protein and biomethane co-production process

Abstract

A process for the co-production of proteins and biomethane comprising at least the steps of: producing biogas by methanation 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 process for the co-production of proteins and biomethane.

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

[0003] Biogas is the gas produced during the decomposition of organic matter in the absence of oxygen (anaerobic fermentation), also known as methanation. This can be a natural decomposition—as observed in marshes or municipal waste landfills—but biogas production can also result from the methanation of waste in a dedicated reactor, with controlled conditions, called a digester or digester, and then in a post-digester, similar to the digester but allowing the methanation reaction to progress further. Biogas is the gas produced during the decomposition of organic matter in the absence of oxygen (anaerobic fermentation), also known as methanation.This can be a natural degradation – as seen in marshes or household waste landfills – but biogas production can also result from the methanization of waste in a dedicated reactor, with controlled conditions, called a methanizer or digester, and then in a post-digester, similar to the digester and allowing the methanization reaction to be pushed further.

[0004] The digester, that is, the reactor dedicated to the methanation of organic matter, is a closed tank, heated or unheated (operation at a fixed temperature, between ambient temperature and 55°C), whose contents, consisting of organic matter, are stirred continuously or sequentially. The conditions in the digester are anaerobic, and the generated biogas is found in the digester's headspace (gaseous space), 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, an appreciable source of renewable energy in a context of scarcity of fossil fuels.

[0006] Biogas mainly contains methane (CH4) and carbon dioxide (CO2) in varying 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 organic matter degraded and the techniques used, the proportions of the components differ, but on average biogas contains, on a dry gas basis, 30 to 75% methane, 15 to 60% CO2, 0 to 15% nitrogen, 0 to 5% oxygen and trace compounds.

[0008] Biogas is used in various ways. After light treatment, it can be used near the production site to provide heat, electricity or a mixture of both (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 application.

[0009] Further purification of biogas allows for its wider use; in particular, further purification of biogas makes it possible to obtain biogas purified to the specifications of natural gas, which can then be substituted for it; this purified biogas is called "biomethane." Biomethane thus complements natural gas resources with a renewable component produced within local areas; it can be used for exactly the same purposes as natural gas of fossil origin. It can supply a natural gas network, a vehicle refueling station, and it can also be liquefied for storage as liquefied natural gas (LNG).

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

[0011] Furthermore, the projected global population of 10 billion by 2030 will generate an increased demand for protein for animal and human consumption, which will be partially met by alternative proteins. Microbial proteins, or single-cell proteins (SCPs), represent one such alternative to animal proteins. SCPs are produced from bacteria, yeasts, molds, or algae. Depending on the type of microorganism used, the substrate can be solid (sucrose, glucose, starch, etc.), liquid (whey, process or wash water from the food industry, etc.), or gaseous (CO2, H2, methane, etc.). The recovered SCPs, often with a protein content of around 70%, are used to enrich nutrient mixtures 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 methanation and the cultivation of a single type of microorganism or a consortium of microorganisms for the purpose of protein production. This coupling is achieved from the perspective of the products (CO2, culture residues, etc.) and from the perspective of the heat flows. The process will therefore be integrated, in order to maximize the value of all the flows, producing three valuable products: biomethane, digestate, and proteins. In addition, metabolites such as Succinic acid could also be used if extracted and purified from the medium, depending on the microorganism involved.

[0013] The process according to the invention, which otherwise conforms to 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% carbon dioxide and up to 7 mol%, in particular up to 5% methane, - introduction of the carbon dioxide-rich stream into a reactor comprising at least one bacterial strain and an aqueous culture medium, - culturing 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 succinogenes, Corynebacterium glutamicum, Propionibacterium 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.

[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 succinogenes, Corynebacterium glutamicum and Propionibacterium acidipropionici. - the culture is carried out in the absence of hydrogen. - the carbon dioxide-rich stream is introduced by sweeping into a sky gaseous from the reactor or by bubbling in a liquid phase of the reactor containing the aqueous culture medium and at least one bacterial strain. - during cultivation, a source of carbohydrate, 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 essential vitamins and amino acids, are added. - the culture is carried out during a stay period of between 8 h and 300 h, in particular between 8 h and 60 h. - biomass includes residues from the culture medium and / or metabolites. - the process includes 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 includes a step of introducing residues from the culture medium separated from the biomass into the methanogenic inputs.

[0015] The present invention proposes a solution for valorizing the CO2-rich vent, otherwise released during the purification of biogas into biomethane, for the growth of protein-rich microorganisms requiring little operational expenditure (no lighting, simple nutrient medium, little heating, valorization 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 ...).

[0016] The process according to the invention is preferably implemented on an agricultural site, with 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 become apparent from the description below.

[0018] Biogas is produced by the methanation of methanogenic inputs in a digester. Methanogenic inputs are defined as any group of organic matter that can be transformed into energy through this methanation process, for example: sewage sludge, manure / slurry, agricultural residues, food waste... [organic waste capable of producing methane by anaerobic fermentation, for example, a substrate that includes organic matter, such as cereal residues, molasses, animal fats, or cattle or poultry manure.] The digester enables the methanation of the inputs according to a well-known anaerobic process. The duration of anaerobic fermentation can be on average from 30 to 80 days, depending on the type of input and the methanation conditions (primarily temperature and humidity). The residue from the methanation process, called digestate, can be recovered for use as fertilizer or soil amendment.

[0019] The biogas produced is then separated in a biogas scrubber, for example a membrane separation unit, comprising at least one membrane stage and allowing the separation of biomethane from 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- thanation.

[0020] Following the purification or separation of the biogas, biomethane and a carbon dioxide-rich stream are obtained, preferably corresponding to the vent of a biogas scrubber. 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 include 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 space above the liquid phase.

[0022] The carbon dioxide-rich stream can be introduced by purging through the gas headspace of the reactor or by bubbling through the liquid phase of the reactor containing the aqueous culture medium and at least one bacterial strain to enhance gas transfer. The culture medium is enriched with carbon dioxide by the injection of the carbon dioxide-rich stream. Carbon dioxide is used as a carbon source in bacterial metabolism. 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, particularly the CO2-rich vent from biogas purification, and for their non-pathogenicity to humans or livestock. In particular, the chosen bacterial strains do not require light or hydrogen to grow and do not produce hydrogen. At least one bacterial strain is chosen from among: Actinobacillus succinogenes, Corynebacterium glutamicum, Propionibacterium acidipropionici, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Thiobacillus denitrificans. Preferably, at least one bacterial strain is chosen from among 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 bacterial strains are cultured in the reactor in such a way as to obtain a biomass containing proteins. The carbon dioxide-rich stream injected into the reactor, especially the carbon it contains, is consumed by the bacterial strains to transform / convert it into biomass containing 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 available essential minerals for the development of 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 that 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 proteins and residues from the culture medium, is recovered. The biomass may also include metabolites such as succinic acid. At the reactor outlet, some or all of the biomass can be recovered, and the proteins and culture medium residues can 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 farming, fish farming) and / or human food, according to the regulations in force.

[0034] The following examples illustrate conditions which favor the culture 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. The TSB was autoclaved at 110°C for 10 min. The lactose culture medium for growth tests and bioreactor 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. The subculture was performed every 18 20 days in TSA.

[0039] Preliminary growth tests

[0040] Culture tests were performed prior to inoculation in the bioreactor to define favorable growth conditions and target the exponential growth phase for inoculation in the bioreactor. A tube containing 10 ml of TSB was inoculated from an isolated Actinobacillus succinogenes colony and incubated for 16 hours at 37–40°C. Subsequently, Erlenmeyer flasks containing 100 ml of lactose culture medium were inoculated with 5 ml of TSB inoculum and incubated at 37°C for 24 hours. 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 a bioreactor

[0042] The TSB inoculum was added at 5% to an Erlenmeyer flask with 100 ml of lactose culture medium. A 50%:50% N2:CO2 mixture was injected hourly (for 8 h), and the pH was regularly adjusted to 6.8 to induce exponential growth of the bacteria before initiating bioreactor testing. 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 1 M NaOH. Pure CO2 or a 96.5%:3.5% (±0.5%) CO2:CH4 mixture was bubbling into the inoculated culture medium using a porous injector at 1 bar every hour (after each sampling).

[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 single-use 70 pL UV cuvette, at 600 nm.

[0045] In addition, 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 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 ratio of the agar with the lowest dilution (FDA, 2001).

[0047] To obtain a total cell concentration value, regular measurements of the dry weight were 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] The pH was monitored manually 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 the standard growth curves ([Fig. 1]). 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 final OD600 of 1.566 and 1.170, respectively (squares and circles, respectively). When pH is controlled and CO2 is injected, bacterial growth is strongly favored (crosses). OD600 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 trials.

[0052] Culture in a bioreactor:

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

[0054] Bacterial growth followed the beginning of standard growth curves, with reproducible results under similar conditions ([Fig.2]): Absorbance at 600 nm (OD6oo) 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. The maximum growth rate (pmax) was also evaluated: it corresponds to the slope of each biomass growth curve in the exponential phase (log plot). Finally, the biomass productivity (P) of the batch fermentation of the culture was calculated by dividing the final biomass (Xf) by the time required to reach it.

[0056] [Table 1] Experimental results characterizing the growth of A. succinogenes 130Z in batch fermentation in the bioreactor, with 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 DM) 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 DM / 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 3.5% CH4 therefore appears to be favorable to the growth of this bacterium. The number of viable cells is lower. A peak in viable cells should be observed at the end of the exponential phase. Since this phase was not reached in the trials, the number of viable cells may not be representative and may explain why the number of viable cells is lower in the trials with CH4.

Claims

Demands

1. A process for the co-production of proteins and biomethane comprising at least the steps of: - producing biogas by 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% carbon dioxide and up to 7 mol%, in particular up to 5% 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, in which: - said at least one bacterial strain is selected from: Actinobacillus succinogenes, Corynebacterium glutamicum, Propionibacterium 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 process 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 culture 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 purging into a gaseous head 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 process according to any one of claims 1 to 5, wherein, during the cultivation, a source of carbohydrate, 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, for example essential vitamins and amino acids, are added.

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

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

10. A process according to any one of claims 1 to 9, comprising a step of recovering part 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 residues from the culture medium separated from the biomass into the methanogenic inputs.

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