Method for converting CO.SUB.2 .by means of biological reduction
11584943 · 2023-02-21
Assignee
- UNIVERSITÉ DE MONTPELLIER (Montpellier, FR)
- Centre National De La Recherche Scientifique (Paris, FR)
- Ecole Nationale Superieure De Chimie De Montpellier (Montpellier, FR)
Inventors
- Laurence Soussan (Montpellier, FR)
- Azariel Ruiz-Valencia (Lyons, FR)
- Djahida Benmeziane (Montpellier, FR)
- José Sanchez-Marcano (Sussargues, FR)
- Marie-Pierre Belleville (Saint Georges d'Orques, FR)
- Delphine Paolucci-Jeanjean (Saint Gely du Fesc, FR)
Cpc classification
C12P7/40
CHEMISTRY; METALLURGY
C12R2001/01
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a process for the recovery of CO.sub.2 by biological reduction comprising a step of bringing a liquid phase containing the bacterium Stenotrophomonas maltophilia into contact with a CO.sub.2-containing gas phase under conditions allowing the production of formate and/or methane from said CO.sub.2. The process according to the invention can be implemented in particular in a closed reactor or a semi-closed reactor or a continuous reactor, electrochemically assisted or not.
Claims
1. A process for the recovery of CO.sub.2 by biological reduction comprising a step of bringing a liquid phase containing the bacterium Stenotrophomonas maltophilia into contact with a CO.sub.2-containing gas phase under conditions allowing the reduction of CO.sub.2.
2. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the step of bringing the liquid phase and the CO.sub.2-containing gas phase into contact is carried out in a semi-closed or continuous reactor, wherein the CO.sub.2-containing gas phase is supplied continuously into the reactor.
3. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein formate and/or methane (CH.sub.4) is produced by CO.sub.2 reduction.
4. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, comprising a preliminary phase of co-culture of Stenotrophomonas maltophilia with at least one methanotrophic bacterium before the step of bringing the liquid phase into contact with the gas phase.
5. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the liquid phase also contains at least one microorganism capable of using the formate to produce organic compounds of interest.
6. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the gas phase is a biogas or CO.sub.2-rich industrial and/or agricultural fumes.
7. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the gas phase comprises at least 30% by volume of CO.sub.2 relative to the total volume of the gas phase.
8. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the gas phase comprises between 30% and 100% by volume of CO.sub.2 with respect to the total volume of the gas phase.
9. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the liquid phase comprises at least 3 g.sub.dry cells/L of Stenotrophomonas maltophilia.
10. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein ammonium and/or ammonia is added in the liquid phase and/or ammonia is added in the gas phase.
11. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein PHB is added in the liquid phase.
12. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein formate is produced, said process comprising an additional step of recovering the formate produced in the form of formate and/or formic acid and/or organic compounds of interest derived from the use of the formate by a microorganism capable of using formate to produce organic compounds of interest.
13. The process for the recovery of CO.sub.2 by biological reduction according to claim 1, wherein the step of bringing the liquid phase into contact with CO.sub.2 is carried out in a closed or semi-closed or continuous reactor.
14. The process for the recovery of CO.sub.2 by biological reduction according to claim 13, wherein the CO.sub.2-containing gas phase is injected into the center of the reactor.
15. The process for the recovery of CO.sub.2 by biological reduction according to claim 13, wherein the air present in a reactor headspace is removed, and the CO.sub.2-containing gas phase is injected into said reactor headspace, so as to place the CO.sub.2 reduction reaction under an atmosphere consisting solely of said gas phase.
16. The process for the recovery of CO.sub.2 by biological reduction according to claim 13, wherein the step of bringing the liquid phase and the CO.sub.2-containing gas phase into contact is carried out in the presence of an intra or extracellular electron and proton donor.
17. The process for the recovery of CO.sub.2 by biological reduction according to claim 13, wherein the reactor contains an electrochemical assistance.
18. The process for the recovery of CO.sub.2 by biological reduction according to claim 13, wherein the CO.sub.2-containing gas phase is injected into the center of the reactor by a gas distributor.
19. A process for the treatment of a biogas or of CO.sub.2-rich industrial fumes and/or CO.sub.2-rich agricultural fumes comprising a step of reduction of the CO.sub.2 by biological reduction comprising a step according to which said biogas and/or fumes are brought into contact with a liquid phase containing the bacterium Stenotrophomonas maltophilia under conditions allowing the reduction of the CO.sub.2.
20. A process for the production of formate from CO.sub.2, comprising a step in which a CO.sub.2-containing gas phase is brought into contact with a liquid phase containing the bacterium Stenotrophomonas maltophilia under conditions allowing reduction of the CO.sub.2 to formate or methane.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
(9) The inventors discovered that Stenotrophomonas maltophilia is capable of producing formate and methane (CH.sub.4) by direct reduction of CO.sub.2 under mild operating conditions. In a particularly interesting manner, the inventors have shown that Stenotrophomonas maltophilia is able to produce formate and methane (CH.sub.4) using CO.sub.2 as the sole carbon source, without the need to supply cofactors, organic molecules, dihydrogen (H.sub.2) or expensive growth factors.
(10) In the context of the invention, CO.sub.2 reduction is understood to mean any reaction allowing the carbon oxidation number of CO.sub.2 to be reduced to a lower degree than in CO.sub.2.
(11) Stenotrophomonas maltophilia is a gram-negative aerobic bacterium of the family Pseudomonadaceae. To date, it has never been used for industrial purposes. However, the inventors have discovered that this bacterium is particularly attractive for the production of molecules of interest from CO.sub.2. Indeed, the inventors discovered that this bacterium has the capacity to produce formate and/or methane from CO.sub.2 as the sole carbon source, after having advantageously been first cultured in autotrophic conditions, i.e. in pure aerobic culture on a usual organic medium of the Lysogeny Broth Miller type, or in co-culture with a methanotrophic bacterium, such as Methylosinus trichosporium OB3b, on a mineral medium brought into contact with a methane/air mixture (1:1 v/v). The co-culture route is particularly advantageous because it guarantees an optimal carbon balance; indeed, methane, of renewable origin, is the sole source of carbon necessary for the culture.
(12) Advantageously, the liquid phase comprises at least 3 g.sub.dry cells/L of the bacterium Stenotrophomonas maltophilia, at least 10 g.sub.dry cells/L, at least 20 g.sub.dry cells/L, 30 g.sub.dry cells/L, 40 g.sub.dry cells/L, 50 g.sub.dry cells/L, 60 g.sub.dry cells/L, 70 g.sub.dry cells/L, 80 g.sub.dry cells/L, 90 g.sub.dry cells/L, 100 g.sub.dry cells/L.
(13) Advantageously, and whatever the culture mode, the cultures are carried out at 30° C., ±10° C. and at atmospheric pressure or slight overpressure (up to 0.5 bar).
(14) According to the invention, the CO.sub.2-containing gas phase brought into contact with the liquid reaction medium containing the bacterium Stenotrophomonas maltophilia can be atmospheric air. The gas phase may otherwise be pure CO.sub.2 or a gas mixture, such as a mixture of CO.sub.2-air, CO.sub.2—N.sub.2, CO.sub.2—O.sub.2, CO.sub.2—CH.sub.4, CO.sub.2—H.sub.2, CO.sub.2—N.sub.2—H.sub.2, CO.sub.2—CH.sub.4—H.sub.2, CO.sub.2—CH.sub.4-air, CO.sub.2—CH.sub.4—N.sub.2 or CO.sub.2—CH.sub.4—O.sub.2. In an embodiment, the gas phase contains or consists of biogas. In another embodiment, the gas phase contains or consists of CO.sub.2-rich industrial or agricultural fumes. In a particular embodiment, the gas phase comprises at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% CO.sub.2. Advantageously, the gas phase comprises at least 30% CO.sub.2. It is also possible to use a gas phase comprising 100% CO.sub.2. In an embodiment, the gas phase comprises between 30 and 100% CO.sub.2.
(15) In one reaction embodiment, the reaction medium contains 20 mM phosphate buffer at pH 7.0±0.1 and 5.0 mM MgCl.sub.2. In another embodiment, the reaction medium contains 1.3 mM KCl, 28.0 mM NH.sub.4Cl, 29.8 mM NaHCO.sub.3 and 5.0 mM NaH.sub.2PO.sub.4. The skilled person can select the appropriate reaction medium for the reduction of CO.sub.2 to formate by Stenotrophomonas maltophilia.
(16) Since formate and/or methane and/or any other product whose carbon oxidation number is lower than that of CO.sub.2 is obtained directly by CO.sub.2 reduction, the yield of formate and/or methane and/or any other product whose carbon oxidation number is lower than that of CO.sub.2 from the process according to the invention depends directly on the CO.sub.2 supply. The skilled person will know how to adapt the CO.sub.2 concentrations to the desired yields.
(17) According to the invention, the CO.sub.2 supply 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 frit (Kim et 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 in a closed reactor (Pen et al. 2014, Bioresource Technology, 174, 42-52).
(18) In an embodiment, the CO.sub.2 recovery process is implemented in a closed (batch) reactor. Alternatively, the process can be implemented in a semi-closed (fed-batch) reactor.
(19) According to the invention, the CO.sub.2-containing gas phase can be injected into the headspace of the closed reactor. In an embodiment, it is possible to remove the air present in the headspace of the closed reactor prior to the injection of the gas phase, so as to place the reaction under an atmosphere consisting solely of the gas phase.
(20) Alternatively or additionally, the gas phase can be injected in the center of the closed or semi-closed reactor, in the core of the liquid phase containing the bacterium Stenotrophomonas maltophilia, in particular by means of a gas distributor.
(21) In the case of a semi-closed (fed-batch) reactor, the CO.sub.2-containing gas phase is supplied continuously, preferentially by bubbling, into the reactor.
(22) In one example embodiment, the pH in the reaction medium, i.e. the liquid phase, is maintained between 5 and 8, preferentially at pH 6.5, ±0.5. Advantageously, the CO.sub.2 dissolved in the water forms a sufficient buffer to allow the pH to be maintained, without external regulation.
(23) Similarly, the temperature in the reaction medium is preferentially maintained between 20° C. and 50° C., preferably between 25° C. and 35° C., and even more preferably at 30° C., ±1.
(24) According to the invention, it is possible, prior to the reaction step, i.e. prior to bringing the bacterium into contact with CO.sub.2 to produce formate and/or methane, to place the bacterium in a culture medium under temperature and pH conditions favorable to its growth.
(25) “Culture medium” is understood to mean the medium in which 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 the bacteria, i.e. a source of carbon, of mineral salts and of water. Standard media are available commercially, described in the scientific literature or in the catalogs of bacterial strain suppliers. The skilled person knows which are the minimum required components without which Stenotrophomonas maltophilia cannot grow and can cultivate such a bacterium without difficulty. The general culture conditions (temperature, composition of the medium, stirring, etc.) allowing the growth or the maintenance of Stenotrophomonas maltophilia, are easily defined by the skilled person.
(26) This preliminary culture step can for example allow the bacterium to accumulate donors of protons and electrons necessary for the CO.sub.2 reduction reaction to formate and/or methane according to equations (1) and (2) and/or (1) and (3):
CO.sub.2+H.sup.++2e−.fwdarw.HCOO.sup.− (equation 1)
CO.sub.2+8H.sup.++8e−.fwdarw.CH.sub.4+2H.sub.2O (equation 2)
HCOO.sup.−+7H.sup.++6e−.fwdarw.CH.sub.4+2H.sub.2O (equation 3)
(27) Alternatively or additionally, it is possible to add to the reactor an exogenous electron and proton donor, such as a biopolymer, so as to provide the bacteria with the electrons and protons necessary for the reaction, even in the absence or after exhaustion of intracellular donors. In particular, it is possible to add polyhydroxybutyrate (PHB) to the reactor. In an embodiment, the PHB is produced beforehand by methanotrophic bacteria from methane, in particular renewable methane (Pieja et al., Distribution and selection of poly-3-hydroxybutyrate production capacity in methanotrophic proteobacteria, Microbial. Ecology, 62(3) (2011) 564-573). Such an embodiment is particularly advantageous from an economic point of view.
(28) In an embodiment, exogenous PHB is added to the reactor. Preferentially between 30 mg/L and 3 g/L exogenous PHB is added in the liquid phase, in particular about 0.3 g/L.
(29) Alternatively or additionally, and in particular in the case of a semi-closed reactor, it can be particularly advantageous to use an electrochemical assistance device. Such a device, housed inside the reactor and in contact with the bacterium, can be used to supply electrons and protons to the bacterium. Electrochemical assistance can in particular be of the bioelectrolysis type (
(30) Different materials can be used for the anode and the cathode, as long as they are electron conductive. For example, the cathode can be made of stainless steel, graphite or felt. The anode can be made of platinum, stainless steel, graphite or felt.
(31) Advantageously, the ratio active cathode surface area/reactor volume is comprised between 1 and 100 m.sup.2/m.sup.3, preferentially about 10 m.sup.2/m.sup.3. “Active surface” is understood to mean the geometrical surface exposed to the anode. The ratio anode projected geometrical surface area/cathode projected geometrical surface area is preferentially comprised between 1 and 10, more preferentially roughly equal to 2. The polarization potential range can for example be from −1.5 V to 0 V vs Ag/AgCl, in particular −1 V to −0.5 V, for example −0.7 V or −0.8 V.
(32) So as to optimize the CO.sub.2 reduction reaction, it is possible to add ammonium and/or ammonia in the liquid phase and/or ammonia in the gas phase, in particular between 5 mmol/L and 100 mmol/L, preferentially between 20 and 30 mmol/L.
(33) Alternatively or additionally, the liquid phase comprises, in addition to the bacterium Stenotrophomonas maltophilia, another microorganism capable of using the formate derived from CO.sub.2 reduction to form more complex metabolites, such as methane, lactate or alcohols. The inventors have shown that such a consortium can lead to a synergy capable of intensifying the flow of CO.sub.2 reduced by the bacterium Stenotrophomonas maltophilia.
(34) All or part of the process according to the invention can be implemented on a laboratory scale or on an industrial scale, i.e. on fermenters, or reactors, of medium capacity (about 0.1 L to 100 L) or of large capacity (100 L to several hundred m.sup.3).
(35) Another object of the invention is the intensification of the CO.sub.2 reduction process by the bacterium Stenotrophomonas maltophilia by means of an addition of exogenous PHB or an electrochemical assistance (bioelectrolysis) as described above.
(36) In a particular embodiment, the CO.sub.2 reduction reaction by the bacterium Stenotrophomonas maltophilia can be promoted by continuously removing the formate produced in the liquid phase of the reactor in the form of formate and/or formic acid. The skilled person knows the possible techniques to continuously remove the formate produced.
(37) In another embodiment of the process according to the invention, the formate-enriched liquid and mineral reaction medium can be used as recovered in a secondary process or bioprocess aimed at converting the formate produced into molecules with higher added value. The skilled person knows the possible techniques for converting the formate produced.
(38) In a particular embodiment, the formate production process according to the invention comprises the steps consisting in:
(39) Introducing Stenotrophomonas maltophilia into a reactor containing a reaction medium; Supply the reaction medium, or liquid phase, with a CO.sub.2-containing gas phase; and optionally
(40) Recovering the formate produced in the reactor by CO.sub.2 reduction, in the form of formate and/or formic acid.
(41) In another embodiment of the process according to the invention, the formate-enriched mineral reaction medium can be used as recovered in a secondary process aimed at converting the formate produced into molecules with higher added value.
(42) In another embodiment of the process according to the invention, the formate produced is recovered in the form of formic acid from the reaction medium.
(43) In a particular embodiment, the step of recovery of formic acid from the reaction medium is carried out by electrodialysis or by liquid-liquid extraction with a co-solvent. These recovery devices allow continuous recovery of formic acid and thus avoid the possible inhibition of bacteria by an excess of product, while obtaining a formic acid free of impurities (such as salts from the reaction medium).
(44) In the case of continuous recovery of the product, a continuous supply of reaction medium can be made to the bioreactor. In the case of an electrodialysis recovery process, a bipolar electrodialysis module is advantageously coupled with a microfiltration module to continuously separate and recover the formate product in its acid form (formic acid). In particular, bipolar electrodialysis can be carried out on a reaction medium without biocatalysts, obtained continuously after separating the bacteria from the medium by a tangential microfiltration module. Bipolar electrodialysis consists in combining a bipolar membrane to acidify the formate to formic acid and monopolar membranes to extract the salts from the reaction medium.
(45) The formate and/or formic acid obtained from such a microbiological process can be advantageously used in any industry likely to have need thereof, and in particular in the leather and textile industry, in perfumery, in the agri-food industry, etc.
(46) Preferentially, the formate and/or formic acid obtained from such a microbiological process is used in a secondary biological process (in situ or ex situ) to obtain organic compounds of higher added value, and in particular methane, lactate or C.sub.1-C.sub.5 alcohols. Alternatively, it is possible to directly use the reaction medium containing the formate and/or formic acid obtained for the implementation of the secondary biological process. If the compounds obtained are alcohols, a stripping process carried out by bubbling CO.sub.2-containing gas can be used to continuously recover the alcohols produced.
(47) In a particular embodiment, it is possible, prior to the reaction step, to co-culture Stenotrophomonas maltophilia with one or more other methanotrophic bacterial species. It is thus possible to produce organic nutrients from methane, said nutrients then being used by Stenotrophomonas maltophilia as a source of carbon and energy. For example, the inventors have demonstrated that co-culture under a methane/air mixture and on a mineral medium of Stenotrophomonas maltophilia with a methanotrophic bacterium, such as Methylosinus trichosporium OB3b, allows the production of acetate that can be used by Stenotrophomonas maltophilia for its growth. The inventors have further shown that the presence of such a methanotrophic bacterium in the liquid phase during the subsequent reaction step does not disrupt the reduction of CO.sub.2 or the production of formate and/or methane by Stenotrophomonas maltophilia.
(48) Alternatively or additionally, it is possible during the reaction step (i.e. bringing Stenotrophomonas maltophilia into contact with the CO.sub.2-containing gas phase) to add to the liquid phase one or more other microorganisms, and in particular bacteria, archaea and/or yeasts, capable of using the formate to produce molecules with higher added value. It is also possible to co-culture Stenotrophomonas maltophilia with one or more of these microorganisms prior to the reaction step, and then to add all the microorganisms obtained to the liquid reaction medium for the reaction step. In an embodiment, the bacterium Microbacterium oxydans is added to Stenotrophomonas maltophilia. In an embodiment, hydrogenotrophic methanogenic archaea are added to obtain methane. In an embodiment, the bacterium Ralstonia eutropha H16 (LH74D) is added to Stenotrophomonas maltophilia in order to obtain C.sub.4-C.sub.5 alcohols. In an embodiment, a methanotrophic bacterium and a methanol dehydrogenase inhibitor are added to Stenotrophomonas maltophilia in order to obtain methanol.
(49) Alternatively, the step of CO.sub.2 reduction reaction to formate by Stenotrophomonas maltophilia is carried out in a first reactor, and the formate produced is recovered to feed a second reactor in which are located the microorganism(s) capable of using the formate to produce molecules with higher added value. The second reactor can be fed directly with the reaction medium of the first reactor, containing the formate produced, or conversely contain a medium to which is added the formate extracted from the reaction medium of the first reactor.
(50) Another object of the invention is the use of Stenotrophomonas maltophilia, for the production of formate and/or methane and/or any other product whose carbon oxidation number is lower than that of CO.sub.2 by CO.sub.2 reduction as described above. The use of Stenotrophomonas maltophilia consumes significant amounts of CO.sub.2 and thus addresses the environmental problems related to the accumulation of CO.sub.2 in the atmosphere while adding value to the CO.sub.2 consumed.
(51) Other aspects and advantages of the present invention will become apparent in the following experimental section, which should be considered for illustrative purposes, without in any way limiting the scope of the protection sought.
EXAMPLES
(52) A] Materials and Methods
(53) 1. Culture of the Bacterium Stenotrophomonas maltophilia
(54) Two routes of S. maltophilia culture were implemented:
(55) (i) Aerobic Co-Culture with the Methanotrophic Bacterium Methylosinus trichosporium OB3b
(56) In this case, the source of carbon and energy used is methane, which is converted by the methanotrophic bacterium into organic nutrients that can be assimilated by the bacterium S. maltophilia for its growth. In particular, acetate, at a concentration of about 35 mg.Math.L.sup.−1, is measured in the culture reactor at the end of growth. Such a synergy between these two types of bacteria has already been reported in nature and for a consortium involving them, the production of acetate by methanotrophs has already been demonstrated under oxygen-limited conditions (C. Costa et al., Denitrification with methane as electron donor in oxygen-limited bioreactors, Appl. Microbiol. Biotechnol., 53(6) (2000) 754-762).
(57) The culture medium is a mineral medium enriched with copper and iron. The basal medium is composed of: 1.06 g/L KH.sub.2PO.sub.4, 4.34 g/L Na.sub.2HPO.sub.4.12H.sub.2O, 1.7 g/L NaNO.sub.3, 0.34 g/L K.sub.2SO.sub.4 and 0.074 g/L MgSO.sub.4.7H.sub.2O. Once prepared, the pH of this basal medium is adjusted to 7.0±0.1 with 0.1 M NaOH or 0.1 M HCl, and then autoclaved. The mineral, copper and iron solutions are prepared independently and sterilized by filtration through cellulose acetate filters with a pore diameter of 0.2 μm before being added to the basal medium. Final mineral concentrations are: 0.57 mg/L ZnSO.sub.4.7H.sub.2O, 0.446 mg/L MnSO.sub.4.H.sub.2O, 0.124 mg/L H.sub.3BO.sub.3, 0.096 mg/L Na.sub.2MoO.sub.4.2H.sub.2O, 0.096 mg/L KI and 7.00 mg/L CaCl.sub.2.2H.sub.2O. Final copper and iron concentrations are: 0.798 mg/L CuSO.sub.4 and 11.20 mg/L FeSO.sub.4.7H.sub.2O.
(58) The cultures are conducted in sealed closed reactors, incubated at 30° C. and under constant rotation (160 rpm). The headspace of the reactors is filled with a mixture of air and methane (1:1 v/v); the volume of gas is three times that of the liquid. The inoculation percentages of S. maltophilia can vary from 10 to 50% v/v. Cultures are stopped when the optical density at 600 nm (OD.sub.600 nm) is constant.
(59) Two successive cultures are conducted before implementing the consortium for the CO.sub.2 reduction reaction.
(60) (ii) Pure Aerobic Culture of the Biocatalyst
(61) The bacterium S. maltophilia was first isolated from a consortium formed with the bacterium M. trichosporium OB3b (obtained according to the protocol described above). The isolation was carried out by successive subcloning of S. maltophilia colonies on Lysogeny Broth (LB) Miller agar medium (Sigma Aldrich, France). The purity and identification of the isolated bacterium were confirmed by biochemical analyses (see point 2 below). The strain is preserved on LB agar plates stored both at 4° C. and in the freezer at −20° C. in its native liquid culture medium to which glycerol (20% v/v) is added. The culture reactor is a closed reactor with an air-permeable plug which is dedicated to sterile cultures. An LB liquid culture medium is inoculated with S. maltophilia, 5% v/v from a frozen aliquot or with all the colonies from an agar plate, then incubated for 24 h at 30° C. and under constant rotation (160 rpm). Cultures are stopped when the stationary phase starts (OD.sub.600 nm is constant).
(62) Two successive cultures are carried out before using the biocatalyst for the CO.sub.2 reduction reaction.
(63) A correlation between the OD.sub.600 nm (−) and the mass concentration [X] of S. maltophilia (g.sub.day cells.Math.L.sup.−1) is established to determine formate productivities; the correlation is obtained on the basis of five independently replicated points:
[X](g.sub.dry cells.Math.L.sup.−1)=0.3035×OD.sub.600 nm (R.sup.2=0.995)
2. Biochemical Analyses
(64) The Stenotrophomonas maltophilia isolate derived from co-culture was first characterized with Gram stain and by mass spectrometry. Gram staining showed that the isolate is a Gram-negative bacillus. Mass spectrometry analysis confirmed that the isolate was pure and identified the bacterium S. maltophilia, which is indeed a Gram-negative bacillus. In mass spectrometry, it should be noted that a score above 1.90 indicates reliable identification and that the score obtained for S. maltophilia is 2.18. Before each culture, a qualitative analysis of the S. maltophilia colonies spread on agar plates is performed in order to detect possible contamination. Furthermore, 16S RNA sequencing of the isolate confirmed that the identified bacterium is indeed Stenotrophomonas maltophilia.
(65) 3. Preparation of the Bacterial Suspension Used in the CO.sub.2 Reduction Tests
(66) At the end of the culture, the cells are collected by centrifugation at 4° C. and 4000 g for 20 min. They are then resuspended in 20 mM phosphate buffer at pH 7.0 and centrifuged again under the same conditions as before and the bacterial pellet is recovered. A reaction medium containing 20 mM phosphate buffer at pH 7.0 and 5 mM MgCl.sub.2 is used to resuspend the bacterial pellet and obtain an OD.sub.600 nm of (i) 10.3±0.5 (i.e. 3.1±0.2 g.sub.cells/L) for closed reactor assays and (ii) 6.6±0.3 (i.e. 2.0±0.1 g.sub.cells/L) for electrolysis-assisted tests. A sample of the prepared suspension is always stored at −20° C. with glycerol (20% v/v) for possible subsequent biochemical analyses. The remainder of the suspension is used immediately for CO.sub.2 reduction tests. The reaction medium does not contain any organic nutrients allowing the growth of the biocatalyst, which is this used as a resting cell.
(67) 4. .sup.13CO.sub.2.sup.3 Reduction Tests in a Closed Reactor
(68) The bacterial suspension obtained in the preceding step is distributed into sealed 60 mL reactors by adding 6 mL of suspension to each reactor. In order to evaluate the ability of the biocatalyst to reduce CO.sub.2, the headspace is filled with a gas mixture containing .sup.13CO.sub.2 which is sterilized by filtration through Teflon filters with a 0.2 μm pore diameter. Different gas mixtures were tested: .sup.13CO.sub.2:atmospheric air (3:7 v/v), .sup.13CO.sub.2: N.sub.2 (3:7 v/v) and pure .sup.13CO.sub.2. For each experiment, a set of several reactors was prepared under identical conditions and incubated at 30° C. with constant stirring (160 rpm). Each kinetics is followed for more than 20 days and up to 35 days; to this end, at each sampling time, one reactor is sacrificed for analysis. Different measurements are carried out: optical density at 600 nm (OD.sub.600 nm), pH, formate determination by gas chromatography coupled with mass spectrometry (GC-MS), characterization of the gas composition of the headspace by GC-MS, determination of intracellular poly-3-hydroxybutyrate (PHB) by GC-MS and determination of ammonium ions (NH.sub.4.sup.+) by ion chromatography (IC). The products derived from .sup.13CO.sub.2 are detected in the reaction medium and in the headspace by GC-MS and NMR. Counts of viable bacteria are also carried out at the beginning and end of the kinetics; to this end, samples are cultured under autotrophic aerobic conditions. Finally, blanks prepared with the reaction medium alone (without bacteria) under a .sup.13CO.sub.2:atmospheric air mixture (3:7 v/v) showed no contamination over the duration of the tests, confirming the sterility of the closed reactor tests.
(69) 5. CO.sub.2 Reduction Tests in a Semi-Closed Reactor, Assisted by Electrolysis
(70) A glass reactor is used. Its useful liquid volume is 60 mL and its headspace represents about 40% of its total volume. A lid is screwed onto the reactor and the tightness of the lid-reactor junction is guaranteed by a gasket. This lid has an inlet port for the gas supply (100% .sup.12CO.sub.2) which is made at the core of the solution by a gas distributor, a port for the gas outlet and 3 ports for the positioning of the electrodes. The reactor is thermostatically controlled at 30° C. under constant stirring (300 rpm).
(71) The electrochemical system is a conventional device with 3 electrodes (working electrode, reference electrode and counter electrode). The working electrode (or cathode) is a graphite coupon with a surface area of 2.5 cm×2.5 cm and a thickness of 0.5 cm (Goodfellow), electrically connected with a titanium rod (Goodfellow). Before each experiment, the graphite coupon is washed for 1 h in a 1 N HCl solution to dissolve any adsorbed species, then in a 1 N NaOH solution to neutralize the acidity and finally rinsed with sterile ultrapure water before being left overnight in 1 L of sterile ultrapure water to drive out any soda residue included in the graphite pores. The titanium rod is cleaned with acetone and then autoclaved. In this device, the ratio of the active surface of the cathode to the volume of the reactor is thus 10.4 m.sup.2/m.sup.3; where the active surface is defined as the geometrical surface exposed to the counter-electrode.
(72) The counter-electrode (or anode) is a platinum grid, previously cleaned and disinfected by flame. In this device, the ratio of the projected geometrical surface of the anode to that of the cathode is about 1.5 so as not to limit the cathodic phenomena.
(73) The potentials are monitored and expressed in relation to an Ag/AgCl reference electrode (potential of 0.240 V/ESH, Radiometer analytical).
(74) The polarization potential of the working electrode is applied with a one-channel potentiostat (Ametek VersaSTAT3) and the current is recorded every 900 s. Chronoamperometry (CA) is periodically stopped to acquire cyclic voltammetries (CVs) between −1.2 and 1.0 V vs Ag/AgCl, at a sweep rate comprised between 1 and 10 mV.Math.s.sup.−1. It should be recalled that a cyclic voltammetry (or cyclic voltamperometry) consists in performing a potential sweep at the working electrode and measuring the current flowing in the electrochemical system. This technique makes it possible to: (i) check the coherence of the CA with the CV, (ii) acquire kinetic information on the system and (iii) detect redox compounds in the suspension.
(75) First, the reactor is filled with a bacterial suspension of S. maltophilia in reaction medium (prepared according to 2.c), in which a continuous bubbling of 100% CO.sub.2 or a CO.sub.2:CH.sub.4 gas mixture (1:1 or 1:2 v/v) is carried out at a flow rate comprised between 2 or 25 ml.Math.min.sup.−1. Then a polarization ranging from −0.7 V to −1.0 V vs Ag/AgCl is applied. Polarization is started at the same time as chronoamperometry. In parallel, a control reactor without electrodes (and thus without polarization) is implemented in the same way as the electrochemical reactor. Samples are taken over time, under a microbiological hood, to measure the optical density at 600 nm (OD.sub.600 nm) and the pH of the liquid medium; the pH was found to be constant and equal to 6.4±0.2 (corresponding to the pKa of the CO.sub.2,H.sub.2O/HCO.sub.3.sup.− pair). The gas composition is analyzed at the reactor inlet and outlet by gas chromatography coupled with a katharometer (GC-TCD) to determine the experimental flow of reduced CO.sub.2.
(76) 6. Physicochemical Analyses
(77) Liquid Sample Processing
(78) The complete reaction medium (with bacteria) and the supernatant (without bacteria) are analyzed. The complete reaction medium is either directly analyzed or frozen at −20° C. upon collection for subsequent analysis. The supernatant is obtained by centrifugation of the complete reaction medium just collected; the centrifugation is conducted for 10 min at 10 000 g and 10° C. and the supernatant is collected as soon as the centrifugation is completed for immediate analysis or freezing at −20° C. The pellet is stored at −20° C. for subsequent analysis.
(79) Analyses Performed by Nuclear Magnetic Resonance (NMR):
(80) For these analyses, 500 μL of liquid sample (reaction conducted with .sup.13CO2, 2.d) is introduced into a 5 mm diameter NMR tube with 50 μL of D.sub.2O and then analyzed by a BRUKER NMR Avance III—500 MHz—CryoProbe Helium for 4 hours. Different standards (labeled and unlabeled sodium formate, sodium acetate, methanol, ethanol, formaldehyde, acetaldehyde, sodium lactate, glycerol, isobutanol, sodium succinate, sodium fumarate, sodium pyruvate, sodium oxaloacetate) prepared in the reaction medium were first analyzed by NMR to obtain the fingerprints of these molecules. On this apparatus, the detection threshold of .sup.13C-labeled molecules is 6 mg.Math.L.sup.−1. However, NMR analyses can only detect .sup.13C isotopes which have a relative abundance of 1.1% compared with their .sup.12C isotope in nature [W. Mook and P. Grootes, International Journal of mass Spectrometry and ion Physics, 1973, 12(3): p. 273-298]. Consequently, a concentration of .sup.13C unlabeled product of about 600 mg.Math.L.sup.1 is needed to detect these compounds.
(81) Analyses Carried Out by Gas Chromatography Coupled with Mass Spectrometry (GC-MS):
(82) Equipment
(83) Gas chromatography (Clarus 580, Perkin Elmer) is conducted with a capillary column (30 m×0.25 mm ID, film thickness 8 μm, Rt-Q-Bond Plot, Restek), coupled with mass spectrometry (Clarus SQ-8-MS, Perkin Elmer) equipped with a quadrupole mass selective electronic impact (EI) detector operated at 70 eV. A sample changer (Turbomatrix Headspace 16S, Perkin Elmer) is used for the injection of headspace obtained after heating liquid samples to 100° C. For the analysis of the composition of the headspace of the closed reactor maintained at 30° C., the samples are taken directly from the reactor with a gas-tight syringe and manually injected into the GC-MS. Helium is used as carrier gas at a flow rate of 1.5 ml/min.
(84) Determination of .sup.12C-Formate and .sup.13C Formate in the Liquid Samples
(85) An esterification method based on the protocol described by Wallage et al (2008) (Formic acid and methanol concentrations in death investigations, Journal of Analytical Toxicology, 32 (2008) 241-248) was used for the quantification of formate. The esterification reaction is carried out between the organic acid present in the liquid sample and the methanol added under acidic conditions. In a GC-MS analysis vial (22 mL), 600 μL of sample, 100 μL of acetonitrile (157.2 mg.Math.L.sup.−1) as internal standard, 100 μL of concentrated sulfuric acid and 100 μL of methanol as derivatizing agent are introduced in this order. The test vial is then placed in the autosampler and heated at 100° C. for 15 minutes in order to complete the esterification reaction. In the case of the analysis of the complete medium (with bacteria), a spreading of the mixture on agar made it possible to verify that these conditions allow the lysis of the bacteria because no re-growth was detected. The temperature of the injector is set at 200° C. and the split at 1:16. In the oven, a temperature gradient is achieved from 40 to 150° C., at a rate of 10° C./min. The ions detected, in single ion recording (SIR) mode, correspond to one of the mass/charge ratios m/z of 60 for .sup.12C-methyl formate and 41 for acetonitrile. In order to determine whether the carbon is .sup.12C or .sup.13C, [M+1] was also evaluated for .sup.13C-methyl formate (m/z of 61). The presence of labeled species is only considered for levels significantly above 1%, which therefore exceed the percentage related to natural isotope abundance. Calibration curves were first established with formate and acetate standards: Na(HCOO) and Na(H.sup.13COO) obtained from Sigma-Aldrich.
(86) Analysis of Gases in the Headspace of the Closed Reactor
(87) The headspace of the closed reactor is analyzed to determine the gaseous species, labeled with .sup.13C or not, present over time. For each analysis, 250 μL of headspace is taken from the reactor incubated at 30° C. using a gas-tight syringe and 50 μL is injected into the GC-MS. The temperature of the injector is set at 200° C. and the split at 1:16. In the oven, a temperature gradient is achieved from 100 to 150° C., at a rate of 10° C./min. In SIR mode, the ions detected are m/z 28 (N.sub.2), m/z 44 (CO.sub.2), m/z 45 (.sup.13CO.sub.2), m/z 16 (.sup.12CH.sub.4) and m/z 17 (.sup.13CH.sub.4).
(88) Determination of Ammonium Ions (NH.sub.4.sup.+) by Ion Chromatography (IC)
(89) NH.sub.4.sup.+ ions are determined in the supernatants. The analysis is performed by injecting 25 μL of sample into a Dionex ICS-1000 (Thermo Scientific) chromatographic device equipped with an IonPAc AS19 (0.4×250 mm) capillary column. The elution program is as follows: 10 mM KOH (from 0 to 10 min), then 10-45 mM KOH (from 10 to 30 min).
(90) Analyses Carried Out by Gas Chromatography Coupled to a Katharometer (GC-TCD)
(91) Equipment
(92) Gas chromatography (Clarus 580, Perkin Elmer) is conducted with a PE-Q column (Perkin Elmer, 30 m) in series with a PE-MOLESIEVE molecular sieve column (Perkin Elmer, 30 m) coupled to a thermal conductivity detector, also called a katharometer (Perkin Elmer). A 10-way loop valve is used to load and inject the gas samples.
(93) Analysis of the Composition of Gases, Labeled or not
(94) For each analysis, a minimum of 20 mL of gas to be analyzed is sent through the loop valve and 20 μL of sample is injected into the columns. Helium is the carrier gas at a flow rate of 10 mL.Math.min.sup.−1. In the oven, a temperature gradient is achieved from 40° C. to 120° C. at a rate of 10° C..Math.min.sup.−1. A calibration is performed to determine the CO.sub.2.
(95) Determination of the Mass % of Intracellular PHB
(96) The method based on PHB digestion and analysis of its monomer (3-hydroxybutyrate) by GC-MS (A. J. Pieja et al, Applied and environmental microbiology, 2011: p. AEM.00509-11; G. Braunegg et al., European journal of applied microbiology and biotechnology, 1978, 6(1): p. 29-37) was implemented, with the difference that the bacterial pellets were frozen instead of being lyophilized before the PHB digestion step.
(97) Bacterial pellets stored at −20° C. are thawed at room temperature and then digested for 3 h at 100° C. in 2 mL of methanol containing 3% concentrated sulfuric acid and 0.1% benzoic acid to which 2 mL of chloroform is added. Re-growth tests on LB medium showed that the bacteria contained in the pellets and treated by this acid digestion were totally inactivated. Once the sample is cooled, 1 mL of demineralized water is added to induce phase separation. The aqueous phase is then removed and 2 μL of organic phase is injected into the GC-MS. Analyses are conducted with the same GC-MS apparatus as that used for formate analysis. A DB-1 column (Agilent J&W) is used for the separation. The carrier gas is helium at a flow rate of 32 mL.Math.min.sup.−1. The oven temperature is programmed at 80° C. for 1 min, up to 120° C. at a rate of 10° C./min, then the temperature is increased to 270° C. at a rate of 45° C./min. The temperature of the injector is set at 200° C. and the split at 30 mL/min.
(98) The use of a PHB standard (Sigma Aldrich) makes it possible to establish a calibration curve giving the ratio of the areas of 3-hydroxybutyrate and benzoate peaks as a function of the mass of PHB introduced.
Peak area ratio (3-hydroxybutyrate/benzoate)=0.1675×m.sub.PHB (R.sup.2=0.9865)
(99) To determine the mass % of intracellular PHB per unit mass of dry cells (w.sub.PHP/w.sub.dry cells) in the samples, the PHB mass measured in the sample by the method described above is divided by the dry mass of bacteria present which is known from OD.sub.600 measurements.
(100) B] Results
(101) I. .sup.13CO.sub.2 Reduction Tests in a Closed Reactor
(102) In order to evaluate the ability of the biocatalyst to reduce CO.sub.2, .sup.13C-labeled CO.sub.2 is used. The purpose of CO.sub.2 labeling is to detect the labeled products derived from .sup.13CO.sub.2 and to distinguish them from those derived from a simple cellular release. To identify these products, the NMR technique is used to analyze the bacterial suspension and its supernatant while the GC-MS technique is used to characterize the reactor headspace. Moreover, this labeling makes it possible to observe the NMR and GC-MS fingerprints of all products having one or more .sup.13C that may result (i) from the reduction of .sup.13CO.sub.2 and/or (ii) from the fixation of .sup.13CO.sub.2 by the cell. The optical density at 600 nm (OD.sub.600 nm) is measured over time to monitor the change in the bacterial mass concentration (in g.sub.dry cells/L). Viable cell counts are also performed to access the bacterial cell concentration (in CFU/mL) and the pH is measured during the reaction. The ammonium ion (NH.sub.4.sup.+) content in the reaction medium is monitored to study the influence of the presence of ammonium on the performance of the CO.sub.2 reduction bioprocess.
(103) 1. Monitoring of .sup.13CO.sub.2 Assimilation Metabolism by NMR and GC-MS
(104) The bacterial suspension of Stenotrophomonas maltophilia is brought into contact with a gaseous atmosphere .sup.13CO.sub.2:atmospheric air (1:1 v/v).
(105) From the start of the reaction, two peaks are visible: that of .sup.13CO.sub.2 (chemical shift δ=124.6 ppm) and that of H.sup.13CO.sub.3.sup.− (chemical shift δ=160.2 ppm). After 8 days of reaction, a peak with a chemical shift corresponding to that of .sup.13C-formate (δ=171.0 ppm) appears.
(106) In order to verify that this peak indeed corresponds to the labeled .sup.13C-formate (H.sup.13COO), a small amount of .sup.13C-formate standard is added to the sample (at a final concentration of 10 mg/L). The amplitude of the peak increased when the standard was added, confirming that this peak is indeed attributable to .sup.13C-formate. No compounds other than .sup.13C-formate are detected by NMR, suggesting that .sup.13C-formate is the result of a direct reduction of .sup.13CO.sub.2. Indeed, .sup.13C-formate has a single carbon, at a lower oxidation state than .sup.13CO.sub.2. Blanks made without bacteria under the same conditions (i.e. under .sup.13CO.sub.2/atmospheric air mixture) show no labeled compounds, except .sup.13CO.sub.2 and H.sup.13CO.sub.3.sup.−, thus confirming the role of bacteria on the reduction of .sup.13CO.sub.2 to .sup.13C-formate.
(107) The reduction kinetics of .sup.13CO.sub.2 by the bacterium Stenotrophomonas maltophilia is reproduced and the liquid medium of the reactor is also analyzed at 35 days (
(108) Furthermore, GC-MS analysis of the headspace of several closed reactors reveals the presence of labeled methane (.sup.13CH.sub.4) in significant amounts after 34 days. Under the conditions tested, the content of this gas nevertheless remains below 1% v/v. Like .sup.13C-formate, .sup.13CH.sub.4 is a single-carbon compound with a lower oxidation state than .sup.13CO.sub.2. This .sup.13CH.sub.4 is therefore derived from the reduction of .sup.13CO.sub.2, either directly (.sup.13CO.sub.2.fwdarw..sup.13CH.sub.4) or indirectly (.sup.13CO.sub.2.fwdarw..sup.13C-formate.fwdarw..sup.13CH.sub.4). Methane is a particularly attractive product because it can be easily recovered by stripping and used as fuel.
(109) In conclusion, the bacterium S. maltophilia is capable of reducing CO.sub.2 to formate and methane. In order to quantify the production of .sup.13C-formate in the liquid reaction medium, .sup.13CO.sub.2 reduction tests are reproduced under similar conditions, considered as reference conditions. The .sup.13C-formate concentration is this time determined by GC-MS; in addition, the optical density at 600 nm (OD.sub.600) and pH are also monitored.
(110) 2. .sup.13CO.sub.2 Reduction Tests in a Closed Reactor Under Reference Conditions
(111) a. Definition of Reference Conditions
(112) The reference conditions are defined as follows: initial bacterial concentration of 3.1±0.2 g.sub.dry cells/L, initial gaseous atmosphere composed of a .sup.13CO.sub.2 atmospheric air mixture (3:7 v/v), aqueous reaction medium composed of 20 mM phosphate and 5 mM MgCl.sub.2. Three independent kinetics are conducted in parallel under these reference conditions.
(113) b. Monitoring of Biomass Concentration and pH
(114) The bacterial mass concentration, denoted [X], is monitored by measuring OD.sub.600. The correlation giving the OD.sub.600 as a function of bacterial mass concentration is given in section A.1]. For all three kinetics monitored, a decrease in biomass concentration is observed (data not shown). The most likely reason to explain this phenomenon is cell lysis. Indeed, a lack of natural nutrients and a CO.sub.2-rich atmosphere exposes bacterial cells to stressful conditions, which are likely to lead to the death and thus cell lysis of part of the bacterial population. Throughout the kinetics, however, no test shows total lysis. Indeed, for all experiments, the cell mass concentration, initially of 3.1±0.2 g.sub.dry cells.Math.L.sup.−1, falls over the first 10 days and then stabilizes at a mean value of 0.5±0.1 g.sub.dry cells/L. This means that only part of the biocatalyst suspension lyses during the first 10 days of the kinetics and then the mass concentration remains stable.
(115) In addition, the counting of viable cells in the bacterial suspension after 35 days confirms on the one hand that cell lysis is occurring and on the other that 15±4×10.sup.4 CFU/mL are still viable; the initial concentration being 60×10.sup.7 CFU/mL. The concentration of viable cells has therefore decreased significantly but still remains high (of the order of 10.sup.5 CFU/mL). It is hypothesized that this cell lysis may be a way for the bacteria to adapt to its new conditions thanks to the organic compounds released in the medium by the lysis of cells (J. M. Navarro Llorens et al., FEMS microbiology reviews, 2010, 34(4): p. 476-495).
(116) During these kinetics, the pH is also monitored (data not shown). Regardless of the experiment, the change in pH is very similar. Indeed, the initial pH is set at 7.0±0.1 and for all the tests, the pH decreases to an average value of 6.4±0.2 over the first 5 days and then remains constant at this value which corresponds to the pKa of the acid-base pair CO.sub.2,H.sub.2O/HCO.sub.3.sup.− induced by the dissolution of CO.sub.2 in the reaction medium. The pH of the medium is thus buffered throughout the kinetics.
(117) c. Monitoring of .sup.13C-Formate Production in the Liquid
(118) The .sup.13C-formate production from .sup.13CO.sub.2 reduction is quantified by GC-MS and its change over the three different kinetics is presented in
(119) Regardless of the kinetics, .sup.13C-formate is produced in significant amounts. Furthermore, GC-MS analysis has shown that .sup.13C-formate is found in complete suspensions (i.e. in the presence of bacteria) but not in the respective supernatants. This shows that the production of this .sup.13C-formate is carried out by the intact bacteria remaining in the reaction medium and not by free enzymes from cell lysis. The .sup.13C-formate appears after only 8 to 10 days, a period which may correspond to a latency phase during which the bacteria adapt to its new .sup.13CO.sub.2 substrate. The fact that a high concentration of bacteria (close to 10.sup.5 CFU/mL) is still viable at the end of the kinetics confirms the presence of bacteria maintaining metabolic activity.
(120) The .sup.13CO.sub.2 thus enters the cells, either in the .sup.13CO.sub.2 form or in the H.sup.13CO.sub.3.sup.− form; indeed, the pH stabilizes around the pKa of the CO.sub.2,H.sub.2O/HCO.sub.3.sup.− pair and the two forms thus coexist in close proportions. Then the cells having survived the reaction conditions and lysis thus use one of their intracellular enzymatic system to catalyze the reaction of reduction of this .sup.13CO.sub.2 to .sup.13C-formate. Two families of enzymes, potentially present inside the bacterium S. maltophilia, are likely to catalyze this CO.sub.2 reduction reaction to formate: the formate dehydrogenases (FDHs) and the nitrogenases. At present, these enzymes are first isolated from their native microorganism and then purified before being used (L. B. Maia et al., Inorganica Chimica Acta, 2017, 455: p. 350-363; Khadka et al. 2016, Inorg. Chem., 55, 8321-8330).
(121) It should be stressed that the maximum formate concentrations produced (10-20 mg.Math.L.sup.−1,
(122) d. Production P.sub.formate of .sup.13C-Formate and Volume Flow F.sub.CO2,vol of Reduced .sup.13CO.sub.2
(123) Regardless of the kinetics considered (
P.sub.formate=ΔC/Δt/C.sub.bacteria (Equation 4)
with Δt: production period (days),
ΔC: difference in .sup.13C-formate concentration over the production period (mg.Math.L.sup.−1 or μmol.Math.L.sup.−1),
C.sub.bacteria: bacterial concentration initially introduced (C.sub.bacteria=3.1±0.2 g.sub.dry cells.Math.L.sup.−1) to take into account the bacterial supply required.
(124) Similarly, if it is considered that one mole of .sup.13C-formate produced corresponds to one mole of reduced .sup.13CO.sub.2, then the flow FCO.sub.2 of reduced .sup.13CO.sub.2 in μmol .sup.13CO.sub.2.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1 will therefore be equal to the formate production P.sub.formate expressed in μmol .sup.13C-formate.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1. The volume flow F.sub.CO2,vol of reduced .sup.13CO.sub.2 expressed in mL .sup.13CO.sub.2.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1 will be calculated according to the following equation:
F.sub.CO2,vol=F.sub.CO2.Math.v.sub.m (Equation 5)
where v.sub.m is the molar volume at 30° C. of the .sup.13CO.sub.2 assumed to be perfect gas (25.19 10.sup.−3 mL.Math.μmol.sup.−1).
(125) Table 1 details the values of ΔC and Δt for each of the three kinetics presented above, as well as the productions of .sup.13C-formate (P.sub.formate) and the flows of reduced .sup.13CO.sub.2 (F.sub.CO2,vol).
(126) TABLE-US-00001 TABLE 1 Production of .sup.13C-formate (P.sub.formate) and flows of reduced .sup.13CO.sub.2 (F.sub.CO2, vol) obtained in a closed reactor P.sub.formate P.sub.formate (mg .sup.13C- (μmol .sup.13C- F.sub.CO2, vol ΔC Δt formate .Math. formate .Math. (mL .sup.13CO.sub.2 .Math. Kinetics (mg .Math. L.sup.−1) (j) (g.sub.dry cells).sup.−1 .Math. d.sup.−1) (g.sub.dry cells).sup.−1 .Math. d.sup.−1) (g.sub.dry cells).sup.−1 .Math. d.sup.−1) 1 (blue) 9.4 7 0.5 10.0 0.3 2 (red) 18.8 7 0.9 20.0 0.5 3 (green) 7.7 7 0.4 8.2 0.2 Average 11.8 ± 5.6 7 0.6 ± 0.3 12.7 ± 6.3 0.3 ± 0.2
(127) e. Production of .sup.13C-Formate from .sup.13CO.sub.2: Reproducibility Study
(128) In order to study the reproducibility of this bioprocess, six other independent .sup.13CO.sub.2 reduction tests were conducted under reference conditions, using bacterial suspensions prepared from different S. maltophilia cultures. For each of these tests, a significant production of .sup.13C-formate was demonstrated. Applying the calculations in the previous section (Eq. 4 and 5) to the results obtained during these kinetics (6 in total), a mean production P.sub.formate of 9.1 μmol .sup.13C-formate.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1 was obtained (i.e. 0.4 mg formate.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1), with a minimum and maximum respectively of 2.3 and 30 μmol .sup.13C-formate.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1 (i.e. 0.1 and 1.4 mg .sup.13C-formate.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1).
(129) Similarly, a mean volume flow F.sub.CO2,vol of reduced .sup.13CO.sub.2 of 0.24 mL .sup.13 CO.sub.2.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1 was obtained, with a minimum and maximum respectively of 0.06 and 0.76 mL .sup.13CO.sub.2.Math.(g.sub.dry cells).sup.−1.Math.d.sup.−1; these differences in flow being certainly related to the differences in physiological state of the bacteria used for the .sup.13CO.sub.2 reduction tests.
(130) f. Production of .sup.13H.sub.4 from .sup.13CO.sub.2: Reproducibility Study
(131) The production of .sup.13CH.sub.4 from .sup.13CO.sub.2 was evaluated at 34 days in 4 independent .sup.13CO.sub.2 reduction tests, conducted under reference conditions, with bacterial suspensions prepared from different S. maltophilia cultures. In all cases, a significant production of .sup.13CH.sub.4 was obtained, with .sup.13CH.sub.4 levels in the reactor headspace varying between 1 to 3% v/v.
(132) 3. Effect of Various Parameters on the Performance in Reduction of .sup.13CO.sub.2
(133) Various parameters that can potentially influence the performance of the CO.sub.2 reduction reaction are studied. To this end, tests are conducted under modified reaction conditions, in comparison with reference conditions. A test under reference conditions is always carried out simultaneously with the study of the influence of a parameter to determine the impact of the modified parameter.
(134) a. Effect of Biomass Concentration
(135) The initial mass concentration of the bacterial suspension used for the .sup.13CO.sub.2 reduction tests is set at 3.1±0.2 g.sub.dry cells/L. Indeed, under the culture conditions used, this concentration is currently the highest possible one making it possible to launch different closed reactors and different series of closed reactors. In the end, the volumes and concentrations of bacteria produced can be increased. A lower concentration (1.6±0.1 g.sub.dry cells/L) is tested to verify that the amount of .sup.13C-formate produced is correlated with the amount of cells; the other reaction parameters remain unchanged. The kinetics are monitored over 25 days.
(136) Table 2 summarizes the results obtained.
(137) TABLE-US-00002 TABLE 2 Initial [X.sub.0] and final [X.sub.f] biomass mass concentrations (in g.sub.dry cells/L), biomass loss Δ[X] at the end of the reaction (in %) and final accumulated .sup.13C-formate concentrations derived from .sup.13CO.sub.2 reduction (in mmol/L and mg/L). [X.sub.0] [X.sub.f] Δ[X] [.sup.13C-formate] [.sup.13C-formate] (g.sub.dry cells/L) (g.sub.dry cells/L) (%) (mmol/L) (mg/L) 3.1 1.1 65 0.65 29.3 1.6 0.7 56 0.03 1.4
(138) Table 2 shows that the concentration of .sup.13C-formate is nearly 20 times higher when the initial concentration of bacteria is doubled. Contrary to what could be expected, the final accumulated concentration of .sup.13C-formate is therefore not correlated to the amounts of cells initially introduced. On the other hand, it is interesting to note that doubling the initial amount of bacteria also doubles the final amount of lysed cells (Table 2, difference between [X.sub.0] and [X.sub.f]). It is therefore possible that the compounds released into the reaction medium by cell lysis could be used by the surviving cells to produce a higher amount of intracellular CO.sub.2-reducing enzyme, which would therefore lead to an increase in .sup.13C-formate production.
(139) In conclusion, the availability of intracellular compounds in the reaction medium seems to be a limiting factor for CO.sub.2 reduction performance when the initial bacterial concentration is too low (1.6 g.sub.dry cells/L).
(140) b. Influence of the Nature of the Gas Mixture
(141) Tests with different gas mixtures are carried out to study the influence of the presence of oxygen (O.sub.2) and nitrogen (N.sub.2) on the CO.sub.2 reduction reaction. The concentration of .sup.13C-formate is monitored.
(142) Different initial gas mixtures containing .sup.13CO.sub.2 were tested: (i) the reference mixture composed of .sup.13CO.sub.2:atmospheric air (3:7 v/v), (ii) a .sup.13CO.sub.2:N.sub.2 mixture (3:7 v/v) to study the influence of oxygen and (iii) pure .sup.13CO.sub.2 to study the effect of nitrogen. These tests were all repeated twice.
(143) The results show that the absence of oxygen and the presence of nitrogen accelerate the appearance of .sup.13C-formate in the medium. Indeed, the production of .sup.13C-formate occurs on average 14±2 days earlier under the .sup.13CO.sub.2:N.sub.2 (3:7 v/v) mixture, compared with the .sup.13CO.sub.2:atmospheric air mixture (3:7 v/v) and with pure .sup.13CO.sub.2. On the other hand, whatever the gas ceiling used, the maximum concentration of .sup.13C-formate accumulated in the medium over a reaction time of 31 days remains of the same order of magnitude (around 0.1 mmol/L). Oxygen is reported to be a potential inhibitor of nitrogenase and FDH activities because it can act as an electron acceptor and lead to the formation of reactive oxygen species (J. Gallon, Trends in Biochemical Sciences, 1981, 6: p. 19-23) but it can also affect the synthesis of nitrogenase (Q. Liu et al., Nature communications, 2015, 6: p. 5933). The decrease in performance observed in the presence of O.sub.2 is therefore unsurprising.
(144) In conclusion, using a .sup.13CO.sub.2:N.sub.2 mixture is favorable to the reaction kinetics of .sup.13CO.sub.2 reduction.
(145) c. Influence of the Composition of the Reaction Medium
(146) New .sup.13CO.sub.2 reduction tests are carried out with an ammonium ion enriched reaction medium (AERM). The AERM contains 2 mmol/L KCl, 28 mmol/L NH.sub.4Cl, 30 mmol/L NaHCO.sub.3 and 5 mmol/L NaH.sub.2PO.sub.4. The .sup.3C-formate and ammonium ion (NH.sub.4.sup.+) concentrations are monitored throughout the reaction (
(147) When ammonium ions are initially present in large amounts, almost half of the initial amount is consumed over the first 8 days, then the concentration seems to stabilize (
(148) Concerning .sup.13C-formate production, it is almost 2.5 times higher at 24 days (or a concentration of 1.45 mmol/L, i.e. about 65 mg/L) and occurs about 7 days earlier (
(149) It is therefore also possible to make upstream adjustments of the culture conditions of the bacterium S. maltophilia and/or genetic modifications of the bacterium to overexpress the quantity of its CO.sub.2-reducing enzymatic system and benefit from a higher intrinsic specific CO.sub.2 reduction activity.
(150) d. Study of the Reversibility of the CO.sub.2 Reduction Reaction
(151) This study showed that the bacterium S. maltophilia is capable of reducing CO.sub.2 to formate. Up to now, the maximum accumulated formate concentration is about 1.5 mmol/L (i.e. 67.5 mg/L). In order to study the reversibility of the reaction of this catalytic system, i.e. its ability to oxidize formate to CO.sub.2, a sodium .sup.13C-formate concentration of 1.5 mmol/L is introduced into a suspension of S. maltophilia prepared as for a CO.sub.2 reduction test (section A] 3). This suspension is then introduced into an NMR tube and analyzed by NMR (section A] 6). If the reduction equilibrium of .sup.13CO.sub.2 is reversible, then oxidation of .sup.13C-formate (H.sup.13COO.sup.−) to .sup.13CO.sub.2 should be able to occur because .sup.13CO.sub.2 is initially a minority:
H.sup.13COO.sup.−.Math..sup.13CO.sub.2 (Equation 6)
(152) The NMR spectrum obtained after 4 h is shown in
(153) Only two peaks are visible, corresponding respectively to the .sup.13C-formate (δ=171.0 ppm) and .sup.13C-hydrogen carbonate (δ=160.2 ppm) ions. This result confirms that when the .sup.13C-formate is predominant over .sup.13CO.sub.2, then the .sup.13C-formate can be oxidized to .sup.13CO.sub.2, which solubilizes as hydrogen carbonate (H.sup.13CO.sub.3.sup.−). Indeed, in the case of this test, the pH of the reaction medium (7.0±0.1) is higher than the pKa of the CO.sub.2,H.sub.2O/HCO.sub.3.sup.− pair (6.4) because the medium is not buffered by the presence of .sup.13CO.sub.2. As was expected, the reaction catalyzed by the bacterium S. maltophilia thus seems to be regulated by the concentration of the most abundant substrate (CO.sub.2 in the direction of reduction and formate in the opposite direction). Moreover, it is interesting to note that the presence of these two NMR peaks only, and thus the absence of other peaks, confirms that the formate produced under these reaction conditions is not used by the bacterium to form biomass or enzymes (section B] I.1). Continuous removal of formate is therefore a possible option to promote the CO.sub.2 reduction reaction.
(154) e. Influence of the Addition of PHB to the Reaction Medium
(155) The enzymatic reduction of CO.sub.2 requires a source of protons and electrons. Ammonium ions (NH.sub.4.sup.+) not being oxidized to nitrate or nitrite ions, it is therefore unlikely that they serve as electron and proton donors for the CO.sub.2 reduction reaction. The source of protons and electrons is most likely accumulated in the cell during the culture phase of the bacteria as an energy reserve. At present, two types of molecules have been identified as being able to play this role: poly-3-hydroxybutyrate (PHB) which is a natural polymer and lipids. As far as PHB is concerned, bacterial cells are indeed able to depolymerize PHB to its monomer (3-hydroxybutyrate) and to oxidize this monomer to acetoacetate in order to recover electrons and protons but also a source of carbon (S. Obruca et al., PLoS One, 2016, 11(6): e0157778).
(156) To verify the role of the PHB, it is initially added to the bacterial suspension and the CO.sub.2 reduction reaction is then carried out under the reference conditions (described in section B] 1.2). However, PHB is a high molecular weight polymer and insoluble in water, which makes impossible its transport and its assimilation in the cell in its polymerized form. However, the bacterium S. maltophilia is capable of excreting depolymerases that can depolymerize PHB to its bacterially assimilable 3-hydroxybutyrate monomer (S. Wani et al., 3 Biotech, 2016, 6(2): p. 179-184; B. Tiwari et al., Bioresource technology, 2016, 216: p. 1102-1105).
(157) To determine the amount of PHB to be added, the intracellular PHB contents of the bacterial suspensions used for the tests is first measured by the method detailed in section A] 6. A mean intracellular PHB mass content of 1.0±0.1% w/w is obtained, corresponding to a PHB concentration of 30±2 mg/L, which is rather common for S. maltophilia species (B. Iqbal et al., Annual Research & Review in Biology, 2016, 9(5): p. 1). A 10-fold higher concentration (300 mg/L, i.e. 0.3 g/L) is then used for testing to avoid PHB limitation.
(158) Two independent series of closed reactors are prepared, using two different bacterial cultures. For each test, kinetics are conducted in parallel under the reference conditions (i.e. without addition of PHB). In all the tests, the gaseous atmosphere of the reactors is thus initially composed of a .sup.13CO.sub.2:atmospheric air mixture (3:7 v/v). For the first series, monitoring is carried out over 20 days and up to 40 days for the second series. The .sup.13C-formate concentrations as well as the intracellular PHB concentrations are measured during the kinetics.
(159) Table 3 presents the results obtained initially then at 20, 30 and 35 days. It is important to point out that PHB measurements for kinetics in which PHB is added are not indicated because they are not reproducible, likely due to a lack of homogeneity in the samples taken from the reactors (indeed, PHB is not soluble in water).
(160) TABLE-US-00003 TABLE 3 Concentrations of .sup.13C-formate ([.sup.13C-formate] in μmol/L) and intracellular PHB ([PHB.sub.intra] in mg/L). Time (days) 0 20 30 35 Series 1 [.sup.13C-formate] (μmol/L) 0 103 — — Reference [PHB.sub.intra] (mg/L) 31 13 — — Without PHB addition Series 1 [.sup.13C-formate] (μmol/L) 0 298 — — With PHB addition [PHB.sub.intra] (mg/L) NM NM — — Series 2 [.sup.13C-formate] (μmol/L) 0 46 7 7 Reference [PHB.sub.intra] (mg/L) 28 13 12 13 Without PHB addition Series 2 [.sup.13C-formate] (μmol/L) 0 4 249 660 With PHB addition [PHB.sub.intra] (mg/L) NM NM NM NM NM means Not Measurable and (—) means Not Measured.
(161) Concerning .sup.13C-formate production, production is much higher when PHB is initially added to the reaction medium. Compared with the reference conditions (without PHB addition), the concentration of .sup.13C-formate produced in the presence of PHB is indeed increased by a factor between 3 and 95 (Table 3, series 1 and 2 respectively). This result shows that the addition of PHB improves the performance of the CO.sub.2 reduction reaction and suggests that it could be an electron and proton donor for this reaction.
(162) A PHB limitation in the reference conditions could explain why the production of .sup.13C-formate is improved when the medium is enriched in PHB. Indeed, under the reference conditions (without PHB addition), the intracellular PHB concentration decreases from 30±2 to 13±1 mg/L regardless of the kinetics and then remains constant (Table 3, series 1 and 2). For both series, it thus appears that about 20 mg/L of intracellular PHB is consumed by the cells during the reaction. Moreover, the PHB limitation is probably not the only parameter to impact the production of .sup.13C-formate. Indeed, under the reference conditions and for the same amount of PHB consumed after 20 days of reaction, the .sup.13C-formate concentration obtained at 20 days is almost twice as high for series 1 as for series 2 (Table 3). This can be explained by the fact that these series were made from two different cultures and that the physiological state of the bacteria was not the same. However, the physiological state can play a role on the intrinsic CO.sub.2-reducing activity of the bacteria but also on its ability to adapt to its new CO.sub.2 substrate.
(163) In conclusion, the amount of intracellular PHB is a limiting factor for the CO.sub.2 reduction reaction and for the production of .sup.13C-formate. The PHB is thus likely a source of electrons and protons for the CO.sub.2 reduction reaction.
(164) 4. Study of Synergistic Effects with Other Bacteria
(165) The bacterium S. maltophilia was put in consortium with bacteria likely to use formate to study their possible synergies.
(166) First, a consortium formed by the bacterium S. maltophilia and the methanotrophic bacterium M. trichosporium OB3b, obtained after culture on methane (according to the protocol described in section A] 1), was implemented under the CO.sub.2 reduction reaction conditions detailed in section A] 4. NMR analysis of the reaction medium was conducted initially and after 8 days. The NMR spectra obtained are presented in
(167) From the beginning (t=0 days) and throughout the reaction, the resonance peaks of the .sup.13CO.sub.2 (δ=124.6 ppm) and its ionic form H.sup.13CO.sub.3.sup.− (δ=160.2 ppm) are still visible (
(168) No other labeled products were found in the NMR spectrum (
(169) This result therefore suggests that the presence of this methanotrophic bacterium in the reaction medium does not interfere with CO.sub.2 reduction and formate production by S. maltophilia. For certain applications, co-culturing the S. maltophilia biocatalyst with the methanotrophic bacterium M. trichosporium OB3b can therefore be considered to guarantee an optimal carbon balance since methane, of renewable origin, would be the sole source of carbon necessary for the culture.
(170) Second, a consortium formed by the bacterium S. maltophilia and the Gram-positive bacillus type bacterium Microbacterium oxydans, obtained after culture on LB medium (according to a protocol similar to that described in section A] 1), was implemented under the CO.sub.2 reduction reaction conditions detailed in section A] 4. NMR analysis of the reaction medium was performed at 5, 8 and 12 days (
(171) Like the initial spectrum obtained with the S. maltophilia/M. trichosporium OB3b consortium, only the peaks of the substrates (.sup.13CO.sub.2 and H.sup.13CO.sub.3.sup.−) are visible at t=0 days.
(172) After 5 days of reaction, a formate peak appears (δ=170.9 ppm,
(173) After 8 days of reaction, the amplitude of the formate peak increases significantly (by a factor of about 10), which means that the concentration of formate produced is higher than at 5 days (
(174) After 12 days of kinetics, the amplitude of the NMR peak relative to the formate decreases significantly (by a factor of about 10) compared with the peak obtained at 8 days (
(175) A possible explanation for these observations is the establishment of a synergy between the two bacteria present. Indeed, S. maltophilia can reduce CO.sub.2 to formate, then excrete the formate produced outside its cell so that M. oxydans can use it and form new compounds necessary for its subsistence such as lactate or other products whose fingerprints are comprised in the peak forests. Nevertheless, NMR analysis of the supernatant revealed only the signals from the .sup.13CO.sub.2 and H.sup.13CO.sub.3.sup.− substrates but not that of the .sup.13C-formate. A probable hypothesis is that the M. oxydans bacterium captures and introduces the formate produced in its cells as soon as the formate has been excreted by the S. maltophilia bacterium, which would explain the significant decrease of the .sup.13C-formate peak observed at 12 days (
(176) II. CO.sub.2 Reduction Test in a Semi-Closed Reactor, Assisted by Electrolysis
(177) 1. Electrochemical Device and its Principle
(178) The polymer poly-3-hydroxybutyrate (PHB) is depolymerized to 3-hydroxybutyrate which is an identified electron and proton donor for the CO.sub.2 reduction reaction (section B] I.3.e]). Nevertheless, the intracellular stock of PHB is finite. The idea is therefore to provide without limitation and continuously the electrons and protons required for the reaction by virtue of an inexpensive in situ electrolytic device, so that the performance of the CO.sub.2 reduction reaction is improved and lasting over time. This electrochemical device therefore aims at replacing intracellular proton and electron donors. A semi-closed electrolysis-assisted reactor (or bioelectrolyzer) was thus implemented to intensify the CO.sub.2 reduction reaction catalyzed by the bacterium S. maltophilia.
(179) In this 3-electrode device (
(180) First, the polarization voltage is chosen on a thermodynamic basis. The oxidation-reduction potential of the acetoacetate/3-hydroxybutyrate pair being −0.53 V vs Ag/AgCl at pH 6.4 (P. A. Loach, Handbook of Biochemistry and Molecular Biology, 1976, 1: p. 122), it is therefore necessary to initially impose a higher potential (by absolute value) than this potential (such as for example −0.7 V vs Ag/AgCl) so that the bacterium has an energetic interest in using the electrons of the electrode rather than those coming from the oxidation of 3-hydroxybutyrate. Nevertheless, if the stock of intracellular PHB, energy source and 3-hydroxybutyrate is depleted during the reaction by the general metabolism of the bacterium, this constraint on the potential will be lifted and a potential lower (by absolute value) than −0.53 V vs Ag/AgCl at pH 6.4 can be implemented. Furthermore, the selected potential is preferentially chosen lower than those of the oxidizer-reducer pairs CO.sub.2/CO.sub.2,reduced involving CO.sub.2 as oxidant so as to favor the reduction of CO.sub.2 at the cathode; the choice of the polarization potential also makes it possible to favor the production of a reduction product CO.sub.2,reduced compared with other possible CO.sub.2 reduction products (among methane and formate for example). In general, a potential greater than or equal to −0.8 V vs Ag/AgCl (for example −0.7 V vs Ag/AgCl) will (i) limit the reduction of the proton to hydrogen (H.sub.2) and (ii) avoid the reduction of phosphates and magnesium ions present in the reaction medium. The minimum potentials below which proton and CO.sub.2 reductions occur can be precisely determined by cyclic voltammetry, under the reaction conditions, to take into account both thermodynamic and kinetic phenomena (overvoltages); overvoltages depending in particular on the material chosen for the cathode.
(181) According to the literature, the nature of the material can also have a significant impact on the establishment of electron transfer between the bacteria and the working electrode (M. Rosenbaum et al., Bioresource Technology, 2010, 102(1): p. 324-333). The chemical composition and the surface structure can indeed induce different electrochemical responses. Among the numerous electrode materials available, graphite is porous and has a surface roughness. Graphite is therefore likely to promote contact between the bacteria and the electrode, which could promote electronic conduction (M. Rosenbaum et al., Bioresource Technology, 2010, 102(1): p. 324-333). Graphite is therefore chosen as the cathode material for the tests presented. In addition, a platinum anode is chosen for these tests in order to reduce the anode overvoltages and not to limit the cathodic phenomena. Other anode materials can also be considered, such as steel. An anode surface larger than the cathode surface is also preferred so as not to limit the reduction reactions at the cathode.
(182) Furthermore, a continuous bubbling of pure CO.sub.2 is also implemented to saturate the suspension with CO.sub.2 and deoxygenate the solution so as to (1) avoid competition with the aerobic metabolic pathway (where O.sub.2 would become an electron acceptor instead of CO.sub.2) and (2) preserve the catalytic activity of the CO.sub.2-reducing enzyme (indeed, oxygen seems to have an inhibitory effect on this enzyme, section B] I.3.b). In order to guarantee an optimal gas-liquid mass transfer, the CO.sub.2 supply is achieved by a porous gas distributor and a volume of liquid (allowing a high volume of gas per volume of liquid per minute, or VVM) is used. A high cathode surface/liquid volume ratio is also desirable to concentrate the product formed by CO.sub.2 reduction at the cathode; a cathode surface/liquid volume ratio of around 10 m.sup.2/m.sup.3 is desirable.
(183) 2. A First Example of a Bioelectrolyzer Fed by Pure CO.sub.2
(184)
(185) The initial zero current (0.0±0.01 A/m.sup.2) corresponds to the baseline (
(186) Then, a small reduction current is observed (to −0.2 A/m2 at 1.2 days). However, this current returns to the baseline before a significant reduction current appears from the second day forward (
(187) In order to experimentally verify the dependence of this reduction current on CO.sub.2, bubbling of an inert noble gas (argon) was carried out with the aim of driving out all or part of the dissolved CO.sub.2. A first argon bubbling at a flow rate of 10 mL.Math.min.sup.−1 is carried out for 3 h and resulted in a near instantaneous reduction of the reduction current by a factor of about 2, from −1.4 to −0.8 A/m.sup.2 (
(188) When a low CO.sub.2 flow rate is reapplied (5 mL.Math.min.sup.−1), then the reduction current increases again, until it returns to its initial stable value, i.e. −1.4 A/m.sup.2
(189) The reaction that occurs at the cathode is therefore:
CO.sub.2+H.sup.++2e−.fwdarw.HCOO.sup.− (Equation 7)
(190) This reduction reaction results from the enzymatic activity of the cell which no longer requires intracellular electron and proton donors.
(191) Cyclic voltammetries (CVs) are also performed during the experiment. In particular, a CV performed at 5.7 days confirms the adequacy of the measured current with the imposed polarization potential (data not shown). This voltammetry also shows a diffusion step for the reduction current ranging from −0.2 V to −0.8 V vs Ag/AgCl, which suggests that a lower polarization voltage per absolute value (for example −0.2 V vs Ag/AgCl) may be sufficient to obtain the same reduction current.
(192) In conclusion, this experiment demonstrates the ability of this bioelectrolytic device to replace the intracellular electron and proton donors necessary for the reduction of CO.sub.2 catalyzed by the bacterium S. maltophilia.
(193) 3. Reproducibility Study
(194) In addition to the test presented above, four other independent bioelectrolyzer tests according to the mode described by the Invention confirmed, by alternating pure CO.sub.2/argon/pure CO.sub.2 bubbling, that the CO.sub.2 is indeed reduced at the cathode and that it generates a significant CO.sub.2-dependent reduction current (data not shown). During these tests, two polarization potentials were tested: −0.7 V and −0.8 V vs Ag/AgCl and CO.sub.2 reduction current densities comprised between −0.5 and −1.5 A/m.sup.2 were obtained.
(195) Furthermore, these tests validated the fact that the CO.sub.2 reduction current stabilizes after a few days of CO.sub.2 bubbling and that after argon bubbling, CO.sub.2 bubbling allows the current to return to its stable value (reached before argon bubbling). This demonstrates the robustness of this bioelectrolysis system.
(196) 4. An Example of a Bioelectrolyzer Fed by a CO.sub.2/CH.sub.4 Mixture
(197) An additional bioelectrolyzer test demonstrated that the presence of methane (CH.sub.4) in the gas mixture that contains CO.sub.2 and that feeds the bioelectrolyzer has no influence on the CO.sub.2-reducing activity of the bacterium S. maltophilia at the cathode (data not shown). Indeed, a CO.sub.2/CH.sub.4 bubbling (1:1 or 1:2 v/v) maintains a reduction current equivalent to that obtained under pure CO.sub.2 under the same conditions. This result is particularly attractive in the treatment of biogas, containing high levels of CH.sub.4 and CO.sub.2.
(198) GC-TCD analysis of the bioelectrolyzer inlet and outlet gases measured an experimental volume flow of reduced CO.sub.2 of 1.3 mL CO.sub.2/min for a 60 mL reactor volume (thus equivalent to a flow of 1.9 m.sup.3 CO.sub.2/d for a 60 L reactor volume). This flow is nearly 60 times higher than the theoretical volume flow of reduced CO.sub.2 (F.sub.CO2,vol) calculable on the basis of the CO.sub.2 reduction current obtained (by Faraday's law). This means that it is not only the bacteria adhering to the cathode that participate in the CO.sub.2 reduction, but also the planktonic bacteria (suspended in the bioelectrolyzer liquid). A possible fixation of this CO.sub.2 by planktonic bacteria cannot be excluded.
(199) Finally, it is important to note that the experimental volume flow of reduced CO.sub.2 in the bioelectrolyzer (i.e. 1.3 mL CO.sub.2/min) is 130 times higher than that obtained in a closed reactor (i.e. 0.6 mL CO.sub.2/d=0.01 mL CO.sub.2/min for an initial bacterial concentration of 2 g.sub.dry cells/L, Table 1). This significant increase in the bioelectrolyzer can be justified by (1) an improved CO.sub.2 mass transfer thanks to the gas supply within the suspension and (2) an electrochemical assistance that provides the bacteria with an inexhaustible source of electrons and protons.