process for the co-production of proteins and biomethane

Abstract

A method for co-producing proteins and biomethane comprising at least the steps of: producing biogas by methanization of methanogenic inputs, separating the biogas produced into biomethane and a carbon dioxide-rich stream containing at least 93 mol% carbon dioxide and up to 7 mol% methane, introducing the carbon dioxide-rich stream into a reactor comprising at least one bacterial strain and an aqueous culture medium, and culturing said at least one bacterial strain in said reactor to obtain a biomass comprising proteins.

Description

Title of the invention: process for the co-production of proteins and biomethane

[0001] The present invention relates to a method for co-producing proteins and biomethane.

[0002] The invention relates more particularly to a method for the co-production of proteins and biomethane using a reactor for the production of proteins of unicellular origin by the integration of carbon dioxide coming from the vent of a biogas purification unit.

[0003] Biogas is the gas produced during the degradation of organic matter in the absence of oxygen (anaerobic fermentation), also called methanization. It can be a natural degradation - it is observed in marshes or household waste dumps - but the production of biogas can also result from the methanization of waste in a dedicated reactor, the conditions of which are controlled, called a methanizer or digester, then in a post-digester, similar to the digester and allowing the methanization reaction to be pushed further. Biogas is the gas produced during the degradation of organic matter in the absence of oxygen (anaerobic fermentation), also called methanization.This can be a natural degradation - we observe it in marshes or household waste dumps - but the production of biogas can also result from the methanization of waste in a dedicated reactor, the conditions of which are controlled, called a methanizer or digester, then in a post-digester, similar to the digester and allowing the methanization reaction to be pushed further.

[0004] The digester, that is to say the reactor dedicated to the methanization of organic materials, is a closed tank, heated or not (operation at a fixed temperature, between ambient temperature and 55°C) and whose contents consisting of organic materials are mixed, continuously or sequentially. The conditions in the digester are anaerobic and the biogas generated ends up in the head space of the digester (gaseous sky), where it is collected. Post-digesters are similar to digesters.

[0005] Due to its main constituents - methane and carbon dioxide - biogas is a powerful greenhouse gas; it also constitutes, at the same time, a significant source of renewable energy in a context of increasing scarcity of fossil fuels.

[0006] Biogas mainly contains methane (CH4) and carbon dioxide (CO2) in variable proportions depending on the method of production, but also, in smaller proportions, water, nitrogen, hydrogen sulfide, oxygen, as well as other organic compounds, in trace amounts.

[0007] Depending on the degraded organic matter and the techniques used, the proportions of the components differ, but on average the biogas contains, on dry gas, 30 to 75% methane, 15 to 60% CO2, 0 to 15% nitrogen, 0 to 5% oxygen and trace compounds.

[0008] Biogas is used in different ways. After light treatment, it can be used close to the production site to provide heat, electricity or a mixture of the two (cogeneration); the high carbon dioxide content reduces its calorific value, increases compression and transport costs and limits the economic interest of its use to this local use.

[0009] Further purification of biogas allows its wider use, in particular, further purification of biogas makes it possible to obtain a purified biogas to the specifications of natural gas and which can be substituted for it; the biogas thus purified is "biomethane". Biomethane thus supplements natural gas resources with a renewable part produced in the heart of the territories; it can be used for exactly the same purposes as natural gas of fossil origin. It can supply a natural gas network, a vehicle filling station, it can also be liquefied to be stored in the form of liquid natural gas (LNG).

[0010] Thus when biomethane is produced, carbon dioxide is also generated in significant quantities. However, carbon dioxide is currently little used and is often released into the atmosphere.

[0011] Furthermore, the world population expected to reach 10 billion by 2030 will generate an increase in the demand for proteins for animal and human food, which will be partly replaced by alternative proteins. Proteins of microbial origin or single-cell proteins (SCPs) represent one of the alternatives to proteins of animal origin. SCPs are produced from bacteria, yeasts, molds or algae. Depending on the variety of microorganism used, the substrate can be solid (sucrose, glucose, starch, etc.), liquid (whey, process or washing water from food industries, etc.) or gas (CO2, H2, methane, etc.). The recovered SCPs, often with a protein content around 70%, are used to enrich nutrient mixtures intended for animal feed. For example, a bioreactor consumes between 1.5 and 2.0 kg of CO2 to produce 1 kg of animal feed made from bacterial SCPs.

[0012] Based on this, the present invention proposes the coupling of methanization and cultivation of a single type of microorganism or a consortium of microorganisms for the purpose of protein production. This coupling is done from the point of view of the products (CO2, crop residues, etc.) and from the point of view of thermal flows. The process will therefore be integrated, to maximize the value of all flows, by producing three valuable products: biomethane, digestate and proteins. In addition, metabolites such as succinic acid could also be recovered if extracted and purified from the medium, depending on the microorganism concerned.

[0013] The method according to the invention, moreover in accordance with the generic definition given in the preamble above, is essentially characterized in that it comprises at least the steps of: - production of biogas by methanization of methanogenic inputs, - separation of the biogas produced into biomethane and a carbon dioxide-rich stream containing at least 93 mol%, in particular at least 95 mol% of carbon dioxide and up to 7 mol%, in particular up to 5% of methane, - introduction of the carbon dioxide-rich flow into a reactor comprising at least one bacterial strain and an aqueous culture medium, - cultivation of said at least one bacterial strain in said reactor to obtain a biomass comprising proteins, in which: - said at least one bacterial strain is chosen from: Actinobacillus suc-cinogenes, Corynebacterium glutamicum, Propionibacterium acidipropionici, Rhodobacter sphaeroides, Rhodobacter capsulatus and Thiobacillus Deni-trificans, and - the culture medium is controlled at a temperature between 30 and 65°C, in particular between 35 and 60°C, and at a pH between 5 and 8, in particular between 6 and 7.5.

[0014] Furthermore, embodiments of the invention may include one or more of the following features: - the carbon dioxide-rich stream comprises at least 95 mol% carbon dioxide, up to 3 mol% methane and up to 2% oxygen, in particular between 0 and 0.5 mol% oxygen. - said at least one bacterial strain is chosen from: Actinobacillus suc-cinogenes, Corynebacterium glutamicum and Propionibacterium acidipropionici. - the culture is carried out in the absence of hydrogen. - the carbon dioxide-rich stream is swept into a sky gaseous phase of the reactor or by bubbling into a liquid phase of the reactor containing the aqueous culture medium and at least one bacterial strain. - during cultivation, a carbohydrate source, for example glucose and / or lactose, and / or mineral salts, for example calcium, magnesium and / or potassium, are added. - during cultivation, one or more growth promoters, for example vitamins and essential amino acids, are added. - the culture is carried out for a residence time of between 8 h and 300 h, in particular between 8 h and 60 h. - the biomass includes residues from the culture medium and / or metabolites. - the method comprises a step of recovering part or all of the biomass and a post-treatment step including separation of proteins and residues from the culture medium. - the process comprises a step of introducing the residues of the culture medium separated from the biomass into the methanogenic inputs.

[0015] The present invention proposes a solution for recovering the CO2-rich vent, otherwise rejected during the purification of biogas into biomethane, for the growth of protein-rich micro-organisms and requiring little operational expenditure (no lighting, simple nutrient medium, little heating, recovery of co-products from an agricultural site e.g. whey as a source of sugars, gaseous effluent (“off-gas” in English) from biogas as a source of CO2, etc.).

[0016] The method according to the invention is preferably implemented on an agricultural site, the proteins being used for animal feed on the agricultural site and the biomethane being used to produce energy on the agricultural site. The digestate produced during biogas production by methanization of methanogenic inputs will also be used as fertilizer in agricultural crops.

[0017] Other features and advantages will appear on reading the description below.

[0018] Biogas is produced by methanization of methanogenic inputs in a digester. By methanogenic inputs we mean any group of organic materials that can be transformed into energy through this methanization process, for example: sewage treatment plant sludge, manure / slurry, agricultural residues, food waste... [organic waste capable of producing methane by anaerobic fermentation, for example a substrate that includes organic matter, for example residues of cereals, molasses, animal fats, cattle or poultry droppings.] The digester allows the methanization of inputs according to an anaerobic process known as such. The duration of anaerobic fermentation can be on average 30 to 80 days depending on the type of inputs and the methanization conditions (temperature, humidity mainly). The residue of the methanization called digestate can be recovered to be used as fertilizer or amendment.

[0019] The biogas produced is then separated in a biogas purifier, for example a membrane separation unit, comprising at least one membrane stage and making it possible to separate the biomethane from the carbon dioxide. For this separation to be possible, the membrane will be more permeable to carbon dioxide than to methane. Note that part of the biogas can be used to generate the heat necessary for the me- thanization.

[0020] At the end of the purification or separation of the biogas, biomethane and a carbon dioxide-rich stream are obtained, preferably corresponding to the vent of a biogas purifier. The carbon dioxide-rich stream contains at least 93 mol% carbon dioxide and up to 7 mol% methane. In particular, the carbon dioxide-rich stream contains at least 95 mol% carbon dioxide and up to 5% methane. The carbon dioxide-rich stream may comprise up to 3 mol% methane and up to 2% oxygen, in particular up to 0.5% oxygen.

[0021] The carbon dioxide-rich stream is introduced into a reactor comprising at least one bacterial strain. The interior of the reactor comprises a liquid phase containing the cell culture medium and at least one bacterial strain, and a gaseous headspace above the liquid phase.

[0022] The carbon dioxide-rich stream may be introduced by sweeping into the gaseous overhead of the reactor or by bubbling into the liquid phase of the reactor containing the aqueous culture medium and at least one bacterial strain to increase gas transfers. The culture medium is made rich in carbon dioxide by injecting the carbon dioxide-rich stream. Carbon dioxide is used as a carbon source in the metabolism of the bacteria. The culture medium is preferably anaerobic or contains less than 2% oxygen.

[0023] The bacterial strains are chosen for their ability to use CO2 as a carbon source, in particular the CO2-rich vent from biogas purification, and for their non-pathogenic nature for humans or livestock. In particular, the bacterial strains chosen do not need light or hydrogen to grow and they do not produce hydrogen. At least one bacterial strain is chosen from: Actinobacillus succinogenes, Corynebacterium glutamicum, Propionibacterium acidipropionici, Rhodobacter sphaeroides, Rhodobacter capsulatus and Thiobacillus Denitrificans. Preferably, at least one bacterial strain is chosen from Actinobacillus succinogenes, Corynebacterium glutamicum and Propionibacterium acidipropionici.

[0024] The reactor or bioreactor comprising at least one bacterial strain and its culture medium is preferably closed without internal lighting and equipped with a heating system, for example of the double-jacket type, to preferably achieve mesophilic conditions (35 - 40°C) or otherwise thermophilic conditions (55 - 60°C).

[0025] The culture medium is controlled at a temperature between 30 and 65°C, in particular between 35 and 60°C, or at a mesophilic temperature (between 35 and 40°C), or thermophilic temperature (between 55 and 60°C), depending on the bacterial strain(s) chosen. The pH of the culture medium is maintained between 5 and 8, in particular between 6 and 7.5, either spontaneously or in a controlled manner, for example by the addition of sodium hydroxide.

[0026] The cultivation of the bacterial strains is carried out in the reactor so as to obtain a biomass comprising proteins. The carbon dioxide-rich flow injected into the reactor, especially the carbon it contains, is consumed by the bacterial strains to transform / convert it into biomass comprising proteins.

[0027] The culture can be carried out with the addition of a carbohydrate source, for example glucose and / or lactose.

[0028] The culture can be carried out with the addition of mineral salts, for example calcium, magnesium and / or potassium, which makes it possible to provide minerals essential for the development of the biomass.

[0029] The culture can be carried out with the addition of one or more growth promoters, for example vitamins and / or essential amino acids which promote bacterial growth, for example biotin.

[0030] The culture can be carried out with effluents available on the agricultural or industrial site. For example, whey can be used as a culture medium.

[0031] At the end of the culture, the biomass comprising the proteins and residues of the culture medium is recovered. The biomass may also comprise metabolites such as succinic acid. At the outlet of the reactor, part or all of the biomass may be recovered, and the proteins and residues of the culture medium may be separated.

[0032] The culture medium residues separated from the biomass can then be introduced into the digester and mixed with the methanogenic inputs.

[0033] The proteins thus produced may be used in animal feed (livestock, fish farming) and / or human food, according to the regulations in force.

[0034] The following examples illustrate conditions that promote the cultivation of a bacterial strain without, however, limiting them.

[0035] Culture media

[0036] Trypticase soy agar (TSA) and trypticase soy broth (TSB) from Dutscher (Brumath, France) were used for strain activation. TSB was autoclaved at 110°C for 10 min. The lactose culture medium for growth and bioreactor tests contained (g / L): lactose (25.0), yeast extract (7.0), KH2PO4 (3.0), MgCl2 (0.2), CaCl2 (0.2) and NaCl (1.0).

[0037] Strain activation

[0038] Actinobacillus succinogenes 130Z (ATCC 55618) from DSMZ (Braunschweig, Germany) were reactivated in TSA and TSB. Seed cultures were incubated at 37°C for 24 to 36 hours. A second subculture was performed, incubated at 37°C for 24 to 48 hours, and then maintained at 4°C. Subculture was performed every 18 to 20 days in TSA.

[0039] Preliminary growth tests

[0040] Culture tests were performed prior to inoculation into the bioreactor, in order to define favorable growth conditions and target the exponential growth phase for inoculation into the bioreactor. A tube containing 10 ml of TSB was inoculated from an isolated Actinobacillus succinogenes colony and placed for 16 hours at 37-40°C. Then, Erlenmeyer flasks containing 100 ml of lactose culture medium were inoculated with 5 ml of TSB inoculum and placed at 37°C for 24 h. Four conditions were tested: (i) free pH; (ii) controlled pH (6.8-7.2); (iii) 50%:50% N2:CO2 injection and free pH; (iv) 50%:50% N2:CO2 injection and controlled pH (6.8-7.2).

[0041] Culture in bioreactor

[0042] The TSB inoculum was added at 5% in an Erlenmeyer flask with 100 ml of lactose culture medium. A 50%:50% N2:CO2 mixture was injected every hour (for 8 h) and the pH was adjusted regularly to 6.8, to place the bacteria in the exponential growth phase before starting the bioreactor tests. Batch fermentation was carried out in a 300 ml Büchiglasuster miniclave reactor (Uster, Switzerland), at 37 °C and 200 rpm, for 24 h. The target pH was 6.8, adjusted with NaOH (1 M). Pure CO2 or a mixture of CO2:CH4 at 96.5%:3.5% (± 0.5%) was injected by bubbling into the inoculated culture medium using a porous injector, at 1 bar, every hour (after each sample).

[0043] Analytical methods

[0044] Cell growth was quantified by measuring the optical density OD600 every hour, with the JENWAY Model Genova Bio spectrophotometer (Stone, UK), using a 70 pL disposable UV cuvette, at 600 nm.

[0045] Additionally, agar plate counts were performed at 24 and 48 h, by taking 1 ml samples, diluting them five times, and incubating them on TSA for 48 h at 37 °C. Equation 1 (FDA, 2001) was used:

[0046] With N: the viable concentration of microorganisms (CFU / mL), C: the sum of colonies on all the agars (CFU), x: the number of agars used, V: the volume of suspension spread on the culture medium (mL), n, : the number of agars retained from the lowest dilution, n2: the number of agars retained from the highest dilution, d: the dilution rate of the agar with the lowest dilution (FDA, 2001).

[0047] To obtain a total cell concentration value, a regular measurement of dry weight was performed. A 10 ml sample was filtered through an MCE membrane of 0.22 pm and placed in an oven at 105 °C for at least 8 h. Before filtration, the filters were placed in the oven for 8 hours to remove moisture.

[0048] pH was manually monitored every hour, on 1 ml samples, with the Mettler Toledo SevenMulti instrument (Schwerzenbach, Switzerland).

[0049] Results

[0050] Preliminary growth tests:

[0051] Under all conditions, bacterial growth follows the shape of standard growth curves ([Fig.l]). Without pH control or CO2 injection (triangles), bacterial growth is minimal, with a final OD600 of 0.828. When pH is controlled or CO2 is added, bacterial growth increases, with a final OD600 of 1.566 and 1.170, respectively (squares and circles, resp.). When pH is controlled and CO2 is injected, bacterial growth is strongly favored (cross). OD60o is 3.040 at the end of the day. The value after 24 h could not be measured because the culture was contaminated overnight. A value around 4.6 is expected. Overall, A. succinogenes must be in the presence of a CO2-rich atmosphere and with pH control for optimal growth. These key parameters should therefore also be controlled during bioreactor tests.

[0052] Culture in bioreactor:

[0053] Bacteria were grown at 37°C, with a target pH of 6.8 and by bubbling pure CO2 or a mixture of CO2 with 3.5 ± 0.5% CH4 every hour. Triplicates were performed: M1, M2 and M3 represent tests with CO2 and tests M4, M5 and M6 with CO2:CH4. M1 data are not reported further because the initial cell number was significantly higher than for the other tests.

[0054] Bacterial growth followed the onset of standard growth curves, with reproducible results under similar conditions ([Fig.2]): Absorbance at 600 nm (OD600) representing the growth of A. succinogenes at 37°C in the bioreactor: CO2 injection, (M2: square, M3: circle); CO2 injection with 3.5% CH4 ± 0.5% (M4: cross, M5: plus, M6: star).

[0055] To quantify the impact of CH4, the final count of total biomass and viable cells was performed. Also, the maximum growth rate (pmax) was evaluated: it corresponds to the slope of each biomass growth curve in exponential phase (in log plot). Finally, the biomass productivity (P) of the batch fermentation of the culture was calculated by the final biomass (Xf) divided by the time required to reach it.

[0056] [Table 1] Experimental results characterizing the growth of A. succinogenes 130Z in discontinuous fermentation in the bioreactor, with the bubbling injection of CO2 or a mixture of CO2:CH4 (3.5% ± 0.5% CH4). CO2 Standard deviation e CO2:CH4 Standard deviation e Xf (g / L MS) 1.17 0.04 2.50 0.35 Vcells xlO6 (CFU / mL) 16.33 21.46 0.51 0.49 Pmax (h 1) 0.22 0.10 0.27 0.04 P (g / L MS / h) 0.13 0.00 0.29 0.06

[0057] The final biomass, maximum growth rates and productivities are also high in the presence of CH4. The presence of CH4 at 3.5% therefore seems favorable to the growth of this bacterium. The number of viable cells is lower. A peak of viable cells should be observed at the end of the exponential phase. Since this phase was not reached in the tests, the number of viable cells may not be representative and may explain why the number of viable cells is lower in the tests with CH4.

Claims

Claims

1. A method for co-producing proteins and biomethane comprising at least the steps of: - producing biogas by methanization of methanogenic inputs, - separating the biogas produced into biomethane and a carbon dioxide-rich stream containing at least 93 mol%, in particular at least 95 mol% of carbon dioxide and up to 7 mol%, in particular up to 5% of methane, - introducing the carbon dioxide-rich stream into a reactor comprising at least one bacterial strain and an aqueous culture medium, and - culturing said at least one bacterial strain in said reactor to obtain a biomass comprising proteins, wherein: - said at least one bacterial strain is selected from: Actinobacillus succinogenes, Corynebacterium glutamicum, Pro-pionibacterium acidipropionici, Rhodobacter sphaeroides, Rhodobacter capsulatus and Thiobacillus Denitrificans,and - the culture medium is controlled at a temperature between 30 and 65°C, in particular between 35 and 60°C, and at a pH between 5 and 8, in particular between 6 and 7.5.,

2. A method according to claim 1, wherein the carbon dioxide-rich stream comprises at least 95 mol% carbon dioxide, up to 3 mol% methane and up to 2% oxygen, in particular between 0 and 0.5 mol% oxygen.

3. A method according to claim 1 or 2, wherein said at least one bacterial strain is selected from: Actinobacillus succinogenes, Corynebacterium glutamicum and Propionibacterium acidipropionici.

4. A method according to any one of claims 1 to 3, wherein the cultivation is carried out in the absence of hydrogen.

5. A method according to any one of claims 1 to 4, wherein the carbon dioxide-rich stream is introduced by sweeping into a gaseous overhead of the reactor or by bubbling into a liquid phase of the reactor. containing the aqueous culture medium and at least one bacterial strain.

6. A method according to any one of claims 1 to 5, wherein, during cultivation, a carbohydrate source, for example glucose and / or lactose, and / or mineral salts, for example calcium, magnesium and / or potassium, are added.

7. A method according to claim 6, wherein one or more growth promoters, e.g., vitamins and essential amino acids, are added.

8. A method according to any one of claims 1 to 7, wherein the cultivation is carried out for a residence time of between 8 h and 300 h, in particular between 8 h and 60 h.

9. A method according to any one of claims 1 to 8, wherein the biomass comprises residues of the culture medium and / or metabolites.

10. A method according to any one of claims 1 to 9, comprising a step of recovering some or all of the biomass and a post-treatment step comprising separation of proteins and residues from the culture medium.

11. A method according to claim 10, comprising a step of introducing the residues of the culture medium separated from the biomass into the methanogenic inputs.

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