxygen, a slight growth retardation was observed (Table 1). Since the methanogenic performance of Methanothermobacter marburgensis strain CLERMONT DSM 34405 is not affected or only slightly affected by oxygen and even air, different modes of subculture from step a) to step b) can be carried out under non-anaerobic conditions without affecting steps c), d), and e).

[0156] Example 2: Evaluation of the methanation catalyst from step a) according to the method of the present invention

[0157] The in-situ methane production method implemented includes the following steps:

[0158] a) Contacting the aggregates, the polyethylene biochip matrix, and the culture medium.

[0159] The aggregates from the methanation unit (Ennezat, 63) are taken out from the anaerobic digester. The liquid or digest sample is sieved (5 mm) to remove solids >5 mm and frozen.

[0160] The culture medium used is derived from the BA medium described by Bu et al., 2018, and its composition is shown in Table 2. The culture medium is adjusted to pH 7.5 with 3M NaOH solution.

[0161] Table 2: List of compounds in the modified BA medium.

[0162] [Table 2]

[0163] Compound Concentration (g / L) <![CDATA[NH4Cl]]> 1,00 NaCl 0,10 <![CDATA[MgCl2·6H2O]]> 0,10 <![CDATA[CaCl2·2H2O]]> 0,05 <![CDATA[Na2S 9H2O]]> 1,0 <![CDATA[K2HPO4·3H2O]]> 0,40 Resazurin 0,0005 L-Cysteine 0,5 <![CDATA[FeCl2·4H2O]]> 0,002000 <![CDATA[H3BO3]]> 0,000050 <![CDATA[ZnCl2]]> 0,000050 <![CDATA[CuCl2·2H2O]]> 0,000038 <![CDATA[MnCl24H2O]]> 0,000050 <![CDATA[(NH4)6Mo7O 24 ·4H2O]]> 0,000050 <![CDATA[AlCl3]]> 0,000050 <![CDATA[CoCl2·6H2O]]> 0,000050 <![CDATA[NiCl2·6H2O]]> 0,000092 EDTA 0,000500 <![CDATA[Na2SeO3 5H2O]]> 0,000100 Biotin 0,00000200 Folic Acid 0,00000200 Pyridoxal Hydrochloride 0,00001000 Thiamine Hydrochloride 0,00000500 Riboflavin 0,00000500 Nicotinic Acid 0,00000500 Calcium D-Pantothenate 0,00000500 Cyanocobalamin 0,00000010 p-Aminobenzoic Acid 0,00000500 Lipoic Acid 0,00000500

[0164] The colonization kinetics of Methanothermobacter marburgensis strain CLERMONT DSM 34405 on the polyethylene biochip matrix was monitored by SEM imaging (FEG SEM, Hitachi Regulus 8230). When the microorganisms associated with the surface switch from the reversible attachment mode (which can be loosened without affecting integrity) to the irreversible attachment mode, the process begins, followed by cell aggregation and its subsequent proliferation. The cells of the biofilm are encapsulated in a matrix of EPS (extracellular polymeric matrix)-type polymers. Through SEM observation, EPS particles, the matrix, cells, and a wireframe structure called fibraeum can be seen.

[0165] Figure 4 , Figure 5 and Figure 6 showed that the strain of Methanothermobacter marburgensis CLERMONT DSM 34405 grew over time when placed in the presence of a polyethylene biochip culture substrate.

[0166] Furthermore, when the strain of Methanothermobacter marburgensis CLERMONT DSM 34405 was placed in the presence of the culture substrate according to the present invention and in the presence of hydrogen and carbon dioxide, its growth seemed to be more efficient. In fact, in the presence of the culture substrate, the optical density of the sample decreased over time ( Figure 2 ).

[0167] Furthermore, the analysis of microbial growth by spectrophotometry (UVisco, V - 1800) at 600 nm showed that in the presence of a polyethylene biochip culture substrate, the number of microorganisms increased threefold after 24 days of colonization ( Figure 3 ).

[0168] The conclusion drawn from these results is that the method according to the present invention can achieve better yields in methanation. This is because the microorganisms grow better when they are attached to the culture substrate.

[0169] Example 3: Equipment for implementing the method according to the present invention

[0170] The non - in - situ methane production method implemented according to the present invention further comprises the following steps:

[0171] b) introducing the mixture obtained in step a) (Example 2) into a gas / liquid bioreactor;

[0172] c) contacting the influent gas in the gas / liquid bioreactor obtained in step b);

[0173] d) reacting the influent gas with at least one methanogenic microorganism;

[0174] e) recovering the effluent gas obtained in step d);

[0175] characterized in that

[0176] - the culture substrate is not immobilized in the gas / liquid bioreactor; and

[0177] - at least one methanogenic microorganism is attached to the culture substrate.

[0178] In this example, Figure 1 the described equipment enables the method according to the present invention to be implemented.

[0179] The bioreactor in stage b) was custom-built from a section of a 2 mm thick 316 stainless steel cylinder. It had an inner diameter of 54 mm and a height of 1400 mm. The height-to-diameter ratio of the bubble column gas / liquid bioreactor was 25.9. The vessel volume was 3.5 L. A glass or stainless steel sintered gas distributor with a known porosity was connected to the reactor using a GL18 three-ring joint. The gas outlet passed through an opening at the top of the column and was connected to the gas circuit via a 1 / 4'G thread. The gas outlet of the reactor was connected to a condenser, where the water circulation temperature was 4 °C (Lauda Eco RE1225 silver). A high-precision volumetric flowmeter (Bioprocesscontrol / Microflow 1100 - 3100) measured the gas flow leaving the condenser. The gas was then analyzed by gas chromatography (Agilent analyzer, Technologies). A medium recirculation system based on a peristaltic pump (AB pump, type PSF2) allowed the liquid to circulate against the direction of bubble rise at a volumetric rate of 10 L / L 反应器 / hour, which was equivalent to 0.166 L / L 反应器 / minute. The liquid recirculation loop included electrodes for measuring redox potential, pH, and temperature (Mettler Toledo INPRO4260i / SG / 12052005381). If necessary, the pH was adjusted to 7.5 by adding an acidic or basic titrant. The temperature was controlled at 55 °C using a thermostatic bath (Lauda Eco RE1225 silver). The recirculation system was equipped with a diaphragm for sampling, adding stock solutions (medium components, Na2S 9H2O), or withdrawing medium volume.

[0180] The inflowing gas in step c) was hydrogen and carbon dioxide. As shown in Table 3 below, it was injected into the bioreactor through a mass flowmeter (Brooks instrument, SLA5800). The total gas flow rate of CO2 gradually increased from 3.61 NL / L 反应器 / day to 18.21 NL / L 反应器 / day, and the total gas flow rate of H2 gradually increased from 14.37 NL / L 反应器 / day to 72.85 NL / L 反应器 / day. If necessary, the value of the H2 / CO2 ratio could be adjusted to 4:1 ± 0.1. The system pressure was 1 bar. The gas mixture was continuously dispersed by a diffuser against the flowing phase of the aqueous phase.

[0181] The effluent gas from step e) mainly consists of methane and water vapor (the water vapor is re-condensed). As shown in Table 3, the total effluent rate of the effluent gas recovered from the bioreactor gradually increases from 5.52 NL / L / day to 18.67 NL / L / day.

[0182] Table 3: Specific inflow rates of CO2 and H2 and specific methane production (expressed in NL / L 反应器 / day) as a function of cultivation time (expressed in days).

[0183] [Table 3]

[0184]

[0185] In view of the results of Example 2, the method according to the invention implemented by the device in Figure 1 has a methane production rate superior to that of conventional processes. This is because the increase in pressure in the system increases the solubility of hydrogen in the culture medium. The presence of a substrate colonized by at least one methanogenic microorganism increases the amount of catalyst per unit reactor volume. The at least one methanogenic microorganism cultured is screened according to its methane-producing ability. The presence of a substrate colonized by at least one methanogenic microorganism increases the residence time of the gas in the culture medium and promotes the solubility of hydrogen in the liquid. This improves the performance of the system by 3.6 times.

[0186] References

[0187] Reference 1: Bu, F.; Dong, N.; Kumar Khanal, S.; Xie, L.; Zhou, Q. Effects of CO on Hydrogenotrophic Methanogenesis under Thermophilic and Extreme-Thermophilic Conditions: Microbial Community and Biomethanation Pathways. Bioresource Technology 2018, 266, 364 - 373, doi:10.1016 / j.biortech.2018.03.092.

Claims

1. A method for off-site methane production, comprising the following steps: a) contacting at least one methanogenic microorganism, a culture substrate, and optionally a first culture medium; and b) introducing the mixture obtained in step a) into a gas / liquid bioreactor optionally containing a second culture medium; c) contacting an influent gas in the gas / liquid bioreactor obtained in step b); d) reacting the influent gas with at least one methanogenic microorganism; e) recovering the effluent gas obtained in step d); characterized in that - the culture substrate is optionally immobilized in the gas / liquid bioreactor; and - at least one methanogenic microorganism is immobilized on the culture substrate.

2. The method according to claim 1, wherein the influent gas is carbon dioxide CO2 and hydrogen H2, preferably, the H2 / CO2 volume ratio of the influent gas is included in the range of 3:1 to 5:1, more preferably 4:

1.

3. The method according to any one of the preceding claims, wherein the at least one methanogenic microorganism is a consortium or a pure methanogenic strain.

4. The method according to any one of the preceding claims, wherein the at least one methanogenic microorganism includes microorganisms belonging to the phylum Euryarchaeota and is selected from the classes Methanobacteria, Methanococci, Methanopyri, or Methanomicrobia.

5. The method according to any one of the preceding claims, wherein the at least one methanogenic microorganism includes at least one strain of the genus Methanothermobacter, preferably a strain of Methanothermobacter marburgensis CLERMONT DSM 34405.

6. The method according to any one of the preceding claims, wherein the gas / liquid bioreactor (11) is selected from pneumatically stirred reactors with a downward liquid circulation.

7. The method according to any one of the preceding claims, wherein the culture substrate (122) includes a porous cube of polyurethane foam impregnated with powdered activated carbon, a rigid polyethylene biochip, sepiolite, volcanic ash, and / or porous glass.

8. An apparatus (1) for off-site methane production, comprising: - a gas / liquid bioreactor (11), which includes a container (12), a gas inlet (13), a gas outlet (14), a liquid inlet (15), and a liquid outlet (16), at least one methanogenic microorganism (121) immobilized on an optionally immobilized culture substrate (122), and a culture medium (123); - a gas injection device (17) for injecting an influent gas into the continuous liquid phase (123) contained in the gas / liquid bioreactor (11); - a liquid injection device (18) for injecting the continuous liquid phase (123) into the gas / liquid bioreactor (11); - a recovery device (19) for recovering the effluent gas of the gas / liquid bioreactor (11).

9. The apparatus according to claim 8, wherein the gas / liquid bioreactor (11) is selected from a bubble column, a mechanically stirred column, a continuous stirred tank reactor (CSTR), an airlift reactor, a submerged fixed bed reactor, a spray bed reactor, a pneumatically stirred reactor with a downward liquid circulation.

10. The device according to claim 8 or 9, wherein the gas / liquid bioreactor (11) is selected from pneumatically stirred reactors and has a downward liquid circulation.

11. The device according to any one of claims 8 to 10, wherein the culture substrate (122) is an organic or inorganic culture substrate of natural or synthetic origin.

12. The device according to any one of claims 8 to 11, wherein the culture substrate (122) comprises a porous cube of polyurethane foam impregnated with powdered activated carbon, a rigid polyethylene biochip, sepiolite, volcanic ash, and / or porous glass, preferably a porous cube of polyurethane foam impregnated with powdered activated carbon and / or a rigid polyethylene biochip.

13. The device according to any one of claims 8 to 12, wherein the at least one methanogenic microorganism (121) comprises at least one strain of the genus Methanothermus, preferably a strain of Methanothermus marburgensis CLERMONT DSM 34405.

14. A kit for obtaining the device (1) according to any one of claims 8 to 13, comprising: - a substrate (122); - at least one methanogenic microorganism (121); - an optional gas / liquid bioreactor (11); - an optional gas injection device (17); - an optional liquid injection device (18); - an optional recovery device (19).

15. The kit according to claim 14, wherein the culture substrate (122) comprises a porous cube of polyurethane foam impregnated with powdered activated carbon, a rigid polyethylene biochip, sepiolite, volcanic ash, and / or porous glass.

16. Use of a strain of Methanothermus marburgensis CLERMONT DSM 34405 for the production of methane.

17. A methanation catalyst comprising at least one methanogenic microorganism (121) immobilized on a substrate (122), preferably the methanogenic microorganism is a strain of Methanothermus marburgensis CLERMONT DSM 34405.

Images