WO2017017355A1

WO2017017355A1 - Microbiological method for producing formate from co2 using a type ii methanotrophic strain

Info

Publication number: WO2017017355A1
Application number: PCT/FR2016/051903
Authority: WO
Inventors: Laurence Soussan; Nakry PEN; José SANCHEZ MARCANO; Marie-Pierre Belleville; Delphine PAOLUCCI
Original assignee: Universite De Montpellier; Centre National De La Recherche Scientifique; Ecole Nationale Superieure De Chimie De Montpellier
Priority date: 2015-07-24
Filing date: 2016-07-21
Publication date: 2017-02-02
Also published as: FR3039168A1

Abstract

The invention relates to a microbiological method for producing formate from CO₂ catalysed by a type II methanotrophic bacterium, particularly by the bacterium Methylosinus trichosporium OB3b. The invention also relates to a method for the recovery of CO₂ catalysed by a type II methanotrophic bacterium, particularly by the bacterium Methylosinus trichosporium OB3b.

Description

Microbiological process for producing formate from CO2 using a class II methanotrophic strain

The invention relates to a microbiological process for the production of formate from CO2 catalyzed by a class II methanotrophic bacterium, and in particular by the bacterial strain Methylosinus trichosporium OB3b. The subject of the invention is also a process for recovering CO2 using a class II methanotrophic bacterium, and in particular the bacterial strain Methylosinus trichosporium OB3b.

BACKGROUND ART Formic acid or methanoic acid is a naturally occurring aliphatic carboxylic acid whose basic form is the formate ion, which is in the form of a colorless liquid with a persistent odor. This molecule was first discovered in ant body distillation products. It is found more generally in the glands of several Hymenoptera insects, as well as in some stinging plants such as nettles. Nowadays it is synthesized, for industrial purposes, mainly by chemical means. Indeed, formic acid has many applications in the leather and textile industry, in the composition of dyes and treatment products, insecticides, solvents, fumigants, anti-mold treatment products, etc. but also in perfumery and the food industry as an aromatic molecule. Recently, it has been shown that formic acid can be used as fuel in fuel cells (Yu et al., 2008, Journal of Power Sources, 182 (1), 124-132) or to store hydrogen which can then be released by dehydrogenation of formic acid (Fellay et al., 2008, Angewandte Chemie, 120 (21), 4030-4032). The possibility of using formic acid as an energy carrier suggests that the need for formic acid will increase in the coming years.

Up to now, formic acid is almost exclusively produced by chemical synthesis under extreme conditions (pressure, temperature and pH) by the hydrolysis of formamide (HC (O) NH2) obtained by reaction of methyl formate (HCO2CH3 ) with ammonia; the methyl formate is synthesized from methanol and carbon monoxide (John et al., 1985 Encyclopedia of Chemical Processing and Design: Volume 23).

Biological synthesis routes have been studied. Nevertheless, the yields are still too low to be industrially interesting. Thus, WO95 / 19425 discloses a microbiological process involving a bacterial strain phenotypically close to the genus Bacillus to produce, in the presence of growth factors, lactate and formic acid from glucose. But, in addition to high glucose consumption, this process requires the use of a large amount of growth factors (yeast extract / trypsin casein). Document FR2745297 also discloses a process for the production of formic acid by a homofermentative lactic acid bacterium having a deficiency in the transport or metabolism of lactose and capable of converting said lactose into formic acid. Here again, the implementation of the process requires a source of fermentable sugar. In addition, the reaction can take place only in the presence of a high concentration of nitrogenous substances and especially yeast extract. It is therefore not economically advantageous to produce formic acid on an industrial scale by the currently available microbiological processes.

A biochemical synthesis route of formate (basic formic acid form) from carbon dioxide (C0 ₂ ) was also studied. Specifically, formate dehydrogenase (FDH) enzymes extracted from microorganisms have been purified and modified to improve their catalysis performance, and used to reduce CO2 to formate (Alissandratos et al., 2013, Appl Environ Microbiol., 79 ( 2), 741-4, Spinner et al., 2012, Catal, Sci., Technol., 2, 19-28). Nevertheless, the addition of an expensive cofactor (NAD) is necessary to perform the bioconversion reaction, making this alternative also incompatible with industrial application. Summary of the invention

By working on the recovery of CO2, the inventors have discovered that class II methanotrophic bacteria, known to use methane as sole source of energy and carbon, are also able to directly and selectively convert CO2 into formate under conditions soft physiological. In particular, the inventors have shown that the bacterium Methylosinus trichosporium OB3b is capable of producing amounts of formate (compared to the other forms of formate synthesis known so far), using CO2 as the sole source of carbon. The use of these class II methanotrophic bacteria, such as the Methylosinus trichosporium OB3b bacterium, to produce formate thus simultaneously makes it possible to transform CO2 and limit the emission of this greenhouse gas into the atmosphere.

The invention therefore relates to a process for producing formate from CO2, characterized in that it comprises a step according to which a class II methanotrophic bacterium is contacted with CO2.

In a particular embodiment, the class II methanotrophic bacterium is Methylosinus trichosporium OB3b.

The method according to the invention may comprise a preliminary step according to which the class II methanotrophic bacterium is cultured in a culture medium in the presence of methane (CH ₄ ) and of air.

The CO2 reduction stage in formate can be carried out in atmospheric air or in a gaseous mixture containing CO2 or in pure CO 2.

Advantageously, the process comprises an additional step of recovering formate and / or formic acid.

The subject of the invention is also a reaction mixture which can be obtained by the implementation of such a process, said mixture comprising at least 150 mg of formate / g of dry cells, preferably at least 250 mg of formate / g. dry cells, more preferably at least 350 mg formate / g dry cells.

The invention also relates to a method for screening a class II methanotrophic bacterium capable of producing formate by reducing CO2, comprising the steps of: (a) placing a class II methanotrophic bacterium in suspension in a medium reaction in the presence of CO2; (b) Check the presence of formate in the reaction mixture; and if formate is detected in step (b)

(c) Select the bacterium.

The subject of the invention is also a class II methanotrophic bacterium, isolated from a bacterial or non-bacterial consortium, obtained or obtainable according to such a screening method.

The invention also relates to the use of a Class II methanotrophic bacterium, and in particular to the bacterium Methylosinus trichosporium OB3b or to a bacterium obtained or obtainable by the screening method, for the production of formate by reduction of CO ₂ . The subject of the invention is also a process for the recovery of C0 ₂ comprising the reduction of C0 ₂ into formate by a class II methanotrophic bacterium, such as the bacterium Methylosinus trichosporium OB3b or a bacterium obtained or obtainable by the screening method. . Such a process advantageously comprises the implementation of the above formate production process. Brief description of the figures

Figure 1: Metabolic pathway of C0 ₂ reduction (case of the serine pathway) in a Class II methanotrophic bacterium;

Figure 2. Kinetic monitoring of the total formate concentration (Figure 2. a) and kinetic monitoring of pH and DOôoonm (Figure 2.b) in a closed reactor; Figure 3. Kinetic observations of the concentration of total formate, pH and ODonoon in a semi-continuous reactor;

Figure 4. Kinetic monitoring of viability of bacteria by cell growth during

the experience in semi-continuous reactor. Detailed description of the invention

The inventors have discovered that methanotrophic bacteria of class II, and in particular the bacterium Methylosinus trichosporium OB3b, are capable of producing formate in large quantities by direct reduction of CO2 under mild operating conditions. This discovery is very interesting on many levels. First, unlike Methylosinus trichosporium IMV 3011 (JY Xin et al., 2007- Journal of Basic Microbiology, 47 (5), 426-435), which produces methanol, the Methylosinus trichosporium OB3b bacterium produces formate, with productions at least 200 times larger than that reported for methanol. Furthermore, Methylosinus trichosporium OB3b is able to selectively produce formate using CO2 as the only carbon source, without the addition of costly cofactors or growth factors. In addition, the inventors have been able to obtain very high concentrations of formate, up to about 20 mM, which is much higher than the amounts obtained so far by the available microbiological processes.

Based on this discovery, the inventors have developed a process for the microbiological production of formate from CO2, comprising a step of contacting a class II methanotrophic bacterium, such as the bacterium Methylosinus trichosporium OB3b,

In the context of the invention, the term "methanotrophic bacteria" means bacteria capable of developing using methane as sole source of carbon and energy. Methanotrophic bacteria of class Π are methanotrophic bacteria belonging to the group of Protobacteria which includes the following four genera: Methylosinus, Methylocystis, Methylocapsa and Methylocella. Methanotrophic class II bacteria use the serine pathway for carbon assimilation.

Advantageously, the method according to the invention is implemented using the bacterium Methylosinus trichosporium OB3b.

As previously indicated, the bacterium Methylosinus trichosporium OB3b is a Group II methanotrophic bacterium (NCBI: Taxonomy ID: 595536; GenBank ADVE00000000; NCJJVIB: 11131). It was originally isolated in 1970 by Roger Whittenbury. His genome was sequenced in 2010 (L. Stein et al., 2010 - Journal of Bacteriology, 6497-6498). In the In the context of the invention, the terms "M. trichosporium OB3b" and "OB3b bacterium" are used interchangeably to refer to the bacterium Methylosinus trichosporium OB3b.

According to the invention, the bacterium is brought into contact with CO2, advantageously dissolved in a reaction medium in which the bacterium is maintained. By "reaction medium" is meant the medium in which the bacterium is maintained, to specifically produce formate, and in which the CO2 supplied will be dissolved, whether or not this medium allows the production of biomass. In the context of the invention, the reaction medium comprises at least water, mineral salts and dissolved CO2.

The CO2 used may be CO2 from atmospheric air, or all or part of a specific CO2 input. In particular, the CO2 can come from pure CO2 or from a gaseous mixture, such as a C0 ₂ -air, CO2-O2, CO2-CH4, CO2-H2, CO2-CH4-H2, C0 ₂ -CH ₄ mixture. air or CO2-CH ₄ -02. In a particular embodiment, the gaseous mixture comprises at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of C0 ₂ . In a particular embodiment, the gaseous mixture is an equivolumic mixture CO2-O2. Since the formate is obtained directly by reduction of CO2, the formate yield of the process according to the invention depends directly on the CO2 input. Those skilled in the art will be able to adapt the concentrations of CO2 to the desired yields. In a particular embodiment, the dissolved CO2 concentration in the reaction medium is between about 30 and about 50 mM. According to the invention, the CO2 contribution can be achieved by any known means and in particular by membrane contactors (Pen et al., 2014, Bioresource Technology, 174, 42-52), a porous gas distribution sinter (Kim et al. al., 2010, Biotechnology and Bioprocess Engineering, 15 (3), 476-480, Duan et al., Bioresource Technology, 102 (15), 7349-7353) or a simple gas-liquid contact (reaction medium) in a closed reactor (Pen et al., 2014, Bioresource Technology, 174, 42-52).

In an exemplary embodiment, it is also possible to add trace molybdenum in the reaction medium, so as to promote the operation of the bacterial FDH enzyme (FIG. 1) and thus the production of CO2. In an exemplary embodiment, the pH in the reaction medium is maintained between 6 and 8, preferably at pH 6.5, +/- 0.5. Similarly, the temperature in the reaction medium is preferably maintained between 20 ° C and 50 ° C, preferably between 25 ° C and 35 ° C, and more preferably at 30 ° C, +/- 1. According to the invention, it is possible prior to the reaction step, that is to say before contacting the bacterium with the CO ₂ to produce the formate, to place the bacteria in a culture medium under conditions temperature and pH favorable to its growth.

By "culture medium" is meant the medium in which the bacteria are grown, to produce bacterial biomass. The culture medium conventionally comprises the chemical elements strictly necessary for bacterial growth in a form usable by bacteria, namely a source of carbon (and especially methane), mineral salts and water. Standard media are available commercially, described in the scientific literature or in the catalogs of suppliers of bacterial strains. Those skilled in the art know what are the minimum components required in the absence of which class II methanotrophic bacteria can not develop, and can without difficulty cultivate such a bacterium, including Methylosinus trichosporium OB3b. Advantageously, the culture medium comprises as sole source of carbon methane. The general conditions of culture (temperature, composition of the medium, agitation, etc.) allowing the growth or maintenance of the bacteria are easily defined by those skilled in the art according to the bacterial strain used.

Without being bound by any theory, the inventors believe that Methylosinus trichosporium OB3b is capable of reducing CO2 to formate by using poly-β-hydroxybutyrate (PHB), an intracellular polymer for storing the energy it produces in large quantities. , as an electron donor for the regeneration of the NAD cofactor (FIG. 1). Also, in a particular embodiment, the method comprises a prior step according to which the bacterium is grown under conditions enabling it to produce greater quantities of PHB, and in particular by maintaining it preferentially in the presence of CH ₄ and under conditions limiting nitrogen, phosphorus and / or oxygen. After the culturing step, the bacterium is advantageously washed from its culture medium and suspended in reaction medium before contacting with CO ₂ .

All or part of the process according to the invention can be implemented on a laboratory scale or on an industrial scale, that is to say on fermenters of average (from about 1 to 100 m ³ ) or large capacity (over 100 m ³ ).

In a particular embodiment of the process of the invention, the step of reducing CO2 by class II methanotrophic bacteria is conducted in a bioreactor, or fermenter. According to the invention, it is possible to use a closed, semi-continuous or continuous reactor, so as to conduct a reaction in "batch", "fed batch" or continuously.

Thus, for example, in a closed reactor, in atmospheric air, the formate concentration can reach 400 mg / L ± 50 mg / L, after 15 days of reaction at 30 ° C; while under a mixture of 50% air and 50% CO2, the formate concentration is greater than 700 mg / L ± 50 mg / L, after 15 days of reaction at 30 ° C. In a semi-continuous reactor, under atmospheric air, 400 mg / L ± 50 mg / L of formate are obtained after 15 days of reaction at 30 ° C.

The process according to the invention makes it possible in particular to obtain at least 150 mg of formate / g of dry cells, preferably at least 250 mg of formate / g of dry cells, more preferably at least 350 mg of formate / g of cells. dry after a reaction time between 10 and 15 days.

In a particular embodiment, the process for producing formate according to the invention comprises the steps of:

Introduce class II methanotrophic bacteria into a bioreactor containing a reaction medium;

Supply the reaction medium with a gas containing CO2; and optionally

Recover the formate produced in the bioreactor by reduction of CO2, in the form of formate and / or formic acid. In one embodiment of the process according to the invention, the product formate is recovered in the form of formic acid from the reaction medium.

In a particular embodiment, the step of recovering formic acid from the reaction medium is carried out by electrodialysis or extraction-liquid with a co-solvent. These recovery devices make it possible to continuously recover the formic acid and thus avoid the possible inhibition of the bacteria by an excess of product, while obtaining a formic acid free of impurities (such as the salts of the reaction medium). In the case of a continuous recovery of the product, a continuous supply of C0 ₂ can be achieved in the bioreactor. In the case of an electrodialysis recovery process, a bipolar electrodialysis module is advantageously coupled with a microfiltration module for continuously separating and recovering the formate produced in its acid form (formic acid). In particular, bipolar electrodialysis can be performed on a reaction medium without biocatalysts, obtained continuously after separating the bacteria from the medium by a tangential microfiltration module. Bipolar electrodialysis involves combining a bipolar membrane to acidify formate into formic acid and monopolar membranes to extract salts from the reaction medium.

The formate and / or formic acid obtained from such a microbiological process can be advantageously used in any industry that may need it, and in particular in the leather and textile industry, in perfumery, in the food industry. food industry, etc. For example, the formate and / or formic acid derived from the process according to the invention can be used in the composition of dyes, insecticides, solvents, fumigants, anti-mold treatment products or as a molecule. aromatic, etc. Furthermore, the formate and / or formic acid from the process according to the invention can be particularly useful as fuel in fuel cells or for storing hydrogen. The subject of the invention is also the reaction mixture that can be obtained by carrying out the process described above. Advantageously, said mixture comprises at least 150 mg of formate / g of dry cells, preferably at least 250 mg of formate / g of dry cells, more preferably at least 350 mg of formate / g of dry cells.

By "reaction mixture" means a reaction medium comprising at least one microorganism and the products of the reaction catalysed by said microorganism. So, according to the invention, the reaction mixture designates a reaction medium comprising a group II methanotrophic bacterium capable of producing formate from CO2, as well as formate. For example, the reaction mixture comprises reaction medium Methylosinus trichosporium OB3b and formate.

The invention also relates to a bioreactor comprising a bacterial suspension of Methylosinus trichosporium OB3b in a reaction medium, a gas supply system in said suspension, and optionally means for supplying the suspension with inoculum and / or mineral salts and / or means for recovering the reaction products.

The invention also relates to the use of a class II methanotrophic bacterium capable of producing formate, such as the bacterium Methylosinus trichosporium OB3b for the production of formate by reducing CO2.

The use of a class II methanotrophic bacterium such as the Methylosinus trichosporium OB3b bacterium to produce formate makes it possible to consume large amounts of CO2. Thus, the invention provides a process for recovering CO2 comprising a step of reducing CO2 to formate by a class II methanotrophic bacterium, and in particular by the bacterium Methylosinus trichosporium OB3b, advantageously implementing the formate production process described in FIG. above.

The invention also relates to a method for screening a class II methanotrophic bacterium capable of producing formate by reducing CO2, comprising the steps of:

(a) Put a class II methanotrophic bacterium in suspension in a reaction medium in the presence of CO2;

(b) Check the presence of formate in the reaction mixture; and in case of presence of formate in the reaction medium;

(c) Select the bacterium.

Advantageously, the step (b) of checking the presence of formate is by comparison with one or two negative controls. A first witness may consist of medium of reaction under CO2 but devoid of bacteria. A second control may consist of reaction medium comprising bacteria but devoid of CO2. The presence of a class II methanotrophic bacterium capable of producing formate by reduction of CO2 in the reaction medium is confirmed if formate is detected in the reaction mixture and in the control (s) there is no formate detected.

In a particular embodiment, the screening method comprises a preliminary step wherein the bacterium is cultured in a culture medium in the presence of methane and air prior to step (a) of reaction.

The subject of the invention is also a Class II methanotrophic bacterium, isolated from a bacterial or non-bacterial consortium, obtained or obtainable by such a screening method.

Other aspects and advantages of the present invention will appear in the following experimental part, which must be regarded as an illustration, in no way limiting the extent of the protection sought.

Examples Materials and methods

1) Bacterial strain and cell culture

The strain Methylosinus trichosporium OB3b (NCIMB, 11131, England) is grown in NMS medium (Nitrate Minerals Salts) modified as described in Pen et al., 2014 (Bioresource Technology, 174, 42-52: "An innovative membrane biorecator for methane biohydroxylation "). The strain is maintained liquid with regular subcultures in fresh NMS medium (Nitrate Minerals Salts) modified as described in Pen et al., 2014 (Bioresource Technology, 174, 42-52: "An innovative membrane biorecator for methane biohydroxylation"). ), i.e. a base medium of 1.060 g / L KH2PO4; 4,340 g / L Na ₂ HPO ₄ .12H ₂ O; 1,700 g / L NaN0 ₃ ; 0.340 g / LK ₂ S0 ₄ and 0.074 g / L MgSO ₄ .7H ₂ 0 enriched with usual minerals (0.570 mg / L ZnSO ₄ .7H ₂ O, 0.446 mg / L MnSO ₄ .H ₂ O; 0.124 mg / LH ₃ B0, 0.096 mg / L Na ₂ MoO ₄ .2H ₂ 0, 0.096 mg / L C0Cl ₂ 6H ₂ O, 0.166 mg / L Kl, 7.00 mg / L CaCl ₂ 2H ₂ O) but also with copper (1.25 mg / L CuSO ₄ .5H ₂ O) and iron (11.20 mg / L FeSO ₄ .7H ₂ O). The cell culture is carried out in a 3 L cell culture reactor (Biostat A plus, Sartorius, France) run at 30 ° C with gas flow rates set at 100 ± 5 mL.min ^"1 for methane and air respectively, and a correlation between the optical density (OD) of the suspension at 600 nm (DOôoonm) and the dry cell concentration was previously established ([dry cell concentration] (g / L) = 0.322 x DOôoonm (-), Pen et al., 2014). reaction and preparation of the bacterial suspension

The experiments are conducted in a sterile reaction medium (pH = 7.0 ± 0.1), composed of 20 mM phosphate mineral salts and 5 mM MgCl ₂ (Xin et al., 2007) in water. ultrapure. At the end of the cell culture step, the bacterial culture is sterile centrifuged for 20 minutes at 4500 rpm and 4 ° C. The supernatant is removed and the bacterial pellets are then resuspended in the reaction medium, such that the OD 50 mm of the bacterial suspension is of the order of 20 (determined from a dilution). The mother suspension thus obtained is then used for the experiments.

3) Bioreactors

Two types of bioreactors have been implemented: a closed bioreactor and a semi-continuous reactor. at. Reactor closed

The closed reactor is a 50 ml sealed flask containing 5 ml of liquid. Two conditions were tested. The head space of the flask (45 ml) is filled in one case with a sterile and equivolumic mixture of atmospheric air and carbon dioxide (99.995%) and in the other case, the gaseous mixture is only constituted sterile atmospheric air; headspace gases are all at atmospheric pressure. It should be noted that the air used contains 0.039% by volume of C0 ₂ . The gases are sterilized by means of hydrophobic Teflon filters (0.2-μιη, Sartorius). The reactor is stirred on a stirring tray (Unimax 1010, Heidolph) in an incubator. The reactor medium is sampled sterilely with a syringe. The reaction is conducted at 30 ± 1 ° C, with stirring of 160 rpm. For each kinetics, a series of identical vials is filled with the same bacterial suspension and incubated. The reaction is stopped at different reaction times. Two types of control are carried out: in one case, the bacterial mixture in the reaction medium is brought into contact with sterile nitrogen (99.995%) and in the other case, the reaction medium alone is brought into contact with one another. sterile CC / air mixture (1: 1 v / v).

Each experiment is reproduced 3 times. The initial average concentration of the suspension in the reactors is 2.5 ± 0.3 g dry cells / L. b. Semi-continuous reactor

The reactor used is a cell culture reactor with a total volume of 3L (Biostat A plus, Sartorius, France). The useful volume is 1.6 L and the initial concentration of the bacterial suspension is 2.1 ± 0.2 g dry cells / L. The reactor is maintained at 30 ° C. with gas flow rates of 100 ± 5 ml. min ^"1 for air and C0 ₂ (case of 1: 1 v / v CC / air mixture) The gases are sterilized by means of hydrophobic Teflon filters (0,2-μιη, Sartorius).

For each experiment (in closed or semi-continuous reactors), samples are taken regularly in order to measure DOOoonm and total formate concentration. In the case of the closed reactor, the sampling performed also makes it possible to measure the pH over time.

4) Bacteria growth tests

In order to determine whether all or part of the bacteria present in the semi-continuous reactor are still alive, samples of the suspension of the reactor are made over time and seeded in fresh culture medium (3% v / v) and then cultured in sealed culture flasks (total volume 100 ml including 35 ml of liquid). The gaseous sky is composed of a methane / air mixture (1: 1 v / v) and the gaseous mixture is renewed every day. The duration of the cultivation is fixed at 7 days. The suspension placed in culture in the flasks is removed (1 ml) initially and after 7 days to measure the initial and final DOooonm.

5) Analysis of the total formate product Immediately after collection, a fraction of the samples are transferred to a 2 mL Eppendorf and centrifuged for 3 min at 4500 rpm and 4 ° C to stop the reaction in the sample. The supernatant is then stored at -20 ° C. for a total formate assay by ion chromatography (Dionex ICS-1000). The column used is a Dionex IonPac AS 19 and the elution is carried out with the following potassium hydroxide (KOH) concentration ramp: 10 mM for 10 min, then 1.75 mM.min- ¹ up to 45 mM and finally 10 mM for 10 min.

Results

1) Closed reactor results The results obtained in a closed reactor with an equivolumic mixture of air and CO ₂ and in air alone are shown in FIG. 2. The controls are also reported there.

The controls reveal no presence of formate (Fig. 2. a). In particular, the control under nitrogen shows that the bacteria do not release formate (as soluble microbial product) during the experiment and the control without bacteria in a CC / air mixture shows that the spontaneous reduction of CO2 in formate does not occur. product not.

The appearance of formate takes place only in the presence of CO2 and bacteria (Fig. 2. a). The evolution of the formate concentration over time has the same pace in both cases tested (under air and under air / CC mixture). Indeed, the concentration increases, after a lag phase of 10 days, to reach a maximum at 15 days (about 350 mg / L in air and 750 mg / L under mixing) then decreases of 27 ± 1% on average in the 2 cases at t = 20 days.

The concentrations of formate reached are all the greater as there is CO2 in the headspace (Fig. 2. a). In addition, the theoretical amount of CO2 available in the headspace under CC / air mixture (8.9 10 ^-1 mmol) is nearly 10 times greater than the maximum amount of formate product (8.3 10 ^-2 mmol). ), which means that there is no CO2 limitation in this case.

All of these elements demonstrate that CO2 is used by the bacterium to make formate and that, under the conditions tested, the bacteria have a limited ability to assimilate CO2 which may possibly be related to an inhibition of bacteria by the bacteria. formate. In both cases (CC-air mixture and air alone), the OD and pH follow-ups (Fig. 2.b) show a sharp decrease in OD at the beginning of the experiment and this parameter tends to stabilize from 5 days around an average value of 5.8 ± 0.2 under mixing and 4.7 ± 0.2 under air alone (the difference observed that can be attributed to the initial concentration difference representing almost one unit OD, ie almost 0.3 g of dry cells / L only). The pH decreases over the first 5 days (from 7.0 to 6.1 mixed and from 7.0 to 6.7 under air) and remains constant throughout the experiment (Fig. 2.b). It should be noted that the average pH tends in both cases towards the pka of the couple CO2 / HCO3 ^" = 6.37 At this pH, the majority species is the basic form (ion formate) because the pKa of the acid The OD at 20 days remains constant around 5-6, which shows that the bacteria are still viable under these conditions.

2) Results in semi-continuous reactor

In this configuration, the CO2 and air inputs are continuous. The results obtained in semi-continuous reactor with equivolumic mixture of air and CO2 are shown in FIG. 3. As in a closed reactor, there is a production of formate and the concentration begins to increase between 5 and 10 days. The maximum production of formate obtained is 215 ± 5 mg / g dry cells. From 30 days, the concentration stabilizes up to 60 days around an average value of 400 mg / L. DOooonm and pH remained constant throughout the experiment (6.5 ± 0.2 for OD and 6.3 ± 0.1 for pH from 3 days). Figure 4 gives the results of the viability tests by growth conducted throughout the experiment with the semi-continuous reactor. The initial OD represents the OD 50 mm of the suspension seeded in fresh medium (t = 0); the final OD is the DOOoonm of the same suspension after 7 days of culture. The initial OD is globally constant over time (Fig. 4) and this is explained by the fact that the OD in the reactor is almost constant (Fig. 3). In all cases, the final OD remains significantly larger than the initial OD. These results thus prove that all or most of the bacteria are still viable after 60 days of operation of the reactor. The above experiments confirm that it is possible to produce large amounts of formate from CO2 using Methylosinus trichosporium OB3b in the presence of CO2. The formate yield is directly a function of the CO2 inputs. In all cases, it is shown that the bacterium is resistant to high concentrations of formate, making it possible to envisage production of formate on an industrial scale.

Claims

1. Process for producing formate from CO ₂ , characterized in that it comprises a step according to which a class II methanotrophic bacterium is contacted with CO 2.

2. Process for producing formate from CO2 according to claim 1, characterized in that the class II methanotrophic bacterium is the bacterium Methylosinus trichosporium OB3b.

3. Process for producing formate from CO2 according to one of the preceding claims, wherein the bacterium is suspended in a molybdenum enriched reaction medium.

4. Process for producing formate from CO2 according to one of the preceding claims, characterized in that it comprises a preliminary step according to which the class II methanotrophic bacterium is cultured in a culture medium in the presence of CH ₄ and air.

5. Process for producing formate from CO2 according to one of the preceding claims, wherein the pH is maintained between 6 and 8, preferably at pH 6.5, +/- 0.5.

6. Process for producing formate from CO2 according to one of the preceding claims, wherein the temperature is maintained between 20 ° C and 50 ° C, preferably between 25 ° C and 35 ° C, more preferably 30 ° C. ° C, +/- 1.

7. Process for producing formate from CO2 according to one of the preceding claims, wherein the bacterium is brought into contact with a gaseous mixture containing CO2, preferably pure CO2 or a gaseous mixture selected from CC-air, CO2 -O ₂ , CO2-CH ₄ , CO2-H ₂ , C0 ₂ -CH ₄ -H ₂ , C0 ₂ -CH ₄ -air and C0 ₂ -CH ₄ -O ₂ .

8. A process for producing formate from CO2 according to one of the preceding claims, comprising an additional step of recovering the formate produced in the form of formate and / or formic acid.

9. A process for producing formate from CO2 according to one of the preceding claims, said method comprising a preliminary step according to which the bacterium is cultured in conditions limiting nitrogen, phosphorus and / or oxygen before the step of contact with C0 ₂ .

10. Reaction mixture obtainable by carrying out the method according to one of claims 1-9, said mixture comprising at least 150 mg of formate / g of dry cells, preferably at least 250 mg of formate / g dry cells, more preferably at least 350 mg formate / g dry cells.

11. A method of screening a class II methanotrophic bacterium capable of producing formate by reduction of CO2, comprising the steps of:

(b) Check the presence of formate in the reaction mixture; and if formate is detected in step (b)

(c) Select the bacterium.

A bacterium obtained or obtainable by the screening method according to claim 11.

13. Use of a class II methanotrophic bacterium for the production of formate by reduction of CO2.

14. Process for recovering CO2 comprising a step of reducing formate CO2 by an IL-class methanotrophic bacterium 15. Process for recovering CO2 according to claim 14, comprising carrying out the process according to one of claims 1 to 1. 9.