Ex-situ biomethanation method

Abstract

The present invention relates to an ex-situ biomethanation method as well as to the device for implementing same. The invention relates to an ex-situ methane production method comprising the steps of placing in contact at least one methanogenic microorganism, a culture substrate and optionally a first culture medium in a gas / liquid bioreactor optionally comprising a second culture medium and producing methane by the reaction of incoming gases with the at least one methanogenic microorganism, characterised in that the culture substrate is optionally immobilised in the gas / liquid bioreactor; and the at least one methanogenic microorganism is fixed to the culture substrate; as well as to the device for carrying out the method.

Description

DESCRIPTION Title: EX-SITU BIOMETHANATION PROCESS Technical field of the invention

[0001] The present invention relates to an ex-situ biomethanation process and to a device enabling its implementation.

[0002] The invention relates to a method for producing methane ex-situ comprising the steps of bringing into contact at least one methanogenic microorganism, a culture support and optionally a first culture medium in a gas / liquid bioreactor optionally comprising a second culture medium and the production of methane by the reaction of incoming gases with the at least one methanogenic microorganism, characterized in that the culture support is immobilized or not in the gas / liquid bioreactor; and at least one methanogenic microorganism is attached to the culture support; as well as the device for implementing the method. Prior art

[0003] Methanation is a reaction of synthesis of methane from dihydrogen and carbon dioxide.

[0004] For several years, methanation has been directly associated with the development of wind and solar energy, which depends on the ability to massively store electricity produced but not consumed. The conversion of electricity into gas, also called Power-to-gas, is a promising solution for converting this excess electricity into hydrogen by electrolysis of water. However, since the hydrogen sector is still under construction, the conversion of this hydrogen into methane by methanation makes it possible to massively store this energy using existing gas infrastructures; this is the concept of power-to-methane. The other advantage of methanation is that it captures and stores carbon dioxide during this conversion, thus reducing the environmental impacts linked to carbon dioxide emissions (biogas from methanization, syngas obtained by pyrolysis or gasification, combustion gas effluent or equivalents).This double advantage which characterizes methanation makes it a technology of the future.

[0005] There are two competing routes for methanation: the catalytic route, which has struggled to become industrialized for many years, and the biological route, which is more robust (with respect to impurities such as CO, NH3, H2S, etc.) and presents a lower environmental footprint, particularly through reduced energy costs.

[0006] However, to date, no in situ or ex-situ biomethanation technology is yet mature or economically viable, and the various dedicated processes require further investigation.

[0007] The major drawback of the biological route lies in the kinetics of methane production, which is slower than the catalytic route. This kinetic barrier results partly from a limitation by the physicochemical processes of transfer of matter from gaseous dihydrogen to the liquid phase. Conventional solutions consist of increasing the pressure and / or intensifying agitation, which can have an antagonistic effect on biological processes due to the sensitivity to mechanical stress of microorganisms, and in particular of hydrogenotrophic methanogenic archaea, and can reduce their productivity. There is a need for the industrialization of a biological process with an acceptable energy cost and therefore a reduced economic cost. Statement of the invention

[0008] The present invention makes it possible to obtain an ex-situ biomethanation process and a device which surprisingly resolve the drawbacks of known biological processes and are an interesting alternative to catalytic processes which are difficult to industrialize. The present invention thus makes it possible to increase the robustness and longevity of the process at lower pressure and temperature compared to the catalytic route, but also to increase the methane content of the outgoing gas, and thus the conversion efficiency, productivity and robustness of the biological processes by reducing the need for external energy input compared to known prior art technologies.

[0009] A first subject of the present invention is a process for the ex-situ production of methane comprising the following steps: a) bringing into contact at least one methanogenic microorganism, a culture support and optionally a first culture medium; b) introducing the mixture obtained in step a) into a gas / liquid bioreactor optionally comprising a second culture medium; c) bringing incoming gases into contact with the gas / liquid bioreactor obtained in step b); d) reacting the incoming gases with the at least one methanogenic microorganism; e) recovery of outgoing gases obtained in step d); characterized in that - the culture medium is immobilized or not in the gas / liquid bioreactor; and - at least one methanogenic microorganism is attached to the culture medium.

[0010] Advantageously, steps a) and b) may occur sequentially or simultaneously.

[0011] Advantageously, steps c), d) and e) can occur sequentially and / or simultaneously.

[0012] By "at least one methanogenic microorganism" is meant herein a consortium of microorganisms comprising at least one methanogenic strain or one pure methanogenic strain.

[0013] Advantageously, the at least one methanogenic microorganism may comprise a microorganism belonging to the Euryarchaeota phylum and may be chosen from the classes Methanobacteria, Methanococci, Methanopyri or Methanomicrobia. Preferably, the at least one methanogenic microorganism may comprise at least one strain of Methanothermobacter. Reference to deposited biological material

[0014] In a particularly advantageous embodiment of the invention, the at least one methanogenic microorganism is the strain of Methanothermobacter marburgensis CLERMONT deposited according to the Budapest Treaty on October 20, 2022; with the DSMZ (Leibniz Institute Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrafte 7B 38124 Braunschweig GERMANY) under the number DSM 34405.

[0015] The at least one methanogenic microorganism may be included in a preculture liquid. The preculture medium may be the same as or different from the first culture medium.

[0016] The term "culture medium" means a medium that allows its colonization by hydrogenotrophic and / or methanogenic microorganisms. The medium may be in the form of beads, chips, pebbles, gel, foam, pellets, rings, towers or biochips. Preferably, the culture medium may be spherical or pseudospherical.

[0017] Advantageously, the culture medium can be an organic or inorganic medium, of natural or synthetic origin.

[0018] Advantageously, the organic culture medium of natural origin can be chosen from: alginate, K-carrageenan, chitosan, sawdust, straw, charcoal, vegetable fibers, corn cob, bagasse, rice, sunflower seed husks, diatomite, mycelium and a mixture of these.

[0019] Advantageously, the organic culture medium of synthetic origin may be a polymer. The polymer may be expanded or unexpanded. The polymer may be chosen from: polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyurethane, polyacrylonitrile, polyvinyl alcohol, polyamide (PA), and polylactic acid (PLA) and a mixture thereof. Preferably, the organic culture medium of synthetic origin is an expanded polymer. Preferably, the culture medium may be chosen from porous cubes of polyurethane foam impregnated with powdered activated carbon and / or rigid polyethylene biochips.

[0020] Advantageously, the inorganic culture medium, of natural or synthetic origin, may be chosen from: magnetite, volcanic rocks, vermiculite, porous glass, silica-based materials, ceramics, nanoparticles and a mixture thereof. Preferably, the culture medium may be chosen from sepiolite, pozzolan and / or porous glass (for example, expanded glass which may be marketed under the name Poraver (registered trademark)). More preferably, the culture medium may be chosen from sepiolite, pozzolan and / or porous glass and be spherical or pseudo-spherical in shape.

[0021] Advantageously, the culture medium can be chosen from porous cubes made of polyurethane foam impregnated with powdered activated carbon, rigid polyethylene biochips, sepiolite, pozzolan and / or porous glass.

[0022] By "immobilized or not" is meant in this case in the case "immobilized" means that all the elements of the solid support are without relative movement between them and in relation to the body of the reactor (case of a descending liquid flow, of an ascending liquid flow at low speed for a solid of density greater than that of the liquid, or of solid supports whose movement is mechanically blocked, for example by means of a grid). Conversely, "non-immobilized" means that the solid supports are in suspension in a liquid flow ascending, in “fluidized bed” mode, moving relative to each other and relative to the reactor body, but that the net speed of all the solid supports is zero relative to the reactor body.

[0023] By "at least one methanogenic microorganism is attached to the culture medium" is meant herein that the microorganism is not free in its environment (culture medium or other) and that when the at least one methanogenic microorganism attached to the medium is brought into contact with a culture medium, a system with two distinct phases appears. Generally, from 0.01 to 100% (by mass) of the at least one methanogenic microorganism is attached to the medium, preferably from 60 to 100% and more preferably from 80 to 100%.

[0024] By "culture medium" is meant a culture medium in which the at least one microorganism can be maintained to generate a mixture of gases, and into which incoming gases will be injected and dissolved, whether or not this medium allows the production of biomass.

[0025] 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 the microorganisms or for the growth of the microorganisms and for microbial activity.

[0026] Advantageously, the culture medium may be a continuous liquid phase. For the purposes of the present invention, the term "continuous liquid phase" means a volume of liquid having physical continuity, as opposed to a discontinuous liquid volume consisting of a set of liquid phases without contact with each other, such as liquid drops percolating in a gas phase.

[0027] Advantageously, the pH of the culture medium may be in a range from 7 to 9. Preferably, the pH of the culture medium may be 8.

[0028] Advantageously, at least one of steps a) or b) may use at least one culture medium. The contacting of step a) may be carried out between at least one methanogenic microorganism and a culture support before the mixture obtained is introduced in step b) into a gas / liquid bioreactor comprising a culture medium. The contacting of step a) may be carried out between at least one methanogenic microorganism, a culture support and a culture medium before the mixture obtained is introduced in step b) into a gas / liquid bioreactor not yet comprising a culture medium. The contacting of step a) can be carried out between at least one methanogenic microorganism, a culture support and a first culture medium before the mixture obtained is introduced in step b) into a gas / liquid bioreactor comprising a second culture medium. The first and second culture mediums may be identical or different.

[0029] Advantageously, the method according to the invention can be continuous, semi-continuous or discontinuous (batch operation).

[0030] In a first variant, the ex-situ methane production process according to the invention comprises the following steps: a) bringing at least one methanogenic microorganism into contact with a culture medium; b) introducing the mixture obtained in step a) into a gas / liquid bioreactor comprising a culture medium; c) bringing incoming gases into contact with the gas / liquid bioreactor obtained in step b); d) reacting the incoming gases with the at least one methanogenic microorganism; e) recovering outgoing gases obtained in step d); characterized in that - the culture medium is immobilized or not in the gas / liquid bioreactor; and - at least one methanogenic microorganism is attached to the culture medium.

[0031] In a second variant, the ex-situ methane production process according to the invention comprises the following steps: a) bringing at least one methanogenic microorganism, a culture support and a culture medium into contact; b) introducing the mixture obtained in step a) into a gas / liquid bioreactor; c) bringing incoming gases into contact with the gas / liquid bioreactor obtained in step b); d) reacting the incoming gases with the at least one methanogenic microorganism; e) recovering outgoing gases obtained in step d); characterized in that - the culture medium is immobilized or not in the gas / liquid bioreactor; and - at least one methanogenic microorganism is attached to the culture medium.

[0032] In a third variant, the ex-situ methane production process according to the invention comprises the following steps: a) bringing at least one methanogenic microorganism, a culture support and a first culture medium into contact; b) introducing the mixture obtained in step a) into a gas / liquid bioreactor comprising a second culture medium; c) bringing incoming gases into contact with the gas / liquid bioreactor obtained in step b); d) reacting the incoming gases with the at least one methanogenic microorganism; e) recovering outgoing gases obtained in step d); characterized in that - the culture medium is immobilized or not in the gas / liquid bioreactor; and - at least one methanogenic microorganism is attached to the culture medium.

[0033] By "gas / liquid bioreactor" is meant herein a fermenter capable of carrying out a biological reaction requiring the dissolution of at least one reagent present in the gaseous state in a culture medium.

[0034] Advantageously, the gas / liquid bioreactor may be chosen from a pneumatically stirred reactor such as a bubble column with upward or downward liquid circulation or an airlift reactor, a mechanically stirred column with upward or downward liquid circulation, a continuous flow stirred tank reactor (CSTR), a flooded fixed bed reactor, a fluidized bed reactor, a watered bed reactor. Preferably, the gas / liquid bioreactor may be chosen from a pneumatically stirred reactor, preferably with downward liquid circulation.

[0035] Advantageously, steps a) and b) of the process according to the invention can be carried out at a temperature between 25 and 70°C. Preferably, the temperature of steps a) and b) can be 55°C.

[0036] Advantageously, steps a) and b) of the process according to the invention can be carried out under a pressure of between 1 and 20 bar (absolute pressure). Preferably, the pressure of steps a) and b) can be between 1 and 3 bar. More preferably, the pressure of steps a) and b) can be 2 bar.

[0037] Advantageously, the method according to the invention may further comprise an intermediate step a'), implemented between steps a) and b) allowing the proliferation of at least one methanogenic microorganism on the support before its introduction into the gas / liquid bioreactor. The proliferation step a') may have a duration of less than or equal to 6 months. Preferably, the duration of step a') may be 30 days.

[0038] Steps a) and a') allow the formation of a methanation catalyst (support colonized by at least one methanogenic microorganism). At the end of step a), or optionally a'), the methanation catalyst may or may not be isolated from the first liquid medium before implementing step b). Thus, the "mixture resulting from step a)" may represent the mixture comprising the catalyst and the first culture medium, or the catalyst only, when the culture medium is absent from step a), or when it has been isolated from it.

[0039] Advantageously, the method according to the invention may further comprise an intermediate step b'), implemented between steps b) and c) allowing the proliferation of at least one methanogenic microorganism on the support in the gas / liquid bioreactor before the introduction of incoming gases. The proliferation step b') may have a duration of less than or equal to 3 months. Preferably, the duration of step b') may be 15 days.

[0040] Advantageously, the incoming gases of step c) of the process according to the invention may be carbon dioxide (CO2) and dihydrogen (H2). The incoming gases are not limited to CO2 and H2. They may in particular comprise any other gas, such as for example methane, carbon monoxide, nitrogen, ammonia and / or hydrogen sulfide. The H2 / CO2 volume ratio may be in a range from 2 / 1 to 6 / 1. Preferably, the H2 / CO2 volume ratio may be 4:1.

[0041] Advantageously, the flow rate of the incoming H2 from step c), d) or e) of the process according to the invention can be within a range from 0.1 to 10 NL / L r reactor / h, which is equivalent to 2.4 to 240 NL / L reactor / day. Preferably, the incoming H2 flow rate may be 10 NL / L reactor / h, which is equivalent to 240 NL / L reactor / day.

[0042] Advantageously, the flow rate of CO2 entering step c), d) or e) of the process according to the invention may be in a range from 0.025 to 2.5 NL / Lreactor / h, which is equivalent to 0.6 to 60 NL / Lreactor / day. Preferably, the flow rate incoming CO2 perhaps 2.5 NL / Lreactor / h, which is equivalent to 60 NL / Lreactor / day.

[0043] Advantageously, the liquid is recirculated in a loop in the reactor, via a recirculation loop in order to homogenize the reaction medium. The liquid recirculation flow rate is in a range from 0.1 to 1 L / L rreactor / min.

[0044] Advantageously, the reaction temperature of step d) of the process according to the invention may be in a range from 25 to 70°C. Preferably, the temperature may be 55°C.

[0045] Advantageously, the pressure inside the bioreactor during steps c), d) and e) of the process according to the invention may be in a range from 1 to 20 bar. Preferably, the pressure of step c), d) and e) may be between 2 and 6 bar or between 1 and 3 bar. More preferably, the pressure of step c), d) and e) may be 2 bar.

[0046] Advantageously, the outgoing gases may comprise methane (CH ). The outgoing gases may further comprise water vapor.

[0047] Advantageously, the volume ratio of methane (Ch) to the other outgoing gases (CO2 and H2) can be within a range from 2 / 3 to 9.9 / 10.

[0048] Advantageously, the flow rate of the outgoing gases may be in a range from 0.025 to 2.5 NL / Lreactor / h, which is equivalent to 0.6 to 60 NL / Lreactor / day. Preferably, the flow rate of the outgoing gases may be 2.5 NL / Lreactor / h, which is equivalent to 60 NL / Lreactor / day.

[0049] Advantageously, the method according to the invention may further comprise a step d) of replenishing the culture medium with nutrients. This step makes it possible to compensate for the reduction in the concentration of nutrients in the culture medium, the latter being consumed by the microorganisms during the production of methane, in step d) of the method.

[0050] A second object of the present invention is a device 1 for ex-situ methane production comprising: - a gas / liquid bioreactor 11 comprising a tank 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 support 122, immobilized or not, and a culture medium 123; - a gas injection means 17 for injecting incoming gases into said continuous liquid phase contained 123 in the gas / liquid bioreactor 11; - a liquid injection means 18 making it possible to inject said continuous liquid phase 123 into the gas / liquid bioreactor 11; - a recovery means 19 for recovering outgoing gases from the gas / liquid bioreactor 11.

[0051] The definitions described above also apply to the device mentioned, where applicable.

[0052] Advantageously, the volume of the tank 12 of the bioreactor can be within a range from 0.01 to 150 m 3 Preferably, the volume of the tank 12 of the bioreactor 11 may be less than 50 m 3 for an agricultural installation and greater than 50 m 3 for an industrial installation. More preferably, the volume of the tank 12 of the bioreactor 11 may be 100 m 3 for an industrial installation. The height / diameter ratio of the tank 12 can be between 10 / 1 and 10 / 3.

[0053] By "gas injection means" is meant any device for injecting incoming gases into the continuous liquid phase contained in a gas / liquid bioreactor.

[0054] Advantageously, the gas injection means 17 may be chosen from fine bubble diffusers such as a porous column bottom diffuser, a perforated tube, a porous membrane made of polymers or ceramic material, a valve bubbler, or from bubble-free membrane contactors such as hollow fiber membranes, or from hydroejectors or static mixers. Preferably, the gas injection means 17 may be a porous column bottom diffuser. The gas / liquid bioreactor 11 may be configured to be continuously supplied with incoming gases, in particular during steps d) and e). The gas injection means 17 is connected to the gas inlet 13 of the bioreactor 11.

[0055] As used herein, “connected” means a direct or indirect connection between two elements of the device.

[0056] By "liquid injection means" is meant any device for injecting a continuous liquid phase into a gas / liquid bioreactor. The liquid injection means 18 is connected to the liquid inlet 15 of the bioreactor 11.

[0057] Advantageously, the device according to the invention may comprise a liquid recirculation loop 24 connected to the liquid outlet 16 and to the liquid inlet 15. The recirculation loop may be connected to the purge means 20. The loop liquid recirculation means may comprise a pump 23 and the liquid injection means 18. The pump may be a peristaltic pump.

[0058] Advantageously, the liquid injection means 18 is chosen from multi-orifice static dispersion systems (in the form of a crown or comb for example). Preferably, the liquid injection means 18 may be a comb-shaped multi-orifice.

[0059] Advantageously, the device 1 may further comprise a purge means 20 making it possible to purge said continuous liquid phase included in the gas / liquid bioreactor 11. The purge means 20 is connected to the liquid outlet 16 of the bioreactor 11.

[0060] The gas / liquid bioreactor 11 can be configured so that the supply of nutrients and / or the purging of the culture medium is carried out continuously or discontinuously.

[0061] By "recovery means" we mean any device for recovering outgoing gases from a gas / liquid bioreactor.

[0062] Advantageously, the recovery means 19 can be chosen from a simple gas outlet, a gas outlet with condenser (in particular to eliminate the residual water vapor contained in the outgoing gases), an outlet associated with a system for recirculating the outgoing gases. Preferably, the recovery means 19 can be an outlet associated with a system for recirculating the outgoing gases. The gas / liquid bioreactor 11 can be configured so that the recovery of outgoing gases is carried out continuously. The recovery means 19 is connected to the gas outlet 14 of the bioreactor 11. The water vapor condensates can either be reinjected into the bioreactor 11 or extracted from the device 1. Since the methanation reaction produces water, this has the advantage of being able to modulate the quantity of water in the device 1.

[0063] Advantageously, the device 1 can comprise a gas outlet 14 equipped with a condenser.

[0064] Advantageously, the device 1 can comprise a gas outlet 14 equipped with a meter.

[0065] Advantageously, the device 1 can comprise a gas loop connected to analyzers, preferably chromatograph type analyzers.

[0066] Advantageously, the device 1 can comprise a loop for recirculating gases from the upper part of the bioreactor 11 to the lower part.

[0067] Advantageously, the device 1 can comprise a mixer of the recirculated gases with the incoming gases.

[0068] Advantageously, the device 1 may comprise a set of probes allowing the verification of the concentration of an incoming gas, the redox potential or the pH in the culture medium 123 (directly in the bioreactor 11). The probe allowing the measurement of the pH advantageously allows the measurement of the temperature.

[0069] Advantageously, the device 1 may comprise gas cylinders for supplying incoming gases. The cylinders may be connected to the gas injection means 17. The gas injection means 17 may further comprise a mixer, so as to control the CO2 / H2 ratio of the incoming gases. Said mixer may be identical to or different from the mixer of the recirculated gases with the incoming gases.

[0070] “Bottle” means a means of storing gas; said means of storage may be substituted by any other compatible means of storage.

[0071] Advantageously, the device 1 may comprise a set of flow meters, preferably mass flow meters. Said mass flow meters allow the adjustment of the inlet flow rates of the incoming gases.

[0072] Advantageously, the device 1 can comprise a gas meter.

[0073] Advantageously, the device 1 may comprise a sampling means. Said sampling means makes it possible to take liquid samples for the analysis of the compounds and to monitor the progress of the process, for example.

[0074] Advantageously, the device 1 may comprise a means for taking a sample of the composition of the outgoing gas mixture for the analysis of the compounds.

[0075] Advantageously, the device 1 can comprise a set of valves.

[0076] Advantageously, the device 1 can comprise a pH regulation system.

[0077] Advantageously, the device 1 may comprise a sub-device housed inside the tank so as to compartmentalize it using a grid. Preferably, the sub-device is present in the case where several culture media of different nature are used.

[0078] The invention also relates to a kit, said kit once assembled making it possible to obtain the device 1 according to the invention, comprising: - a support 122; - at least one methanogenic microorganism 123; - optionally a gas / liquid bioreactor 11; - optionally a gas injection means 17; - optionally a liquid injection means 18; - optionally a means of recovery 19.

[0079] The invention also relates to a methanation catalyst comprising a support 122 obtained in step a) of the process according to the invention. The catalyst comprises at least one methanogenic microorganism 123 attached to a support 122.

[0080] Another subject of the present invention relates to the use of the strain of Methanothermobacter marburgensis CLERMONT DSM 34405 for the production of methane.

[0081] Another subject of the present invention relates to the use of the strain of Methanothermobacter marburgensis CLERMONT DSM 34405 for the implementation of a biomethanation process.

[0082] Other advantages will become apparent in the light of the following examples, given for illustrative and non-limiting purposes, with reference to the appended figures. Brief description of the figures

[0083] [Fig. 1] Figure 1 represents a device 1 for the production of methane according to the present invention comprising: - a gas / liquid bioreactor 11 comprising a tank 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 support 122, a culture medium 123, a gas injection means 17, a gas recovery means 19 and a liquid recirculation loop 24, connected to the liquid inlet 15 and to the liquid outlet 16, comprising a pump 23 and a liquid injection means 18 for injecting said continuous liquid phase 123 into the gas / liquid bioreactor 11 - two sources of incoming gas connected by two valves 21 by means of gas injection 17; - a chromatograph 22 connected by a valve 21 to the recovery means 19; - a purge means 20 connected to the recirculation loop 24 and comprising a valve 21.

[0084] [Fig. 2] Figure 2 represents the evolution over time of the growth of bacteria and archaea in a stirred bioreactor, in the absence (■ without support) or in the presence of polyethylene culture supports at a low filling rate (• TR_low) or high (▲ TR_high), in the presence of the modified basal BA medium 100 RPM and at 55°C, with supplementation in H2 and CO2.

[0085] [Fig. 3] Figure 3 represents the evolution of the total number of archaea and bacteria in the absence (without support, left) or in the presence of culture supports at a low filling rate (Low_TR, center) or high (High_TR, right) of polyethylene culture supports. The culture in a stirred bioreactor is carried out in the presence of the modified basal BA medium, at 100 RPM and at 55°C, with supplementation of H2 and CO2.

[0086] [Fig. 4] Figure 4 represents an image of a methanation catalyst in polyethylene biochips taken by SEM at 0 days of growth of Methanothermobacter marburgensis Clermont DSM 34405, in modified BA medium, at 55°C with H2 and CO2 supplementation.

[0087] [Fig. 5] Figure 5 represents an image of a methanation catalyst in polyethylene biochips taken by SEM at 14 days of growth of Methanothermobacter marburgensis Clermont DSM 34405, in modified BA medium, at 55°C with H2 and CO2 supplementation.

[0088] [Fig. 6] Figure 6 represents an image of a methanation catalyst in polyethylene biochips taken by SEM at 34 days of growth of Methanothermobacter marburgensis Clermont DSM 34405, in modified BA medium, at 55°C with H2 and CO2 supplementation. EXAMPLES

[0089] Other advantages, aims and particular characteristics of the present invention will emerge from the examples which follow, given for explanatory and in no way limiting purposes.

[0090] In the examples that follow, the different parameters were measured using the techniques detailed below:

[0091] Measurement of the tolerance of at least one methanogenic microorganism to exposure to oxygen (O2)

[0092] Several open air exposures lasting 1 min, 10 min, 30 min, 60 min and 180 min are carried out in a laminar flow hood (PSM II Cytosafe, Faster).

[0093] For each exposure time, three flasks containing 30 mL of culture medium comprising at least one methanogenic microorganism are inoculated with 3 mL of a culture medium comprising at least one methanogenic microorganism in the exponential growth phase. To restore anaerobiosis, the C>2 is removed by purging the flasks with the H2 / CO2 mixture, the pressure is then adjusted to 1.5 bar. The cultures are incubated at 55°C for 10 days. The ability to grow again after exposure to C>2 is monitored by CP assay and spectrophotometry (Cell Density Meter, Fisherbrand).

[0094] Measurement of gas phase composition

[0095] The composition of the gas phase is continuously analyzed by gas chromatography (Agilent Technologies 3000A® analyzer) equipped with two separation modules and a thermal conductivity detector (TCD). Module A, equipped with a 5Â molecular sieve, separates hydrogen, nitrogen, oxygen, and methane under argon flow. Module B, equipped with a PoraPlot U column (Agilent), separates carbon dioxide and hydrogen sulfide under hydrogen flow. A hydrophobic filter is placed at the analyzer inlet to protect it from moisture. Gas analyses of the bioreactor are carried out every hour.

[0096] Measurement of the content of metabolites and organic acids

[0097] The content of metabolites and organic acids was analyzed by liquid chromatography (HPLC Agilent Technologies, 1260 Infinity). Separation was ensured by two ion exclusion columns (Rezex ROA 300x7, 8 nm, Phenomenex, USA) connected in series and heated to 50°C. The detector used to identify the different compounds was a refractometer (HP series 1100). The liquid phase consisted of a sulfuric acid solution (2 mM) circulating at a flow rate of 0.7 mL / min. Before injection, the samples were deproteinized. A volume of 1 mL of culture sample was mixed with 125 pL of Ba(OH)2 8H2O (0.3 M) and 125 μl of ZnSO 7H2O (5% w / v) and centrifuged (5 min; 10,000 g). The supernatant is then filtered at 0.22 pm and transferred to an HPLC vial stored at 4°C before injection.

[0098] Measurement of the growth of microorganisms

[0099] The evolution of the growth of microorganisms is monitored by spectrophotometry (llvisco, V-1800) at 600 nm.

[0100] Microscopic observation

[0101] A volume of 0.5 ml of a culture on BA (Basal anaerobic) medium described by Bu et al., (reference 1) then modified, was collected and fixed with a formaldehyde solution (2%, final concentration). The cells were then pelleted by centrifugation at 18000 g (20 min, 14°C) on 400-mesh carbon-coated copper grids (Formvar, Pelanne Instruments, France) and contrasted by immersion in 20 μL of 2% uranyl acetate. After rinsing with distilled water and drying on absorbent paper, observations were carried out using a JEOL 2100 plus transmission electron microscope (TEM) (Akishikma, Tokyo, Japan, UCA Partner CYSTEM Platform) equipped with a Gatan CMOS RIO 9 camera (Gatan Ametek, Pleasanton, USA) at an accelerating voltage of 80 kV. Samples from culture media were fixed with a solution comprising 2.5% glutaraldehyde and 0.15% ruthenium red, in 0.2M sodium cacodylate buffer, pH 7.4 overnight at 4°C. After rinsing in the same buffer (3x10min), they are post-fixed for 1h at room temperature with 1% osmic acid in 0.2M sodium cacodylate buffer, pH 7.4. Then the samples are rinsed with distilled water for 20 minutes and dehydrated by ethanol baths of increasing degree (25° to 100°), 10 minutes / bath. The last dehydration step is carried out with a VA / ethanol 1007 hexamethyldisilazane mixture for 10 minutes then pure hexamethyldisilazane (evaporation overnight in a fume cupboard). The samples are then deposited on a metal pad using double-sided carbon tape. A chrome metallization (5nm) is carried out with the Quorum Q150 TES Plus metallizer. Observations were performed using the Hitachi Regulus 8230 scanning electron microscope at an accelerating voltage of 1 kV and with the secondary electron detector.

[0102] Example 1: Evaluation of the tolerance of the strain Methanothermobacter marburgensis Clermont DSM 34405 to exposure to oxygen (O2)

[0103] The simplification of a consortium from a methanization unit (Ennezat, 63) by selection of hydrogenotrophic microorganisms allowed the isolation of a new methanogenic strain overproducing methane.

[0104] To isolate the archaeal strain with the highest content in the bioreactor, a 50 mL sample of the consortium from the digestate of a methanization unit (Ennezat, 63) is taken under sterile and anaerobic conditions. In some cases, the sample is cultured under selective conditions for methanogenic hydrogenotrophic strains, in the presence of a H2 / CO2 mixture in a 4 / 1 ratio at a temperature of 55°C for a period of 6 weeks. After a 72-hour incubation at 55°C (bioreactor temperature), subculture is carried out in a modified BA medium (10% inoculation: 0.5 mL of inoculum in 5 mL of culture medium). Serial dilutions (from 10-1 to 10-10) are carried out while respecting the physicochemical conditions of the bioreactor (55°C, 1.5 bar and pH=7.5). The tube of the last positive dilution (turbidity and CH4 production) serves as inoculum for inoculating the next series.Growth is monitored by spectrophotometry (Cell Density Meter, Fisherbrand) and gas determination by micro-chromatography (Agilent analyzer, Technologies 3000A®) equipped with two separation modules and a thermal conductivity detector (TCD). Module A equipped with a 5Â molecular sieve allows the separation of hydrogen, nitrogen, oxygen and methane, under argon flow. Module B equipped with a PoraPlot U column (Agilent) allows the separation of carbon dioxide and hydrogen sulfide, under hydrogen flow, in the gas phase. Isolation on solid medium is then carried out using the Roll-tube method (Hungate, 1969). For this, 0.1 g of agar-agar (BactoTM Agar) is added to 5 mL of modified BA medium (Hungate tube), under N2 flow, then autoclaved. The agar medium is then liquefied in a water bath at 100°C, then placed at 45°C in order to slow down its gelation.After inoculation by the last series of positive dilution in culture medium, the agar medium is uniformly arranged in a thin layer on the inner face of the tube by rotating the Spinner tube and using an ice cube on the outer face to solidify the medium. The tubes are then incubated at 55°C for 168 hours in a vertical position to avoid contamination of the colonies due to condensation. The tubes showing the presence of a mother entity of microorganism (colony), visible to the naked eye, comprising a large number of identical microorganisms, and a production of CP, are opened in an anaerobic chamber with controlled atmosphere (atmospheric pressure) to collect colonies using a sterile Pasteur pipette. The colonies are resuspended in 5 mL of culture medium. The purity of the liquid cultures is. confirmed by carrying out several successive serial dilutions (10-1 to 10-5) and verifications under an optical microscope (Labophot, Nikon). Analyses can be carried out on the strain thus isolated.

[0105] Table 1: Influence of exposure to oxygen on the growth and metabolic activity of the isolate Methanothermobacter marburgensis CLERMONT DSM 34405 [Table 1]

[0106] It appears that exposure to oxygen according to the method described above had no significant effect on the growth and hydrogenotrophic and methanogenic activities of the strain Methanothermobacter marburgensis CLERMONT DSM 34405 between 0 and 60 min of exposure. A slight growth delay is observed after 180 min of exposure to oxygen (Table 1). Since the methanogenic performance of the strain Methanothermobacter marburgensis CLERMONT DSM 34405 is not or only slightly affected by exposure to oxygen and therefore air, the different methods of transition from step a) to step b) can be carried out in non-anaerobic conditions without impact on steps c), d) and e).

[0107] Example 2: Evaluation of the methanation catalyst from step a) of the process according to the invention

[0108] The ex-situ methane production process implemented comprises the following step: a) bringing into contact a consortium, a culture support in polyethylene biochips and a culture medium.

[0109] The consortium from a methanization unit (Ennezat, 63) is taken from the methanization tank. The liquid sample or digestate is sieved (5 mm) to remove solids > 5 mm and frozen.

[0110] The culture medium used is derived from the BA medium described by Bu et al., 2018 and its composition is described in Table 2. The culture medium is adjusted to pH 7.5 with 3 M NaOH solution.

[0111] Table 2: List of compounds in the modified BA medium. [Table 2]

[00112] The colonization kinetics of polyethylene biochip supports by Methanothermobacter marburgensis CLERMONT DSM 34405 is monitored by SEM imaging (FEG SEM, Hitachi Regulus 8230). The process begins when the microbes associated with the surface switch from a reversible attachment mode (which can be detached without compromising integrity) to an irreversible attachment mode, followed by cell aggregation and subsequent proliferation. The biofilm cells are enclosed in a matrix of EPS (extracellular polymeric substances) polymers. SEM observation allows visualization of EPS grains, the matrix, the cells as well as thread-like structures called fibraeum.

[0113] Figures 4, 5 and 6 illustrate that the strain Methanothermobacter marburgensis CLERMONT DSM 34405 develops over time when placed in the presence of a polyethylene biochip culture medium.

[0114] Furthermore, it appears that the strain Methanothermobacter marburgensis CLERMONT DSM 34405 develops more efficiently when it is placed in the presence of a culture medium according to the invention in the presence of dihydrogen and carbon dioxide. Indeed, the optical density of the samples over time is lower in the case where a culture medium is present (figure 2).

[0115] Furthermore, an analysis of the growth of microorganisms by spectrophotometry (UVisco, V-1800) at 600 nm indicates that the number of microorganisms after 24 days of colonization is three times higher in the presence of a polyethylene biochip culture support (figure 3).

[0116] These results allow us to conclude that the process according to the invention makes it possible to obtain a better yield in methanation. Indeed, there is better growth of microorganisms when they are fixed on a culture support.

[0117] Example 3: Device for implementing the method according to the invention

[0118] The ex-situ methane production process implemented according to the invention also comprises the following steps: b) introduction of the mixture obtained in step a) (example 2) into a gas / liquid bioreactor; c) bringing incoming gases into contact with the gas / liquid bioreactor obtained in step b); d) reaction of the incoming gases with the at least one methanogenic microorganism; e) recovery of outgoing gases obtained in step d); characterized in that - the culture medium is not immobilized in the gas / liquid bioreactor; and - at least one methanogenic microorganism is attached to the culture medium.

[0119] In the present example, the device as described in Figure 1 allows the implementation of the method according to the invention.

[0120] The bioreactor for step b) was custom-built from a section of a 2 mm thick 316 stainless steel cylinder. It has an internal diameter of 54 mm and a height of 1400 mm. The height / diameter ratio of the gas / liquid bubble column bioreactor is 25.9. The volume of the tank is 3.5 L. A glass or stainless steel fritted gas distributor of known porosity is coupled to the reactor using a GL18 tri-ring connector. The gas outlet is through an opening at the top of the column, coupled to the gas circuit by a VÀG thread. The gas outlet of the reactor is connected to a condenser in which water at 4°C circulates (Lauda Eco RE1225 silver). A high-precision volume flow meter (Bioprocess control / Microflow 1100-3100) measures the gas flow leaving the condenser. The gas is then analyzed by gas chromatography (Agilent analyzer, Technologies 3000A®).A culture medium recirculation system based on the use of a peristaltic pump (AB pump, Type PSF2) allows liquid circulation against the current of the ascending gas bubbles, at an hourly volumetric speed of 10 L / Lreactor / h, which is equivalent to 0.166 L / L. r reactor / min. The liquid recirculation loop includes an electrode for measuring the redox potential, pH and temperature (Mettler Toledo INPR04260i / SG / 120 52005381). The pH is regulated to 7.5 if necessary by the addition of acidic or basic titrants. The temperature of 55 °C is controlled using a thermostatic bath (Lauda Eco RE1225 silver). The recirculation system is equipped with a septum for taking samples, adding stock solutions (culture medium constituents, Na2S 9H2O) or withdrawing culture medium volumes.

[0121] The incoming gases of step c) are dihydrogen and carbon dioxide. They are injected into the bioreactor at a total gas flow rate gradually increasing from 3.61 to 18.21 NL / L r reactor / day for CO2 and gradually increasing from 14.37 to 72.85 NL / L reactor / day for H2 using flow meters mass flow (Brooks instrument, SLA5800), as shown in Table 3 below. The H2 / CO2 ratio is adjusted, if necessary, to obtain a value of 4 / 1 ±0.1. The system pressure is 1 bar. The gas mixture is distributed continuously by a diffuser, countercurrent to the flow of the aqueous phase.

[0122] The exit gases from step e) consist mainly of methane and water vapor (the water vapor being recondensed). They are recovered from the bioreactor at a total exit gas flow rate gradually increasing from 5.52 to 18.67 NL / Lreactor / day, as shown in Table 3 below.

[0123] Table 3: Evolution of the specific incoming flow rate of CO2 and H2 and the specific production of CH4 (expressed in NL / Lreactor / day) as a function of the age of the culture (expressed in days). [Table 3]

[0124] In view of the results of Example 2, the method according to the invention implemented by the device of Figure 1 makes it possible to obtain a methane yield higher than conventional methods. Indeed, the increase in pressure in the system increases the solubility of hydrogen in the culture medium. The presence of supports colonized by at least one methanogenic microorganism increases the quantity of catalyst per unit of reactor volume. The at least one methanogenic microorganism cultivated is selected for its ability to produce methane. The presence of supports 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. It increases the system's performance by a factor of 3.6. Bibliographic references

[0125] 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

CLAIMS

1. A method for producing methane ex-situ comprising the following steps: a) bringing into contact at least one methanogenic microorganism, a culture support and optionally a first culture medium and; b) introducing the mixture obtained in step a) into a gas / liquid bioreactor optionally comprising a second culture medium; c) bringing incoming gases into contact with the gas / liquid bioreactor obtained in step b); d) reacting the incoming gases with the at least one methanogenic microorganism; e) recovering outgoing gases obtained in step d); characterized in that - the culture medium is immobilized or not in the gas / liquid bioreactor; and - at least one methanogenic microorganism is attached to the culture support.

2. Method according to claim 1, in which the incoming gases are carbon dioxide, CO2, and dihydrogen, H2, preferably in a volume ratio H2 / CO2 of between 3 / 1 and 5 / 1, and more preferably 4 / 1.

3. Method according to any one of the preceding claims, in which the at least one methanogenic microorganism is a consortium or a pure methanogenic strain.

4. A method according to any one of the preceding claims, wherein the at least one methanogenic microorganism comprises a microorganism belonging to the kingdom of Euryarchaeota and selected from the classes Methanobacteria, Methanococci, Methanopyri or Methanomicrobia.

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

6. A method according to any preceding claim, wherein the gas / liquid bioreactor (11) is selected from a pneumatically stirred and downward liquid circulation reactor.

7. A method according to any preceding claim, wherein the culture medium (122) comprises porous cubes made of polyurethane foam impregnated with powdered activated carbon, rigid polyethylene biochips, sepiolite, pozzolan and / or porous glass.

8. Device (1) for ex-situ methane production comprising: - a gas / liquid bioreactor (11) comprising a tank (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 support (122), immobilized or not, and a culture medium (123); - gas injection means (17) for injecting incoming gases into said continuous liquid phase contained (123) in the gas / liquid bioreactor (11); - a liquid injection means (18) for injecting said continuous liquid phase (123) into the gas / liquid bioreactor (11); - a recovery means (19) for recovering outgoing gases from the gas / liquid bioreactor (11).

9. Device according to claim 8, in which the gas / liquid bioreactor (11) is chosen from a bubble column, a mechanically stirred column, a continuous flow stirred tank reactor (CSTR), an airlift reactor, a flooded fixed bed reactor, a watered bed reactor, a pneumatically stirred reactor, with downward liquid circulation.

10. Device according to claim 8 or 9, in which the gas / liquid bioreactor (11) is chosen from a reactor with pneumatic stirring and downward liquid circulation.

11. Device according to any one of claims 8 to 10, in which the culture medium (122) is organic or inorganic, of natural or synthetic origin.

12. Device according to claim 8 to 11, wherein the culture support (122) comprises porous cubes of polyurethane foam impregnated with powdered activated carbon, rigid biochips of polyethylene, sepiolite, pozzolan and / or porous glass, preferably porous cubes of polyurethane foam impregnated with powdered activated carbon and / or rigid biochips of polyethylene.

13. Device according to any one of claims 8 to 12, wherein the at least one methanogenic microorganism (121) comprises at least one strain of Methanothermobacter, preferably the strain of Methanothermobacter marburgensis CLERMONT DSM 34405.

14. Kit for obtaining the device (1) according to any one of claims 8 to 13, comprising: - a support (122); - at least one methanogenic microorganism (123); - optionally a gas / liquid bioreactor (11); - optionally a gas injection means (17); - optionally a liquid injection means (18); - optionally a means of recovery (19).

15. A kit according to claim 14, wherein the culture medium (122) comprises porous cubes of polyurethane foam impregnated with powdered activated carbon, rigid polyethylene biochips, sepiolite, pozzolan and / or porous glass.

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

17. Methanation catalyst comprising at least one methanogenic microorganism 123 fixed on a support 122, preferably the methanogenic microorganism is a strain of Methanothermobacter marburgensis CLERMONT DSM 34405.

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