Microbial products comprising a mixture of bacillus / peribacillus strains for improving biogas production

Abstract

The present invention relates to a mixture of Bacillus / Peribacillus strains or a combination thereof with another microbial product or with fermented molasses, useful for improving biogas production by anaerobic digestion of a methanogenic substrate. The invention also relates to the use of this mixture, or of a combination thereof, for increasing the production of biogas and to a method for preparing biogas by anaerobic digestion of a methanogenic substrate in the presence of this mixture, or of a combination thereof.

Description

[0001] Microbial Products Comprising a Mixture of Bacillus / Peribacillus Strains to Enhance Biogas Production - Parent Patent Application

[0002] This patent application claims priority from French patent application number FR 25 02716 filed on March 18, 2025. The content of the French patent application is incorporated by reference in its entirety.

[0003] Technical Field

[0004] The present invention relates to the field of biogas production. More particularly, the invention relates to a method for increasing the methanogenic potential of substrates used in methanization by adding a specific microbial product.

[0005] Context of the invention

[0006] Anaerobic digestion is a natural process of organic matter decomposition in an oxygen-free environment through the action of numerous microorganisms. This decomposition process, known as anaerobic fermentation, produces biogas, a mixture composed primarily of methane (CH4), carbon dioxide (CO2), and water vapor (H2O), along with traces of other undesirable compounds, including hydrogen sulfide (H2S). Anaerobic digestion also produces digestate, which is the portion of waste remaining at the end of the process.Anaerobic digestion can occur spontaneously in certain environments, such as in natural ecosystems (marshes and rice paddies), large tropical hydroelectric reservoirs or dams, and landfills containing waste or organic matter, or it can be implemented voluntarily in dedicated industrial units, particularly to treat sewage sludge and industrial or agricultural organic waste.

[0007] Like natural gas, biogas can be used to produce heat and electricity through cogeneration. However, biogas has the advantage of being much more environmentally friendly than its fossil fuel equivalent, as it emits 10 times less carbon dioxide than natural gas while allowing for the same uses (heating, cooking, industrial processes, etc.). Biogas can also be purified by removing components other than methane so that its characteristics are similar to those of natural gas. It is then called "biomethane." The biomethane thus obtained can be injected into the natural gas distribution network. Biomethane is also used as a vehicle fuel. Compared to other fuels, the combustion of biomethane, or compressed natural gas (bioCNG), is less polluting, generating less carbon dioxide, nitrogen oxide, and particulate matter.Digestate, also produced by methanization, is one of the few sources of mineral nitrogen of organic origin. It can be used as a natural fertilizer, as a substitute for chemical fertilizers.

[0008] Anaerobic digestion offers numerous advantages: valorization of organic waste, substitution of fossil fuels in industry and transportation, reduction of carbon dioxide emissions, fertilizer production, improved energy independence, and creation of jobs and local economic activity. Biogas is classified as a renewable energy source because it is produced from organic waste that would not otherwise be utilized. Biogas production is therefore part of a circular economy model.

[0009] According to the International Energy Agency, global biogas production is expected to increase by 33% between 2022 and 2028. GRDF, the French gas distribution company, indicated that biogas production capacity from methanization should reach 50 terawatt-hours per year (TWh / year) in France by 2030, compared to 12 TWh / year at the end of 2023. From 3% at the end of 2023, the share of biogas in distribution networks is expected to rise to 20% within seven years, thus reducing the share of fossil gas by the same amount. At the end of March 2024, France recorded an increase in its methanization capacity, with 22 new units added compared to the previous year, bringing the total number of sites to 674.

[0010] However, there is still a need to improve the processes of biogas production by methanization.

[0011] Summary of the invention

[0012] The present inventors have demonstrated the beneficial effects of a mixture of Bacillus and Peribacillus strains on biogas production through the anaerobic digestion of a methanogenic substrate. They have shown that the use of this mixture, alone or in combination with another microbial product or fermented molasses, during the anaerobic digestion of cattle manure results in an increased rate of biogas production and an improved biogas yield, i.e., an increase in the methanogenic potential of the cattle manure. Significant effects were observed, in particular, when the mixture of Bacillus / Peribacillus strains was combined with the fungal strain Trichoderma atroviride TAA2025, and when the mixture of Bacillus / Peribacillus strains was combined with the live yeast strain Saccharomyces cerevisiae BG1.Beneficial effects have also been demonstrated on the methanogenic potential of other substrates, such as mixtures of cattle manure and plant fibers; pig slurry and plant fibers; or plant fibers alone. Furthermore, in continuous methanization of livestock effluents, the “BG1 + BG6” mixture showed a significant 50% increase in biomethane production compared to the control.

[0013] Consequently, the present invention relates to a process for producing biogas from a methanogenic substrate, characterized in that it comprises an anaerobic digestion step of said methanogenic substrate in the presence of:

[0014] a mixture of Bacillus / Peribacillus BG6 strains as defined in this document;

[0015] or in the presence of a combination or composition:

[0016] of the mixture of Bacillus / Peribacillus BG6 strains; and

[0017] of the Trichoderma atroviride TAA2025 strain defined in this document; or in the presence of a combination or composition:

[0018] of the mixture of Bacillus / Peribacillus BG6 strains; and

[0019] of the live yeast strain of Saccharomyces cerevisiae BG1 defined in this document;

[0020] or in the presence of a combination or composition:

[0021] of the mixture of Bacillus / Peribacillus BG6 strains; and

[0022] of fermented molasses.

[0023] In some embodiments, the anaerobic digestion of the methanogenic substrate is carried out in the presence of the Bacillus / Peribacillus BG6 mixture at a content between 0.002% and 0.0000002% or between 0.00002% and 0.0000002% of the weight of the methanogenic substrate, in particular a content of about 0.00002% of the weight of the methanogenic substrate.

[0024] In some embodiments, the anaerobic digestion of the methanogenic substrate is carried out in the presence of the mixture of Bacillus / Peribacillus BG6 strains and the live yeast strain Saccharomyces cerevisiae BG1, where the content of the Saccharomyces cerevisiae BG1 strain is between 0.1% and 0.001%, in particular is about 0.010% of the weight of the methanogenic substrate, and the content of the mixture of Bacillus / Peribacillus strains is between 0.000002% and 0.0010% or between 0.00002% and 0.0005% of the weight of the methanogenic substrate, in particular is about 0.00002% of the weight of the methanogenic substrate.

[0025] In some embodiments, the anaerobic digestion of the methanogenic substrate is carried out in the presence of the mixture of Bacillus / Peribacillus BG6 strains and the Trichoderma atroviride TAA2025 strain, wherein the content of the Bacillus / Peribacillus BG6 strain mixture is between 0.000002% and 0.0002% or between 0.00002% and 0.0002% by weight of the methanogenic substrate, in particular 0.00002% by weight of the methanogenic substrate, and the content of the Trichoderma atroviride TAA2025 strain is between 0.000001% and 0.000000001% or between 0.000005% and 0.000000001% by weight of the methanogenic substrate, in particular about 0.000005% by weight of the substrate methanogen.

[0026] In some embodiments, the anaerobic digestion of the methanogenic substrate is carried out in the presence of the mixture of Bacillus / Peribacillus BG6 strains and fermented molasses, where the fermented molasses is fermented sugar beet molasses, fermented sugar cane molasses, or a mixture thereof.

[0027] In some embodiments, the content of fermented molasses is between 0.01% and 5% or between 0.05% and 2% of the weight of methanogenic substrate, in particular about 1% of the weight of methanogenic substrate, and the content of mixture of Bacillus / Peribacillus BG6 strains is between 0.000002% and 0.0002% of the weight of methanogenic substrate, in particular about 0.00002% of the weight of methanogenic substrate.

[0028] In some embodiments, the anaerobic digestion of the methanogenic substrate is further carried out in the presence of an inoculum.

[0029] The present invention also relates to a composition or combination (e.g., a kit) for improving biogas production by anaerobic digestion of a methanogenic substrate, characterized in that it comprises, or consists of:

[0030] the mixture of Bacillus / Peribacillus BG6 strains; and

[0031] or in what it includes, or consists of:

[0032] the mixture of Bacillus / Peribacillus BG6 strains; and

[0033] the Trichoderma atroviride TAA2025 strain; or in that it comprises, or consists of:

[0034] the mixture of Bacillus / Peribacillus BG6 strains; and

[0035] the live yeast strain Saccharomyces cerevisiae BG1;

[0036] or in what it includes, or consists of:

[0037] the mixture of Bacillus / Peribacillus BG6 strains; and

[0038] fermented molasses.

[0039] In some embodiments, the composition or combination comprises, or consists of, the Trichoderma atroviride TAA2025 strain and the Bacillus / Peribacillus BG6 strain mixture in relative weight amounts between 1:0.2 and 1:40000, in particular between 1:0.4 and 1:20000 or between 1:0.4 and 1:40 or between 1:0.4 and 1:20, more particularly are about 1:4.

[0040] In some embodiments, the composition or combination comprises, or consists of, the mixture of Bacillus / Peribacillus BG6 strains and the live yeast strain Saccharomyces cerevisiae BG1, wherein the relative amounts by weight of the mixture of Bacillus / Peribacillus BG6 strains and the BG1 strain are between 1:10 and 1:10000 or between 1:100 and 1:5000, in particular between 1:100 and 1:1000, more particularly are about 1:500.

[0041] In some embodiments, the composition or combination comprises, or consists of, a mixture of Bacillus / Peribacillus BG6 strains and fermented molasses, wherein the fermented molasses is fermented sugar beet molasses, fermented sugar cane molasses, or a mixture thereof.

[0042] In some embodiments, the composition or combination comprises, or consists of, a mixture of Bacillus / Peribacillus BG6 strains and fermented molasses in relative weight amounts between 1:500 and 1:500000 or between 1:5000 and 1:50000, in particular are about 1:50000.

[0043] The present invention also relates to a method for preparing a composition defined above, comprising a mixing step of:

[0044] of the mixture of Bacillus / Peribacillus BG6 strains and the Trichoderma atroviride TAA2025 strain;

[0045] Or :

[0046] of the mixture of Bacillus / Peribacillus BG6 strains and the live yeast strain Saccharomyces cerevisiae BG1; or:

[0047] of the mixture of Bacillus / Peribacillus BG6 strains and fermented molasses.

[0048] The present invention also relates to the use of a composition or combination defined above in a process for producing biogas by anaerobic digestion of a methanogenic substrate.

[0049] In some embodiments, this use aims to increase biogas production yield.

[0050] In some embodiments, this use is intended to increase the rate of biogas production.

[0051] A more detailed description of some preferred embodiments of the invention is given below.

[0052] Figure Captions

[0053] Figure 1: Diagram of anaerobic digestion kinetics and definition of parameters. Vmax is the maximum rate of substrate degradation per tonne of raw material (RM), estimated slope of the line defined by the first points of the kinetics (tangent); Pmax = maximum methane production per tonne of raw material; Biogas composition at the end of the test expressed as a percentage (% of CH4 and % of CO2); and MO = organic matter.

[0054] Figure 2: (A) Daily biomethane production throughout the trial in chemostat 1 (control - green), chemostat 2 (blue - BG1 + BG6 mixture), and chemostat 3 (yellow - TAA2025 + BG6 mixture). (B) Biomethane production (methanogenic potential) during the last 30 days of the trial in chemostat 1 (control - green), chemostat 2 (blue - BG1 + BG6 mixture), and chemostat 3 (yellow - TAA2025 + BG6 mixture).

[0055] Description of Implementation Methods

[0056] As mentioned above, the present invention relates to a mixture of Bacillus / Peribacillus strains used alone or in combination, its use in the preparation of biogas from a methanogenic substrate, and biogas production processes using this mixture. I - Mixture of Bacillus / Peribacillus Strains, Combinations and Compositions The microbial product at the basis of the present invention is a mixture of Bacillus / Peribacillus strains. In the context of the present invention, this mixture can be used alone or in combination with other microbial products or with fermented molasses. The present invention therefore relates not only to the mixture of individual Bacillus / Peribacillus strains, but also to its combinations and compositions.

[0057] A. Mixture of Bacillus / Peribacillus Strains

[0058] The microbial product is a mixture of bacteria of the genera Bacillus and Peribacillus. Bacillus are Gram-positive bacteria belonging to the Bacilloid family. Peribacillus also belong to the Bacillus family—they are rod-shaped bacteria that exhibit Gram-positive or Gram-variable staining. More specifically, in the context of the present invention, the mixture of Bacillus / Peribacillus strains comprises or consists of:

[0059] the strain of Bacillus licheniformis, which was deposited by ENVERA LIC, LLC (220 Garfield Avenue, West Chester, PA 19380, USA) with the Agricultural Research Service Culture Collection (NRRL) (1815 N. University Street, Peoria, IL 61604, USA) on November 20, 2024, under accession number NRRL B-68474;

[0060] the strain of Bacillus licheniformis, which was deposited by ENVERA at the NRRL on November 20, 2024, under accession number NRRL B-68477;

[0061] the strain of Bacillus megaterium or Priestia megaterium, which was deposited by ENVERA at the NRRL on November 20, 2024, under accession number NRRL B-68473;

[0062] the Bacillus pumilus strain, which was deposited by ENVERA at the NRRL on November 20, 2024, under accession number NRRL B-68475;

[0063] the strain of Peribacillus butanolivorans or Bacillus butanolivorans, which was deposited by ENVERA with the NRRL on November 20, 2024, under accession number NRRL B-68478; and

[0064] the strain of Bacillus velezensis, which was deposited by ENVERA at the NRRL on November 20, 2024, under accession number NRRL B-68476.

[0065] Bacillus and Peribacillus strains may be in spore form, predominantly in spore form, in vegetative form, or predominantly in vegetative form. In some embodiments, Bacillus and Peribacillus strains are found in vegetative form or predominantly in vegetative form. The “vegetative form” of a bacterium refers to the form of a bacterium under favorable conditions. The expression “predominantly in vegetative form” means that at least 70% of the cells are in vegetative form, preferably at least 80%, and more preferably at least 90%. An example of favorable conditions is a non-limiting culture medium at a temperature and pH favorable to bacterial growth. A non-limiting culture medium contains all the nutrients necessary for cell growth.

[0066] In some embodiments, Bacillus and Peribacillus strains are found in spore form or predominantly in spore form. The “spore form” of a bacterium refers to the form of a bacterium under unfavorable conditions. The expression “predominantly in spore form” means that at least 70% of the cells are in spore form, preferably at least 80%, and more preferably at least 90%. The spore form is a resistant form that allows cells to withstand a hostile environment such as nutrient deprivation (i.e., a nutrient-limiting environment), water stress, significant variations in pH or temperature, or passage through the digestive tract.Unfavorable cell conditions can be achieved, for example, by: not renewing the cell culture medium, stopping feeding into the culture medium, using a limiting culture medium, changing the temperature and / or pH, and controlling aeration and / or agitation to maintain a Cl pressure above 20% in the culture medium and a CO2 pressure below 1.5% in the outgoing gases or their combination. It is also possible to obtain predominantly spore-forming cells by adding glutamic acid to the culture medium in an amount of approximately 0.75 g / L.

[0067] In some embodiments, Bacillus / Peribacillus stem cells are predominantly in spore form.

[0068] Within the mixture, the different Bacillus / Peribacillus strains may be present in varying respective amounts. For example, the mass ratios of strains B-68474, B-68477, B-68473, B-68475, B-68478, and B-68476 may be 30:30:5:10:15:10. Alternatively, in some embodiments, the Bacillus / Peribacillus strain mixture contains equivalent (i.e., identical) respective amounts of the different strains. Preferably, the Bacillus / Peribacillus strain mixture is the product designated BG6 (IxlO 8 CFU / g or IxlO 9 CFU / g or IxlO 10 CFU / g) produced by ENVERA LIC, LLC (220 Garfield Avenue, West Chester, PA 19380, USA). This product is in the form of a fine powder.

[0069] A container containing a mixture of Bacillus / Peribacillus strains must be kept tightly closed in a dry, well-ventilated place at a temperature between approximately 15°C and approximately 30°C.

[0070] The term “between”, when used in this document to refer to a range of numerical values, encompasses the numerical values ​​at each endpoint of the range. For example, a temperature between 15°C and 30°C includes the temperature of 15°C and the temperature of 30°C.

[0071] The term “approximately”, when used in this document with reference to a recited numeric value, includes the recited numeric value and numeric values ​​at plus or minus 10% of the recited value.

[0072] B. Combinations and Compositions Comprising the Bacillus / Peribacillus Strain Mixture

[0073] As mentioned above, the Bacillus / Peribacillus strain mixture, such as BG6, described here, can be used alone or in combination with another microbial product or with fermented molasses. As used here, the term “combined” means that two products are used simultaneously in a single application (in this case, a biogas production reaction). The term “combination” is used here when the two products are used simultaneously in a biogas production reaction by methanation without prior mixing, or when the two products are part of the same package, such as a kit, and are mixed just before use in a biogas production reaction by anaerobic digestion.Thus, a combination of two products can be presented as a kit comprising, or consisting of, a mixture of Bacillus / Peribacillus strains and another microbial product or fermented molasses, the two products being contained in two separate containers. The term “composition” is used here when the mixture of Bacillus / Peribacillus strains and another microbial product or fermented molasses are part of a mixture, and therefore are present in a single container. A combination, or composition, comprising the mixture of Bacillus / Peribacillus strains according to the invention comprises or consists of:

[0074] the mixture of bacterial strains of the genus Bacillus / Peribacillus, such as BG6, and the live yeast strain Saccharomyces cerevisiae BG1; or

[0075] the mixture of bacterial strains of the genus Bacillus / Peribacillus, such as BG6, and the fungal strain Trichoderma atroviride TAA2025; or

[0076] the mixture of bacterial strains of the genus Bacillus / Peribacillus, such as BG6, and fermented molasses.

[0077] The present invention relates to each of these combinations and compositions. In a combination according to the invention, the two products (i.e., the Bacillus / Peribacillus strain mixture described herein and another microbial product or fermented molasses) can be used in any relative amounts. Similarly, in a composition according to the invention, the two components can be present in any appropriate relative amounts. Those skilled in the art can determine such relative amounts. The relative amounts (by weight) between the Bacillus / Peribacillus strain mixture and the microbial product (as defined above) or fermented molasses depend heavily on the nature of the components of the composition. Specific examples of relative amounts are provided in the paragraphs concerning compositions comprising the Bacillus / Peribacillus strain mixture.

[0078] A two-component composition according to the invention can be prepared by any method known in the art, for example by simple mixing. The mixtures are made from the ingredients in powder form, in compacted solid form, mixed in water-soluble sachets, or in liquid form.

[0079] In general, a composition according to the invention must be kept in a hermetically sealed container in a dry, well-ventilated place, protected from light and at a temperature between 0°C and 40°C, and preferably between about 15°C and about 30°C.

[0080] 1. Mixture of Bacillus / Peribacillus strains and Saccharomyces cerevisiae BG1 Live Yeast Strain

[0081] In some embodiments, the mixture of Bacillus / Peribacillus strains, such as BG6, is used in combination with the live yeast strain Saccharomyces cerevisiae BG1. The live yeast strain Saccharomyces cerevisiae BG1 was deposited by the Applicant (Lesaffre et Compagnie) with the CNCM (National Collection of Microorganism Cultures, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on January 21, 2025, under accession number CNCM 1-6169.

[0082] The term “yeast strain,” as used here, refers to a relatively homogeneous population of yeast cells. A yeast strain is obtained from the isolation of a clone, a clone being a population of cells derived from a single yeast cell. The term “live yeast,” as used here, refers to yeast with an active or reactivatable metabolism, or yeast capable of self-replication, as opposed to dead yeast in which metabolism has been irreversibly halted.

[0083] Live yeast Saccharomyces cerevisiae BG1 (2xlO 10 (CFU / g or more) is available from SI Lesaffre / BU Leaf (137 rue Gabriel Péri, 59703 Marcq-en-Baroeul, France). Dry yeast is defined as any yeast with a dry matter content greater than 90%, preferably between approximately 92% and 98%, for example, a dry matter content between 94.5% and 96.5%. One advantage of dry yeast is its long shelf life.

[0084] The container holding the yeast must be kept tightly closed in a cool, dry place. The product is stable at room temperature.

[0085] In a combination or composition comprising the live yeast strain Saccharomyces cerevisiae BG1 and the Bacillus / Peribacillus strain mixture, the relative amounts of the two components may be any appropriate relative amounts. These relative amounts can be readily determined by those skilled in the art. In some embodiments, the relative amounts by weight of the Bacillus / Peribacillus strain mixture and the BG1 strain may be between 1:10 and 1:10,000 (i.e., between 1:10 and 1:110). 4 In particular, the relative quantities by weight of the mixture of Bacillus / Peribacillus strains and the BG1 strain can be between 1:10 and 1:5000 (i.e., between 1:10 and 1:5000). 3). More specifically, the relative weight amounts of the Bacillus / Peribacillus strain mixture and the BG1 strain can range from 1:100 to 1:1000. In certain particular embodiments, the relative weight amounts of the Bacillus / Peribacillus strain mixture and the BG1 strain are approximately 1:500.2. Bacillus / Peribacillus and Trichoderma atroviride TAA2025 Strain Mixture In certain embodiments, the Bacillus / Peribacillus strain mixture, such as BG6, is used in combination with the Trichoderma atroviride TAA2025 fungal strain. The Trichoderma atroviride strain TAA2025 was deposited by the Applicant (Lesaffre et Compagnie) with the CNCM (National Collection of Microorganism Cultures, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on February 5, 2025 under accession number CNCM 1-6171.

[0086] Trichoderma atroviride is a filamentous fungal species commonly found in soil. This species is of commercial interest due to the secondary metabolites it produces, which are used in industry. Trichoderma atroviride is used as a biofungicide active ingredient in plant protection product formulations, particularly in viticulture for controlling grapevine trunk diseases, as a biocontrol agent.

[0087] The Trichoderma atroviride TAA2025 strain can be found in spore form, predominantly in spore form, in vegetative form, or predominantly in vegetative form.

[0088] In some embodiments, the cells of Trichoderma atroviride TAA2025 are predominantly in spore form. For example, the spores of Trichoderma atroviride TAA2025 (5xl0 9 - 5xlO 10CFU / g - marketed by AGRAUXINE (7 avenue du Grand Périgné, 49070 Beaucouze, France) - are in the form of a powder of fine particles having an average diameter of less than 100 µm. In the context of the present invention, Trichoderma atroviride TAA2025 with a concentration of approximately 1100 is generally used. 10 CFU / g.

[0089] A container holding the Trichoderma atroviride TAA2025 strain must be kept tightly closed in a dry, cool, and well-ventilated place, away from direct sunlight or other heat sources. Storage can be carried out at a temperature between -2°C and 20°C. A temperature of approximately 4°C promotes a longer shelf life.

[0090] In a combination or composition consisting of a mixture of Bacillus / Peribacillus strains, such as BG6, and the fungal strain Trichoderma atroviride TAA2025, the relative amounts of the two components may be any appropriate relative amounts. These relative amounts can be readily determined by a person skilled in the art. In some embodiments, the relative amounts by weight of the Trichoderma atroviride TAA2025 strain and the Bacillus / Peribacillus bacterial mixture may be between 1:0.2 and 1:40,000 (i.e., between 1:2 x 10⁻³⁴). 1 and 4xl0 4 In particular, the relative weight quantities of the Trichoderma atroviride TAA2025 strain and the Bacillus / Peribacillus bacterial mixture can range from 1:0.4 to 1:20,000 (i.e., between 1:4 x 10 -1 and l:2xl0 4). In particular, the relative amounts by weight of the Trichoderma atroviride TAA2025 strain and the Bacillus / Peribacillus bacterial mixture can be between 1:0.4 and 1:40 or between 1:0.4 and 1:20. In certain specific embodiments, the relative amounts by weight of the Trichoderma atroviride TAA2025 strain and the Bacillus / Peribacillus bacterial mixture are 1:4.

[0091] 3. Mixture of Bacillus / Peribacillus Strains and Fermented Molasses

[0092] In some embodiments, the mixture of Bacillus / Peribacillus strains is used in combination with fermented molasses. The term “molasses,” as used here, has its well-known meaning in the arts and designates a mixture resulting from the refining of sugar extracted from sugar beets or sugar cane. As used here, the term “fermented molasses” designates a co-product of molasses obtained by its fermentation by bacteria, yeasts, or fungi, said fermentation yielding so-called “noble” products such as baker’s yeast, ethyl alcohol, citric acid, and glutamic acid.

[0093] Preferably, in the context of the present invention, fermented molasses is obtained by fermenting molasses with yeasts.

[0094] The fermented molasses used in the implementation of the present invention can be obtained from sugar beet molasses, sugar cane molasses, or a mixture of sugar beet molasses and sugar cane molasses. Alternatively, the fermented molasses is prepared by mixing fermented sugar beet molasses and fermented sugar cane molasses.

[0095] Preferably, when the fermented molasses is a mixture of fermented sugar beet molasses and fermented sugar cane molasses, it comprises predominantly fermented sugar beet molasses. For example, the mixture may contain at least about 60%, 70%, 80%, 90%, or 95% fermented sugar beet molasses, by dry weight of the mixture. Advantageously, when the fermented molasses is a mixture of fermented sugar beet molasses and fermented sugar cane molasses, the mixture contains at least about 90% fermented beet molasses by weight of the mixture.

[0096] Preferably, when the fermented molasses is a mixture of fermented sugar beet molasses and fermented sugar cane molasses, said mixture contains between approximately 60% and approximately 95% fermented sugar beet molasses, preferably between approximately 70% and approximately 90% fermented sugar beet molasses, and more preferably between approximately 80% and approximately 90% fermented sugar beet molasses, by weight of the mixture, the remainder being fermented sugar cane molasses. Preferably, the mixture is such that the total percentages of fermented sugar beet molasses and fermented sugar cane molasses are 100%.

[0097] Fermented molasses itself generally contains more than 90% water, but this water can advantageously be concentrated to reduce the water content and obtain higher dry matter percentages. Thus, the dry matter percentage of the fermented molasses used in the context of the present invention can be between approximately 50% and approximately 80%. Preferably, the dry matter percentage of the fermented molasses is between approximately 55% and approximately 75%, and particularly between approximately 55% and approximately 65%, such as, for example, approximately 60%.

[0098] The fermented molasses used in the implementation of the present invention may have the following distribution of nitrogenous matter:

[0099] nitrogen from total amino acids: 25% to 50% of total nitrogen;

[0100] Betaine nitrogen: 0% to 40% of total nitrogen; and

[0101] ammoniacal nitrogen: 2% to 3% of total nitrogen.

[0102] Expressed in g / kg of dry matter, the ranges of average amino acid content of fermented molasses used in the context of the present invention may be: aspartic acid: 6-8; threonine: 0.5-3; serine: 1-4; glutamic acid: 10-50; proline: 3-4; glycine: 4-5; alanine: 2, 5-3, 5; valine: 2, 5-3, 5; methionine and cysteine: 0.5-3; isoleucine: 1.5-2, 5; tyrosine: 2-3; leucine: 3-4; phenylalanine: 1-2; lysine: 0.5-2, 5; histidine: 0.5-2; and arginine: 0.2-1. As a by-product of fermentation, fermented molasses has a low sugar content. "Low sugar content" here means fermented molasses containing less than 5%, less than 4%, less than 3%, less than 2%, or, more specifically, less than 1% by weight of sugars relative to the total mass of the dry extract. Preferably, the fermented molasses is free from fermentable sugars.

[0103] The fermented molasses used in the present invention may be raw fermented molasses or fermented molasses that has undergone one or more chemical or physicochemical treatments. For example, the fermented molasses may have undergone depotassium reduction (i.e., a process to reduce the potassium content) or demineralization. A depotassium reduction treatment consists, for example, of acidifying the raw fermented molasses with a H2SO4 solution followed by neutralization with ammonia.

[0104] The treatments that can be applied to fermented molasses qualitatively and quantitatively alter its mineral content. Thus, raw fermented molasses can have a crude ash content ranging from 14% to 22% by weight relative to the raw product and a potassium content ranging from 5% to 18% by weight relative to the raw product. By comparison, in depotassium-reduced or demineralized fermented molasses, the crude ash content varies from 5% to 14% by weight relative to the raw product, and the potassium content is generally less than 4% by weight relative to the raw product.

[0105] Due to a high ash content, raw fermented molasses has a density that can vary from 1.10 to 1.50. Preferably, the density of fermented molasses varies from 1.20 to 1.40, and particularly from 1.25 to 1.35.

[0106] In some embodiments, a fermented molasses used in the context of the present invention has a viscosity between approximately 10 mPa·s and 5000 mPa·s, preferably between approximately 10 mPa·s and 900 mPa·s. The viscosity is determined using a cone-plate rheometer at a temperature of 20°C and a shear rate of 20 s⁻¹. 1 For example, viscosity can be determined according to the detailed protocol below, where the measurements are carried out:

[0107] in a Kinexus Pro+ rheometer from Netzsch, featuring a cylindrical cassette (integrated cuvette), with a Mobile C34 having a smooth surface geometry, made of stainless steel, with a diameter of 33.64 mm, standard DIN; by placing approximately 30 ml of sample in the cuvette; and

[0108] by choosing the following parameters: Temperature (Pelletier effect) = 20°C, Initial shear rate = 0.1 s' 1 , End shear speed = 500 s'1 Number of points per decade of shear rate = 10 points.

[0109] The viscosity used is the viscosity recorded for a shear rate of 20 s' 1 .

[0110] Preferably, the fermented molasses used in the context of the present invention has a pH between 2 and 12, preferably between 4.5 and 8.

[0111] In some embodiments, fermented molasses is mixed with a yeast extract or used in combination with a yeast extract. The yeast extract is preferably a yeast fraction, preferably a soluble yeast fraction.

[0112] Generally, a distinction is made between yeast hulls (insoluble fraction) and yeast extracts (soluble fraction). Typically, yeast hulls or yeast extracts are obtained by a process comprising an autolysis or enzymatic hydrolysis step, primarily by proteases, followed by a separation step of the soluble fraction from the insoluble fraction. The isolated insoluble fraction corresponds to the yeast hulls, and the soluble fraction to the yeast extract. The insoluble and / or soluble fractions can then be dried. The processes for obtaining yeast hulls and yeast extracts are well-known in the art (see, for example, the reference work “Yeast Technology”, 2 eme edition, 1991, G. Reed and TW Nagodawithana, Van Nostrand Reinhold Ed., New York).

[0113] Yeast extract can be in dry form, preferably as a fine, water-soluble powder, in liquid form, as a paste, or as granules. Yeast extract is considered to be in dry form when its dry matter content is at least 85%. If its dry matter content is less than 70% by mass, it is considered to be in liquid form. From 70% to less than 85% by mass, the yeast extract is considered to be in paste form. The yeast extract used is preferably in dry form, and more preferably as a fine, water-soluble powder. Yeast extract consists mainly of protein, preferably at least 55% protein.

[0114] In some embodiments, fermented molasses is mixed with, or used in combination with, a yeast extract, where the yeast extract is a downgraded yeast extract, i.e., a yeast extract that cannot be marketed for its original use due to non-compliance with parameters for the originally intended application, such as non-compliant particle size, contamination, an incorrect salt content, or exceeding the best-before date. The use of downgraded yeast extracts is particularly advantageous, as it allows for the industrial and commercial valorization of yeast extracts destined for destruction. In some embodiments, the yeast extract is downgraded due to microbiological contamination, typically by bacteria such as E. coli.E. coli, Salmonella, Staphylococcus, or Clostridium, by yeasts or fungi (molds). In some embodiments, the yeast extract is a yeast extract unsuitable for use in food preparation.

[0115] The dry weight of the yeast extract preferably represents at least 90% of the total weight of the extract, and even more preferably at least 95% of the weight of the extract. In other words, the majority of the mass of the extract preferably consists of solid matter once the water has been removed.

[0116] The yeast extract advantageously has a total nitrogen content of between 1% and 20% or between 1% and 15% by dry weight of yeast extract. The total nitrogen content of the yeast extract may, in particular, be between 5% and 20% or between 5% and 15% by dry weight of yeast extract.

[0117] In some embodiments, fermented molasses is mixed with a yeast extract or is used in combination with a yeast extract during the anaerobic digestion step, the total content of fermented molasses and yeast extract being in particular between 0.01% and 15% by weight of substrate.

[0118] In some embodiments, the ratio by dry weight of fermented molasses: yeast extract is between 80:20 and 99:1, preferably between 85:15 and 99:1.

[0119] In a combination or composition comprising or consisting of a mixture of Bacillus / Peribacillus strains, such as BG6, and fermented molasses, the relative amounts of the two components may be any appropriate relative amounts. These relative amounts can be readily determined by a person skilled in the art. In some embodiments, the relative amounts by weight of the mixture of Bacillus / Peribacillus strains and fermented molasses may be between 1:500 and 1:500,000 (i.e., between 1:5 x 10⁻¹). 2 and l:5xl0 5 In particular, the relative quantities by weight of the mixture of Bacillus / Peribacillus strains and fermented molasses can be between 1:5000 and 1:50000 (i.e., between 1:5 x 10 3 and l:5xl0 4In certain particular embodiments, the relative quantities by weight of the mixture of Bacillus / Peribacillus strains and fermented molasses can be approximately 1:50,000 (i.e., between 1:5 x 10 4 ).

[0120] It is also envisaged to use combinations or compositions described above in the production of biogas by methanation of a methanogenic substrate, such that such combinations comprise, or are made up of, three or four components:

[0121] the mixture of Bacillus / Peribacillus strains (such as BG6), the Trichoderma atroviride strain TAA2025, and the live yeast strain Saccharomyces cerevisiae BG1; or

[0122] the mixture of Bacillus / Peribacillus strains (such as BG6), the Trichoderma atroviride strain TAA2025, and fermented molasses; or

[0123] the mixture of Bacillus / Peribacillus strains (such as BG6), the live yeast strain Saccharomyces cerevisiae BG1, and fermented molasses; or

[0124] the Trichoderma atroviride TAA2025 strain, the mixture of Bacillus / Peribacillus strains (such as BG6), the live yeast strain Saccharomyces cerevisiae BG1, and fermented molasses.

[0125] In particular, in certain embodiments, the anaerobic digestion of the methanogenic substrate is carried out in the presence of the Trichoderma atroviride TAA2025 strain, the live Saccharomyces cerevisiae BG1 yeast strain, the Bacillus / Peribacillus BG6 strain mixture, and fermented molasses, wherein the content of the Trichoderma atroviride TAA2025 strain is between 0.000000001% and 0.000001% or between 0.000001% and 0.00001% of the weight of the methanogenic substrate, in particular 0.000005% of the weight of the methanogenic substrate; where the content of the live yeast strain Saccharomyces cerevisiae BG1 is between 0.001% and 0.1% or between 0.001% and 0.05%, in particular about 0.012%, of the weight of methanogenic substrate;where the content of the Bacillus / Peribacillus BG6 strain mixture is between 0.000002% and 0.0002% or between 0.00002% and 0.0002% by weight of the methanogenic substrate, in particular 0.00002% by weight of the methanogenic substrate, and where the content of the fermented molasses is between 0.01% and 5% or between 0.05% and 2% by weight of the methanogenic substrate, in particular about 1% by weight of the methanogenic substrate.;

[0126] In some embodiments, the composition or combination comprises, or consists of, the Trichoderma atroviride TAA2025 strain, the Saccharomyces cerevisiae BG1 live yeast strain; the Bacillus / Peribacillus BG6 strain mixture, and fermented molasses in relative weight amounts of 1:2000:4:200000.

[0127] II - Use of Bacillus / Peribacillus Strain Mixtures and their Combinations and Compositions in Biogas Production

[0128] The present invention relates to the use of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, as described above, in the production of biogas by methanation (or anaerobic digestion) of a methanogenic substrate. The present invention also relates to a process for producing biogas from a methanogenic substrate, the process comprising the anaerobic digestion of said methanogenic substrate in the presence of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, according to the invention. The presence of the Bacillus / Peribacillus strain mixture, or the combination or composition thereof, increases the rate of biogas production (i.e., increases the kinetics of anaerobic digestion) and / or increases the yield of biogas production.

[0129] A. Biogas Production

[0130] As used here, the term “biogas” refers to a gaseous mixture produced by the fermentation of organic matter in the absence of oxygen. This gaseous mixture is primarily composed of methane (CH4) (50 to 70%) and carbon dioxide (CO2) (20 to 50%). Its composition varies depending on the substrate used. Indeed, depending on the nature of the substrate, biogas may also contain varying amounts of water (H2O), and traces of nitrogen (N2), ammonia (NH3), and hydrogen sulfide (H2S).

[0131] Generally, a biogas production process according to the invention comprises an anaerobic digestion step of a methanogenic substrate in the presence of the Bacillus / Peribacillus strain mixture or a combination or composition thereof described herein. 1. Methanogenic Substrate

[0132] The terms “methanogenic substrate,” “organic substrate,” “methanation substrate,” and “substrate” are used interchangeably here and refer to any substrate commonly used in a biogas production process. It is well established in the field that, to be suitable for methanization, a substrate must be rich in biodegradable (fermentable) organic matter, excluding woody materials, and must not contain elements that disrupt digestion (undesirable substances, inhibitors, etc.). a. Nature of the Methanogenic Substrate

[0133] A methanization process according to the present invention applies to any organic matter that is capable of naturally fermenting, including, but not limited to:

[0134] materials of agricultural origin, such as livestock manure (cattle or horse manure, cattle or pig slurry, etc.), crop residues, cereal waste, intermediate crops or intercrops, in particular energy-producing intermediate crops (CIVE), or sorting rejects of fruits or vegetables;

[0135] agri-food materials, such as vegetable or animal fats, co-products or by-products from factories producing / processing starch, vegetable proteins, citric acid, fish, meat (including slaughterhouses), milk, fruits and vegetables, sugar / alcohol co-products (beet / sugar factories, distilleries, breweries...), wine-growing effluents;

[0136] materials of industrial origin, such as industrial wastewater, production waste, effluents from the paper and green chemistry industries...;

[0137] materials managed by local authorities, such as the fine fraction of green waste (leaves, grass clippings, etc. from pruning and maintaining public and private green spaces, or from the trimming of hedges and roadside trees), wastewater treatment plant sludge, grease, and septic tank waste; and

[0138] Bio-waste from selective collection from individuals or produced by large producers linked to the distribution of foodstuffs (fairground markets, wholesale markets, retailers...), to catering (commercial and collective catering) and to food trade (waste from hypermarkets, supermarkets and food shops).

[0139] The amount of biogas produced by methanization can depend on the nature of the substrate used. Indeed, under given conditions, each type of organic matter generates its own specific amount of biogas, known as its methanogenic potential. This methanogenic potential is determined by the intrinsic characteristics, as well as the storage conditions, of these materials. Fats, and in particular oils, generally have the highest biogas yield potential, while other organic materials such as carbohydrate biopolymers, like cellulose or hemicellulose, have a slower degradation rate and a much lower biogas yield.

[0140] In practice, for so-called farm methanization units, the most used raw materials in methanization processes are animal waste (manure and slurry from cattle and pigs in particular).

[0141] Industrial-scale biogas plants primarily use mixtures of food waste, sewage sludge, flotation fats, fats and waste from the agri-food industry, and green waste as substrates. The drawback of mixing these different raw materials is that it often results in a substrate with a low biogas rate and yield because the mixture is poor in fermentable carbon compounds.

[0142] In certain preferred embodiments of the present invention, the methanogenic substrate consists of organic products or by-products of animal or plant origin, and more particularly of animal or plant waste, notably selected from animal excrement (in particular selected from manure and slurry), agri-food waste, sewage sludge, flotation fats, and green waste. In certain specific embodiments of the invention, the methanogenic substrate is bovine manure, pig slurry, a plant substrate, or combinations thereof.

[0143] In certain specific embodiments, the substrate consists of bovine manure. The substrate can also consist of a mixture of cattle manure and cattle slurry, where the manure and slurry are present in any suitable mass ratio, ranging from 1:99 to 99:1, for example 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:65, 50:50, 55:45, 60:40, 65:45, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5, particularly between 10:90 and 20:80, such as approximately 10:90, approximately 11:89, approximately 12:88, approximately 13:87, approximately 14:86, approximately 15:85, approximately 16:84, approximately 17:83, approximately 18:82, approximately 19:81, or approximately 20:80.

[0144] In other particular embodiments, the substrate consists of bovine manure and plant fibers, such as corn, beet pulp, and triticale (an annual plant of the grass family). The mass ratio of cattle manure to plant fiber can be any suitable mass ratio from 1:99 to 99:1, for example 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:65, 50:50, 55:45, 60:40, 65:45, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5, particularly between 30:70 and 55:45, such as approximately 30:70, approximately 31:69, approximately 32:68, approximately 33:67, approximately 34:66, approximately 35:65, approximately 36:64, approximately 37:63, approximately 38:62, approximately 39:61, approximately 40:60, approximately 41:59, approximately 42:58, approximately 43:57, approximately 44:56, approximately 45:55, approximately 46:54, approximately 47:53, approximately 48:52, approximately 49:51, approximately 50:50, approximately 51:49, approximately 52:48, approximately 53:47, approximately 54:46, or approximately 55:45.For example, the substrate may consist of 50% by weight of cattle manure, 15% by weight of maize, and 35% by weight of triticale; or of 35% by weight of cattle manure, 15% by weight of maize, and 50% of triticale.

[0145] In other particular embodiments, the substrate consists of pig slurry and plant fibers, such as corn, beet pulp, and triticale. The mass ratio of bovine slurry to plant fiber can be any suitable mass ratio from 1:99 to 99:1, for example 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:65, 50:50, 55:45, 60:40, 65:45, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5, particularly between 20:80 and 30:70, for example approximately 20:80, approximately 21:79, approximately 22:78, approximately 23:77, approximately 24:76, approximately 25:75, approximately 26:74, approximately 27:73, approximately 28:72, approximately 29:71, or approximately 30:70. For example, the substrate may consist of 50% by weight maize, 25% by weight triticale, and 25% pig slurry.

[0146] In other specific embodiments, the substrate consists solely of plant fibers, such as maize, beet pulp, and triticale. For example, the substrate may consist of 15% by weight maize, 35% by weight beet pulp, and 50% triticale.

[0147] b. Form of the Methane-Producing Substrate

[0148] In the context of a biogas production process according to the invention, a methanogenic substrate can be used in any suitable form, for example, as a liquid, paste, or solid. The terms “paste” and “pasty product” are used interchangeably here. They refer to an incompressible, deformable medium of medium or high apparent viscosity, in one or more phases. Experts in the field know that pasty products belong to an intermediate range between liquid and solid media. In some embodiments, when anaerobic digestion is desired under given viscosity conditions of the methanogenic substrate, the viscosity of the substrate can be adjusted by mixing it with water.

[0149] c. Pretreatment of the Methane-producing Substrate

[0150] In some embodiments, the methanogenic substrate is used as recovered before being subjected to anaerobic digestion.

[0151] However, it is well known in the field that a pretreatment phase may be necessary to accelerate the methanogenic conversion of certain materials. Thus, in some embodiments, the methanogenic substrate undergoes pretreatment before anaerobic digestion. This pretreatment may include at least one step selected from: grinding, composting, thermodynamic treatment, enzymatic treatment, or a combination of these techniques. Pretreatment breaks down the substrate's macromolecules into monomers, thereby shortening the hydrolysis phase of anaerobic digestion (see below).

[0152] 2. Anaerobic Digestion

[0153] Biogas production is achieved through the anaerobic digestion of a methanogenic substrate. The terms “anaerobic digestion,” “methanization,” and “biomethanization” are used interchangeably here and refer to a sequence of reactions by which microorganisms decompose a substrate (biodegradable material) in the absence of oxygen. Methanization generates a gaseous product (including methane) and a residual sludge, the digestate. Methanization is distinct from composting, which is a process of transforming organic waste by microorganisms and small animals (earthworms) in the presence of oxygen (an aerobic reaction).

[0154] Anaerobic digestion, which involves a complex microbial ecosystem, comprises the following main stages:

[0155] hydrolysis, where the substrate macromolecules, such as nucleic acids and biopolymers (polysaccharides, lipids, proteins), are degraded into water-soluble fragments (monomers) such as monosaccharides (glucose), fatty acids, amino acids, and nitrogenous bases;

[0156] Acidogenesis, where the monomers from the hydrolysis step are primarily fermented into organic acids and alcohols, and where hydrogen (H2) and carbon dioxide (CO2) are also produced; and acetogenesis, where the various intermediates from the previous phases are transformed into acetate, hydrogen (H2), and carbon dioxide (CO2). During this step, two metabolic pathways have been identified: the homoacetogenic pathway, which leads only to the production of acetate, and the heteroacetogenic pathway, which results in the production of acetate, H2, and CO2; and

[0157] Methanogenesis, which constitutes the final stage of the anaerobic digestion process, consists of the mineralization of the products of acetogenesis into methane. Two metabolic pathways are involved in this final transformation: the acetoclastic methanogenesis pathway, which produces methane and carbon dioxide from acetate, and the hydrogenotrophic methanogenesis pathway, which uses hydrogen (H2) and CO2 to produce methane.

[0158] a. Digester

[0159] The conditions for implementing anaerobic digestion are well known in the art. Anaerobic digestion operations are typically carried out in closed reactors called digesters, biodigesters, or methanizers. A methanizer generally consists of a large, airtight tank with a lid, into which the methanogenic substrate to be treated is placed.

[0160] Digesters come in various shapes and sizes. Anaerobic digestion according to the present invention can be implemented using any suitable digester. Several types of digesters exist, each designed for specific operating conditions and substrate types. The main types of digesters include, but are not limited to:

[0161] continuous digesters (flow type digesters), such as the continuous stirred-tank reactor which keeps the substrate in motion to ensure homogeneity and efficiency of the digestion process, or the plug flow digester which is designed for more solid waste and allows materials to move slowly from inlet to outlet with or without mixing along the way;

[0162] Batch type digesters, such as the batch digester, which processes one substrate load at a time, allowing complete digestion before a new load is added; multi-phase digesters, such as the separate-phase digester, which separates the different stages of anaerobic digestion (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) into different reactors to optimize conditions for each phase;

[0163] dry digestion systems, such as the dry digester, which is suitable for solids with a dry matter content greater than 15% and which operates with little or no liquid recirculation;

[0164] Wet digesters are used for waste with a low dry matter content (less than 15%), generally in liquid or semi-liquid form, requiring a constant supply of water or liquid:

[0165] Temperature-controlled digestion systems, such as the psychrophilic digester which operates at temperatures between 15°C and 25°C; or the mesophilic digester which operates at average temperatures of 25°C to 40°C, which is the most common; or the thermophilic digester which operates at higher temperatures, generally between 45°C and 60°C, for faster anaerobic digestion; and

[0166] Fixed dome digesters, where the biogas chamber is integrated into the digester with a fixed dome; and

[0167] floating drum digesters, in which the biogas container moves up or down depending on the amount of biogas produced.

[0168] A person skilled in the art is able to select the most appropriate digester according to the nature and form of the methanogenic substrate used, and the situation (farms or livestock farms; methanization plants; wastewater treatment and sludge treatment plants; etc...).

[0169] A digester is generally integrated into a methanization unit which may include several pieces of equipment, such as, for example: a storage and sorting area for materials, a gasometer to store the gas produced, a cogeneration boiler, a technical room to monitor the various operations, etc.

[0170] b. Anaerobic Digestion Reaction

[0171] In a biogas production process according to the invention, the anaerobic digestion step of a methanogenic substrate in the presence of the Bacillus / Peribacillus strain mixture or a combination or composition thereof as described above generally takes place under controlled temperature and / or pressure conditions known to those skilled in the art or that they can easily determine. Mesophilic digestions, which are carried out at a temperature between approximately 30°C and approximately 40°C, are the most commonly used. Thermophilic digestions, which are carried out at a temperature between approximately 50°C and approximately 65°C, are more energy-intensive but allow for the acceleration of the hydrolysis step of solid organic substrates. Generally, the pressure within a digester is between 0.2 and 0.5 bar, which is slightly higher than atmospheric pressure.Depending on the volume of substrate subjected to digestion and the type of substrate, the anaerobic digestion stage can last, for example, from about 15 minutes to about 30 days, or more than 30 days.

[0172] In general, during the anaerobic digestion of the methanogenic substrate in the presence of the Bacillus / Peribacillus strain mixture or a combination or composition thereof as described herein, the concentration of the Bacillus / Peribacillus strain mixture or combination or composition can be any appropriate concentration. Such a concentration can easily be determined by a person skilled in the art based on the nature and quantity of the substrate and the operating conditions of the anaerobic digestion.

[0173] In certain embodiments, when a mixture of Bacillus / Peribacillus strains, such as BG6, is used alone in biogas production by anaerobic digestion of a methanogenic substrate, the content of the Bacillus / Peribacillus strain mixture can be between 0.002% and 0.0000002% by weight of the methanogenic substrate. In particular, the content can be between 0.00002% and 0.0000002% by weight of the methanogenic substrate. In some specific embodiments, the content of the Bacillus / Peribacillus strain mixture is approximately 0.00002% by weight of the methanogenic substrate.

[0174] In some embodiments, when the Bacillus / Peribacillus strain mixture, such as BG6, is used in combination or composition with the live yeast strain Saccharomyces cerevisiae BG1 in biogas production by anaerobic digestion of a methanogenic substrate, the content of the BG1 strain may be between 0.1% and 0.001%, for example about 0.010% of the weight of the methanogenic substrate, and the content of the Bacillus / Peribacillus strain mixture may be between 0.000002% and 0.0010% of the weight of the methanogenic substrate. In particular, the content of the BG1 strain can be about 0.010% of the weight of methanogenic substrate and the content of the mixture of Bacillus / Peribacillus strains can be between 0.00002% and 0.0005% of the weight of methanogenic substrate.In certain particular embodiments, the content of strain BG1 is about 0.010% of the weight of the methanogenic substrate and the content of the mixture of Bacillus / Peribacillus strains is about 0.00002% of the weight of the methanogenic substrate.

[0175] In some embodiments, when the Bacillus / Peribacillus strain mixture, such as BG6, is used in combination or composition with the Trichoderma atroviride TAA2025 strain in biogas production by anaerobic digestion of a methanogenic substrate, the content of the Bacillus / Peribacillus strain mixture may be between 0.000002% and 0.0002% or between 0.00002% and 0.0002% by weight of the methanogenic substrate and the content of the Trichoderma atroviride TAA2025 strain may be between 0.000001% and 0.000000001% or between 0.000005% and 0.000000001% by weight of the methanogenic substrate. In certain particular embodiments, the content of the Bacillus / Peribacillus strain mixture is about 0.00002% by weight of the methanogenic substrate and the content of the Trichoderma atroviride TAA2025 strain is about 0.000005% by weight of the methanogenic substrate.

[0176] In some embodiments, when the Bacillus / Peribacillus strain mixture, such as BG6, is used in combination or composition with fermented molasses in biogas production by anaerobic digestion of a methanogenic substrate, the content of the fermented molasses may be between 0.01% and 5%, preferably between 0.05% and 2%, and more preferably about 1% by weight of methanogenic substrate, and the content of the Bacillus / Peribacillus strain mixture may be between 0.000002% and 0.0002% by weight of methanogenic substrate.

[0177] In some embodiments, the Bacillus / Peribacillus strain mixture, or combination or composition thereof, is added before the methanogenic substrate is added to the digester. In other embodiments, the Bacillus / Peribacillus strain mixture, or combination or composition thereof, is added concurrently with the addition of the methanogenic substrate to the digester. In still other embodiments, the Bacillus / Peribacillus strain mixture, or combination or composition thereof, is added after the methanogenic substrate has been added to the digester. c. Inoculum

[0178] In some embodiments, the only microorganisms added to the digester for the anaerobic digestion of the methanogenic substrate are the Bacillus / Peribacillus strain mixture or combination or composition thereof described above. In other embodiments, anaerobic digestion according to the present invention is carried out in the presence of microorganisms other than the Bacillus / Peribacillus strain mixture or combination or composition thereof described herein. The microorganisms are preferably used in the form of an inoculum. As used herein, the term “inoculum” refers to a population of microorganisms which, when introduced into a fermentation medium, is capable of promoting or assisting in the initiation of the decomposition and degradation of a methanogenic substrate. The inoculum may be in powder or liquid form.It should be noted here that an inoculum will be introduced at the start of each batch anaerobic digestion reaction, whereas it will only be introduced once at the initiation of a continuous anaerobic digestion.

[0179] An inoculum used in anaerobic digestion according to the invention comprises at least one methanogenic microorganism. The terms “methanogenic microorganism” and “microorganism with methanogenic metabolism” are used interchangeably herein. They refer to microorganisms that produce methane as a metabolic byproduct of life under anoxic conditions, through methanogenesis.

[0180] An inoculum used in anaerobic digestion according to the invention may comprise any methanogenic microorganism or combination of methanogenic microorganisms known to catalyze at least one of the steps of anaerobic digestion (hydrolysis, acidogenesis, acetogenesis, or methanogenesis).

[0181] Methanogenic microorganisms belong to the class Archaea, which are unicellular prokaryotic microorganisms. Methanogenic archaea belong to 7 phylogenetic orders: Methanococcales, Methanopyrales, Methanobacteriales, Methanosarcinales, Methanomicrobiales, Methanocellales, and Methanoplasmatales.

[0182] Methanogenic archaea can be classified into three groups based on the substrate used for methane production: hydrogenotrophic methanogens, which use carbon dioxide as a carbon source, in the presence of hydrogen as a reducing agent for methane formation. Apart from the genus Methanosaeta, all methanogenic archaea are capable of carrying out this reaction. The most represented genera are Methanosarcina, Methanobacterium, Methanobrevibacter, Methanospirrilium, Methanogenium, and Methanocorspusculum;

[0183] Methylotrophic methanogens use methylated compounds as a carbon source in the presence of carbon dioxide. The main methylotrophic archaea belong to the orders Methanobacteriales and Methanoplasmatales, as well as the genus Methanosarcina; and

[0184] Acetotrophic or aceticlastic methanogens utilize acetate in the presence of hydrogen. Acetoclastic methanogenic archaea are dominated by the order Methanosarcinales, and in particular the two genera: Methanosaeta and Methanosarcina.

[0185] Methanogenic microorganisms are characterized by the presence of a coenzyme F420 responsible for blue-green autofluorescence when cells are exposed to ultraviolet light at a wavelength of approximately 350-420 nm. Thus, cells or colonies of methanogenic microorganisms can be rapidly identified under an epifluorescence microscope.

[0186] Thus, in certain embodiments, an inoculum used in anaerobic digestion according to the invention comprises at least one methanogenic microorganism belonging to the class Archaea, in particular to a phylogenetic order selected from the group consisting of the phylogenetic orders Methanococcales, Methanopyrales, Methanobacteriales, Methanosarcinales, Methanomicrobiales, Methanocellales, and Methanoplasmatales. In certain embodiments, a methanogenic microorganism present in an inoculum used in anaerobic digestion according to the invention is a hydrogenotrophic methanogen, and belongs in particular to a genus selected from the group consisting of the genera Methanosarcina, Methanobacterium, Methanobrevibacter, Methanospirrilium, Methanogenium, and Methanocorspusculum.In certain embodiments, a methanogenic microorganism present in an inoculum used in anaerobic digestion according to the invention is a methylotrophic methanogen, and belongs in particular to the order Methanobacteriales or the order Methanoplasmatales or to the genus Methanosarcina. In certain embodiments, a methanogenic microorganism present in an inoculum used in anaerobic digestion according to the invention is an acetotrophic or aceticlastic methanogen, and belongs in particular to the order Methanosarcinales, especially to the genus Methanosaeta or the genus Methanosarcina.

[0187] Those skilled in the art know that a typical inoculum used in anaerobic digestion processes generally comprises methanogenic microorganisms mixed with non-methanogenic microorganisms. Thus, an inoculum used in a biogas production process according to the invention may comprise non-methanogenic bacteria, for example, non-methanogenic bacteria belonging to the Firmicutes and Bacteroidetes groups.

[0188] In some embodiments, the inoculum used in the anaerobic digestion of a methanogenic substrate for the production of biogas according to the invention is digested sludge from a wastewater treatment plant digester (for sewage or industrial effluents), or from another anaerobic digester. This type of inoculum is considered suitable because it exhibits relatively high methanogenic activity and a sufficiently heterogeneous microbial ecosystem to adapt to the treatment of several types of substrates. Alternatively, the inoculum may be digested cattle manure or digested pig manure, or may be derived from other fermenting animal waste.

[0189] Generally speaking, a person skilled in the art knows how to determine the inoculum content to be introduced into a digester according to the nature of the anaerobic digestion reaction (continuous mode or batch mode).

[0190] In some embodiments, the inoculum is added before the Bacillus / Peribacillus strain mixture, or combination or composition thereof, is added to the digester. In other embodiments, the inoculum is added concurrently with the addition of the Bacillus / Peribacillus strain mixture, or combination or composition thereof, to the digester. In still other embodiments, the inoculum is added after the addition of the Bacillus / Peribacillus strain mixture, or combination or composition thereof, to the digester.

[0191] B. Implementation of Anaerobic Digestion for Biogas Production In certain embodiments, a process for producing biogas from a methanogenic substrate according to the present invention comprises the following steps: introduction, into a digester, of the mixture of Bacillus / Peribacillus strains or of the combination or composition thereof described herein, of a methanogenic substrate, and optionally of an inoculum;

[0192] Anaerobic digestion of the methanogenic substrate to produce biogas; and optionally, collection of the biogas produced.

[0193] The methanogenic substrate, the Bacillus / Peribacillus strain mixture, or combination or composition thereof, the inoculum, and the digester have been described above; as have the order of addition and the relative quantities of the various reagents in the methanation reaction. The temperature and / or pressure conditions and the duration of the anaerobic digestion reaction have also been described above.

[0194] In some embodiments, the methanogenic substrate, the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, and optionally F inoculum, are the only reactants in the methanation reaction. In other embodiments, the viscosity of the methanogenic substrate can be reduced by mixing it with water.

[0195] The collection of biogas produced by a digestion process according to the invention can be carried out by any method known to a person skilled in the art.

[0196] In addition to carbon dioxide and water vapor, biogas produced by anaerobic digestion can contain several pollutants: sulfur compounds, organohalogen compounds, and volatile organic compounds (VOCs). In some embodiments, a production process according to the invention further includes a biogas purification (or cleaning) step for the purpose of recovering methane. The purification (or cleaning) can be carried out by any method or combination of methods known in Fart. Existing cleaning technologies include, but are not limited to:

[0197] Adsorption: Pressure Swing Adsorption (PSA) purifies biogas by binding its constituent molecules to supports (the adsorbent being composed of a series of molecular filters or zeolites). Depending on the treatment pressure (generally varying between 4 and 7 bar), different compounds are separated from the gas: carbon dioxide, water, hydrogen sulfide, and oxygen.

[0198] Absorption: Purification by absorption is achieved by counter-current contact between the biogas and a solvent which absorbs carbon dioxide and other soluble gases. Depending on the nature of the solvent used, we distinguish between water scrubbing, chemical absorption (or amine scrubbing) and physical absorption (glycol scrubbing).

[0199] Membrane separation: the separation of carbon dioxide from biogas is due to the difference in permeability of the membranes with respect to the compounds of biogas: carbon dioxide passes through the membrane faster than methane, there is a concentration of methane on one side of the module.

[0200] Cryogenic purification: Cryogenics, or cold distillation, takes advantage of the different boiling points of the gaseous compounds in biogas (-78°C at atmospheric pressure for carbon dioxide and -160°C at atmospheric pressure for methane). In this technique, the biogas is cooled to -165°C, allowing the methane to be extracted in its liquid phase.

[0201] Regardless of the purification process, each treatment includes at least the following three steps: desulfurization (which removes hydrogen sulfide (H2S)); dehydration (which removes water); and decarbonation (which removes carbon dioxide).

[0202] III - Advantages of Bacillus / Peribacillus Strain Mixtures and their Combinations and Compositions in Biogas Production

[0203] The present inventors have experimentally demonstrated that the presence of the Bacillus / Peribacillus strain mixture, such as BG6, or a combination or composition thereof described above, during the anaerobic digestion of a methanogenic substrate positively influences biogas production. In particular, they have demonstrated that the presence of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, during the anaerobic digestion of a methanogenic substrate improves the biogas production kinetics (i.e., increases the rate of biogas production) and / or improves the biogas production yield (i.e., increases the amount of biogas produced for a given amount of substrate entering the biogas production process, or, in other words, increases the methanogenic potential of the substrate).

[0204] In a first aspect, the present invention relates to the use of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, described above, to increase the rate of biogas production by anaerobic digestion of a methanogenic substrate. In a second aspect, the present invention relates to the use of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, described above, in a biogas production process by anaerobic digestion from a methanogenic substrate to increase the biogas production yield from the substrate.

[0205] In a third aspect, the present invention relates to the use of the mixture of Bacillus / Peribacillus strains or a combination or composition thereof described above, in a process of biogas production by anaerobic digestion of a methanogenic substrate to increase the rate of biogas production and to increase the yield of biogas production from the substrate.

[0206] In another aspect, the present invention relates to the use of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, described above in a biogas production process by anaerobic digestion of a methanogenic substrate to reduce biogas production costs, particularly energy costs. Indeed, the use of the Bacillus / Peribacillus strain mixture, or a combination or composition thereof, leads to increased homogeneity of the material during digestion, thereby reducing the energy required for effective mechanical mixing of the decomposing material.

[0207] A. Increased Biogas Production Rate by Anaerobic Digestion In the context of the present invention, the terms “increased biogas production rate” and “improved biogas production kinetics” are used interchangeably. They refer to an increase in the quantity of biogas produced from a given quantity of substrate within a given time period. This increased biogas production rate may occur with or without a concomitant increase in the methanogenic potential of the substrate.

[0208] The rate of biogas production can be increased throughout the entire anaerobic digestion reaction or only during a portion or stage of the anaerobic digestion reaction (e.g., during hydrolysis, acidogenesis, acetogenesis, or methanogenesis). For example, the rate of biogas production can be increased during the initiation of anaerobic digestion. The term “initiation of anaerobic digestion” refers to the beginning of the process during which microorganisms (i.e., the Bacillus / Peribacillus strain mixture or a combination or composition thereof described above, with or without an inoculum) begin to degrade the methanogenic substrate.

[0209] In the context of the present invention, an increase in the rate of biogas production is said to occur when the overall rate of biogas production or the rate of a stage of biogas production is increased by at least 20%, preferably by at least 50%, more preferably by at least 80%, for example 100% or more, compared to the overall rate of biogas production in the absence of the mixture of Bacillus / Peribacillus strains or a combination or composition thereof during the anaerobic digestion reaction.

[0210] Similarly, we speak of an increase in the rate of biogas production when the presence of a mixture of Bacillus / Peribacillus strains, or a combination or composition thereof, reduces the anaerobic digestion time of a methanogenic substrate by at least 3%, preferably at least 5%, more preferably at least 10%, and even more preferably at least 20%, compared to the standard anaerobic digestion time, all other things being equal. For example, for a 30-day anaerobic digestion in the presence of substrate alone, the mixture of Bacillus / Peribacillus strains, or a combination or composition thereof, reduces the anaerobic digestion time by at least 1 day, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days or more.

[0211] B. Increased Biogas Production Yield by Anaerobic Digestion In the context of the present invention, the terms “increased biogas production by anaerobic digestion of a methanogenic substrate” and “increased methanogenic potential of a substrate” are used interchangeably. They refer to an increase in the quantity of biogas produced from a given quantity of substrate. This increase in the methanogenic potential of the substrate may occur with or without a concomitant increase in the biogas production rate.

[0212] The terms “methanogenic potential” and “methanogenic power” are used interchangeably here. Also known as BMP (acronym for “Biochemical Methane Potential”), methanogenic potential corresponds to the amount of methane produced by a given quantity of substrate during its complete biodegradation under anaerobic conditions during the methanation process. This volume of methane, relative to the quantity of fresh, dry (DM) or volatile (VM) substrate, is generally expressed under standard temperature and pressure conditions (0°C, 1013 hPa). This indicator is well known to those in the field and is used, in particular, for sizing methanation units, dosing inputs and monitoring their quality, as well as for assessing potential carbon losses during storage.Determining this potential, for each input or for mixtures of inputs, is a central and essential element for any consideration concerning methanization processes, from the technical and economic analysis of a project, the sizing of treatment and recovery facilities, to the evaluation of the performance of a process.

[0213] In the context of the present invention, the determination of the methanogenic potential of a substrate can be carried out using any suitable method known in the art. The BMP calculation is performed in the laboratory. Depending on the biodegradability of the analyzed substrate, the BMP calculation is lengthy: from two to six weeks.

[0214] In some embodiments, the methanogenic potential of a substrate can be determined according to a method comprising the following steps:

[0215] 1. Sample preparation: Substrate samples are collected and prepared for analysis. They are usually ground or homogenized to obtain a uniform particle size, and placed in reaction flasks;

[0216] 2. Inoculation: A known quantity of inoculum, for example anaerobic sludge or a culture of methanogenic microorganisms, is added to the reaction flasks to seed the substrate samples and initiate the anaerobic digestion process. It should be noted that this step is not repeated in a continuous process, which does not require reseeding except for the very first time at process start-up; 3. Flask conditioning: The reaction flasks are sealed, and anaerobic conditions are maintained inside these flasks to simulate the environment in an anaerobic digester;

[0217] 4. Measurement of gas volumes: the bottles are placed in a gas production measuring device, where the volumes of gas produced are regularly measured over time.

[0218] 5. Calculation of BMP: The cumulative methane production is measured over a given period, generally several weeks, typically 3 weeks. The BMP is then calculated by dividing the amount of methane produced by the amount of substrate used, and generally expressing it in units of volume per weight of dry substrate (for example: mL of methane per gram of dry substrate).

[0219] A method for determining the effect of the mixture of Bacillus / Peribacillus strains or one of its compositions described here is detailed in the Examples section below.

[0220] In the context of the present invention, we speak of increasing the yield of a methanogenic substrate into biogas, or increasing the methanogenic potential of a methanogenic substrate into biogas, when the methanogenic potential of the substrate into biogas is increased by at least 50%, preferably by at least 75%, more preferably by at least 100% or more, compared to the methanogenic potential of the substrate into biogas in the absence of the mixture of Bacillus / Peribacillus strains or a combination or composition thereof during the anaerobic digestion reaction.

[0221] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by an ordinary specialist in the field to which this invention belongs. Likewise, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

[0222] Examples

[0223] The following examples describe certain embodiments of the present invention. However, it is understood that the examples are presented for illustrative purposes only and do not in any way limit the scope of the invention.

[0224] Example 1: Effects of the mixture of Bacillus / Peribacillus strains and their composition on the kinetics and yield of biogas production by anaerobic digestion of bovine manure

[0225] Materials and Methods

[0226] Measurement of Biochemical Methane Potential (BMP) a. Definition of Parameters

[0227] MB = Raw Material, the methanogenic substrate has undergone no treatment. MS = Dry Material, the methanogenic substrate is dried to determine the proportion of dry elements present. MO = Organic Matter, the methanogenic substrate undergoes heat treatment to define the proportion of organic matter.

[0228] MO / MS represents the proportion of Organic Matter in the Gross Product.

[0229] b. Description of the BMP Test

[0230] The methanogenic potential was evaluated in 1-liter bottles using a batch method (controlled incubation in bottles). An identical and exact quantity of the sample to be evaluated was initially introduced into each bottle and constituted the sole input for the test. The test ended when all of the product had been degraded. Each sample was tested five times to increase the representativeness of the values ​​obtained.

[0231] The sample to be evaluated may include:

[0232] Cattle manure and an inoculum of sewage sludge diluted (max. 6%) with water, inoculum dry matter: 2-3%; or

[0233] Cattle manure, inoculum, and fermented molasses (PI - a mixture of fermented beet molasses and fermented cane molasses in a 90 / 10 weight / weight ratio); or

[0234] Cattle manure, the inoculum, and a competing product (P3 - commercial enzyme-based preparation: Trichoderma longibrachiatum containing cellulase, xylanase, and beta-glucanase); or

[0235] Bovine manure, inoculum, and the mixture of Bacillus / Peribacillus strains or a composition thereof to be tested.

[0236] For each test, two types of checks were carried out:

[0237] Negative controls, also known as "blank" controls, are tests where the sample contains only the inoculum. These negative controls allow us to determine the proportion of biogas production resulting from the residual activity of the inoculum. These values ​​are then subtracted from those obtained for the tested samples.

[0238] Positive controls, containing the inoculum and powdered raw glucose, are used. The glucose is completely degraded by microorganisms involved in the anaerobic digestion process. These positive controls allow for the evaluation of the inoculum's biological activity, thus validating the test. In the studies presented below, a positive control was systematically performed to verify the inoculum's activity, validating the test for each series of experiments. Prior to determining the methanogenic potential, the sample is characterized in terms of its dry matter (DM) and organic matter (OM) content to allow for the adjustment of the inputs for each sample according to the protocol. The objective is to add 3 g of OM to each bottle containing the substrate to be tested.

[0239] The samples tested can be in three forms: fresh raw, dry raw, and freeze-dried ground. Each form has advantages and disadvantages in terms of ease of preparation and representativeness. In the examples below, the samples tested are in liquid form for fermented molasses, in fresh raw form for the substrate (cattle manure), and in dry form for the other ingredients.

[0240] The BMP test preparation was carried out as follows:

[0241] Weigh the mass of sample required for the addition of 3 g of OM from the latter. For this, it is necessary to take into account the characteristics of the test sample (MS, OM) and also its form (fresh raw, dry raw, freeze-dried ground, etc...);

[0242] Top up with water so that the gross mass of sample and water represents 50 g;

[0243] Add 700 g of inoculum, preferably homogenized in the storage container and shake with a magnetic stirrer;

[0244] Perform the initial pH measurement;

[0245] To remove the bottle with the septum and the screw cap;

[0246] Place the bottle in an oven at 37.5 °C;

[0247] Note the start time of the manipulation when all bottles in a series of manipulations are put to study.

[0248] Biogas production measurements were taken regularly during the anaerobic digestion reaction.

[0249] Biogas production measurements were carried out as follows:

[0250] Remove the bottles from the oven in batches of 10 maximum;

[0251] Shake each bottle before measuring for 30 seconds while making a zero-point motion;

[0252] Using a needle, puncture the septum, taking care to keep the puncture hole as far away as possible from the previous ones; Record the volume of biogas produced on the Ritter drum meter as well as the percentage of methane and the percentage of carbon dioxide displayed on the infrared sensors;

[0253] Repeat the process for each bottle;

[0254] When the series of 10 bottles is measured, place them back in the oven at 37.5°C.

[0255] The test ends between 30 and 40 days after its launch when the cumulative production of biogas linked to the degradation of the tested substrate is almost zero.

[0256] The final pH is measured for each bottle to ensure that the medium has not undergone acidosis.

[0257] Results

[0258] A. Preliminary Evaluation of the Effects of Different Microbial Products and Fermented Molasses on the BMP of Bovine Manure

[0259] The microbial products tested in this preliminary assessment are:

[0260] the strain of the fungus Trichoderma atroviride TAA2025 (referred to as TAA2025 hereinafter),

[0261] the live yeast Saccharomyces cerevisiae BG1 (designated BG1),

[0262] the mixture of bacterial strains of the genus Bacillus / Peribacillus (designated BG6), the fermented molasses defined above (designated PI), and

[0263] a product of the competition defined above (designated P3).

[0264] The methanogenic potential of cattle manure was determined through anaerobic digestion at 37.5°C, which continued for 38 days. Anaerobic digestions were conducted both with and without each of the different products being tested. The concentrations of the various products used in the anaerobic digestion reactions are presented in Table 1 below.

[0265] Table 1. Contents of the microbial products tested and of the fermented molasses in the anaerobic digestion of bovine manure. Each of the products was tested at a high (E), medium (M) and low (F) content.

[0266] Products Tested Content Level Content (expressed as a % by weight of manure)

[0267] E 0.000500%

[0268] TAA2025 M

[0269]

[0270] 0.000050%F 0.000005%

[0271] E 0.01200%

[0272] BG1 M 0.00625%

[0273] F 0.00200%

[0274] E 0.00200%

[0275] BG6 M 0.00020%

[0276] F 0.00002%

[0277] E 2.0%

[0278] PI M 1.0%

[0279] F 0.1%

[0280] P3 Recommended content 0.0010%

[0281]

[0282] The kinetics of the anaerobic digestion reaction were monitored for each sample, and key parameters were determined (see Figure 1). Kinetic data and biogas composition are presented in Table 2 below.

[0283] Table 2. Kinetic data of anaerobic digestion of bovine manure in the absence or presence of different microbial products and fermented molasses at a high (E, according to Table 1), medium (M, according to Table 1) or low (F, according to Table 1) content; and composition of the biogas produced.

[0284] V max V max degradation degradation. Product Composition (m 3 of CH4 (m 3 of CH4 / at 80% to 90% biogas produced microbial / day / T MO) day / T MB) (days) (days)

[0285] % CH4% CO2aucun 10,46 + 0,99 1,63 16j. 27 j. 48% 24% E 11,80 + 1,43 1,87 23 j. 26 j. 48% 23% TAA2025 M 14,16 + 2,58 2,21 23 j. 27 j. 48% 25% F 19,40 + 1,02 3,03 22j. 28 j. 49% 25% E 10,97 + 0,13 2,21 16j. 22 j. 49% 24% BG1 M 12,77 + 1,08 1,96 16j. 22 j. 49% 24% F 10,39 + 0,91 1,63 18 j. 23 j. 48% 23% E 11,07 + 0,82 1,73 23 j. 30 j. 47% 24% BG6 M 10,81 + 1,61 1,57 23 j. 30 j. 48% 25% F 18,11 + 1,37 2,66 24j. 29 j. 49% 25% E 6,68 + 2,22 1,08 15 j. 23 j. 48% 24% PI M 12,95 + 3,32 2,25 23 j. 30 j. 49% 25% F 12.56 + 2.86 2.34 21 j- 26 j. 48% 24%

[0286]

[0287] P3 8,12 + 1,03 1,27 20j. 28 j. 47% 23%

[0288]

[0289] The results in Table 2 show that each of the microbial products tested (Trichoderma atroviride TAA2025, live yeast Saccharomyces cerevisiae BG1, and Bacillus / Peribacillus strain mixture) increases the initial rate of substrate (cattle manure) degradation, whereas this is not the case for the competing product P3. In particular, an increase in the initial rate of degradation of 85.5% is observed for Trichoderma atroviride TAA2025 at a content of 0.000005% by weight of cattle manure; of 73.14% for the Bacillus / Peribacillus strain mixture at a content of 0.00002% by weight of cattle manure; and of 22.08% for the live yeast Saccharomyces cerevisiae BG1 at a content of 0.00200% by weight of cattle manure.The results in Table 2 also show that at least Trichoderma atroviride TAA2025 and the live yeast Saccharomyces cerevisiae BG1 reduce the time it takes for cattle manure to degrade by anaerobic digestion, whereas this is not the case for the competing product P3. In particular, 90% substrate degradation is achieved in one day less with Trichoderma atroviride TAA2025 at a concentration of 0.000500% by weight of cattle manure; and in four to five days less with the live yeast Saccharomyces cerevisiae BG1 at all concentrations tested, compared to manure alone.

[0290] It is also worth noting that the composition (%CH4, %COi) of the biogas formed does not vary in any way depending on the presence or absence of microbial products, nor on the nature of the microbial product used.

[0291] The values ​​of the methanogenic potential of the manure calculated in the preliminary assessment are presented in the following Table 3.

[0292] Table 3. Methanogenic potential of cattle manure in the absence or presence of different microbial products and fermented molasses at a high (E, according to Table 1), medium (M, according to Table 1) or low (F, according to Table 1) content.

[0293] Potential Potential Microbial Products Methanogen Gain (%) Methanogen (m 3 / T MB) (m W MO) none 20.25 129.34

[0294] E 31.91 47.58% 204.10 TAA2025 M 35.75 76.54% 228.28

[0295] F 49.81 145.98% 318.05

[0296]

[0297] E 36.48 80.15% 232.93

[0298] BG1 M 29.54 45.88% 188.65

[0299] F 27.08 33.73% 172.90

[0300] E 27.73 36.94% 177.05

[0301] BG6 M 31.19 54.02% 199.19

[0302] F 42.08 107.80% 268.70

[0303] E 16.25 -19.75% 103.74

[0304] PI M 33.50 65.43% 213.91

[0305] F 27.38 35.21% 174.84

[0306] P3 24.84 22.67% 158.63

[0307]

[0308] The results in Table 3 show that each of the microbial products tested (Trichoderma atroviride TAA2025, live yeast Saccharomyces cerevisiae BG1, and Bacillus / Peribacillus strain mixture) increases the methanogenic potential of cattle manure, and the increase in methanogenic potential is significantly higher than that observed for the competing product P3. The best results were obtained for the low content (0.000005% by weight of cattle manure) of Trichoderma atroviride TAA2025; the low content (0.00002% by weight of cattle manure) of Bacillus / Peribacillus; and the high content (0.012%) of BGL. These results were used in the subsequent study to select the contents of the microbial products and the respective contents of the components of the compositions including the Bacillus / Peribacillus strain mixture.

[0309] B. Measurement of the Effects of Different Microbial Products, Fermented Molasses, and Compositions Including Bacillus / Peribacillus Strain Mixture on the Methanegenic Potential of Bovine Manure

[0310] The products tested in this study are:

[0311] the mixture of bacterial strains of the genus Bacillus / Peribacillus (referred to as BG6 hereafter),

[0312] the fungal strain Trichoderma atroviride TAA2025 (designated TAA2025), the live yeast strain Saccharomyces cerevisiae BG1 (designated BG1), fermented molasses as defined in the preceding Example (designated PI), a competition product as defined in the preceding Example (designated P3), a mixture of bacterial strains of the genus Bacillus IP eribacillus and fermented molasses (designated BG6 + PI); a mixture of bacterial strains of the genus Bacillus IP eribacillus and the live yeast strain Saccharomyces cerevisiae BG1 (designated BG6 + BG1); and

[0313] a mixture of bacterial strains of the genus Bacillus Iperibacillus and the fungal strain Trichoderma atroviride TAA2025 (BG6 + TAA2025)

[0314] The methanogenic potential of cattle manure was determined through anaerobic digestion at 37.5°C, which continued for 35 days. Anaerobic digestions were conducted both in the absence and presence of each of the different microbial products. The concentrations of the various microbial products used in the anaerobic digestion reactions are presented in Table 4 below.

[0315] Table 4. Contents of the products and compositions tested in the anaerobic digestion of bovine manure. Each product and composition was tested at a high (E), medium (M), low (F), and possibly very low (TF) content. For the compositions, the table presents the respective contents of the components of the compositions including the Bacillus Iperibacillus strain mixture.

[0316] Microbial Products Concentration Content (expressed as a % by weight of manure)

[0317] E 0.000005%

[0318] TAA2025 M 0.000001%

[0319] F 0.000000001%

[0320] E 0.024%

[0321] BG1 M 0.012%

[0322] F 0.009%

[0323] E 0.0002000%

[0324] M 0.0000200%

[0325] BG6

[0326] F 0.0000020%

[0327] TF 0.0000002%

[0328] E 1.0%

[0329] PI M 0.5%

[0330] F 0.1%

[0331] P3 Recommended content 0.0010%

[0332] BG6 0.000020%

[0333] BG6 + TAA2025

[0334] TAA2025 0.000005%

[0335] BG6 0.00002%

[0336] BG6 + PI

[0337] PI 1%

[0338]

[0339] BG6 0.00002%

[0340] BG6 + BG1

[0341] BG1 0.01%

[0342]

[0343] The kinetics of the anaerobic digestion reaction were monitored for each sample, and key parameters were determined (see Figure 1). Kinetic data and biogas composition are presented in Table 5 below.

[0344] Table 5. Kinetic data of anaerobic digestion of bovine manure in the absence or in the presence of different microbial products or fermented molasses at a high (E, according to Table 4), medium (M, according to Table 4), low (F, according to Table 4), or very low (TF, according to Table 4) content or in the presence of different compositions including the mixture of Bacillus / Peribacillus strains and whose respective component contents are presented in Table 4.

[0345] V max V max Degradation Degradation Microbial products (m 3 of CH4 (m 3 of CH4 / at 80% to 90%

[0346] / day / T MO) day / T MB) (days) (days) none 3.38 + 0.18 0.96 + 0.05 22d. 30d.

[0347] E 5.77 + 2.06 1.51 + 0.36 19d-24d.

[0348] TAA2025 M 9.11 + 0.71 2.59 + 0.21 20d. 25 days.

[0349] F 10.54 + 0.94 2.99 + 0.27 20d. 25 days.

[0350] E 6.35 + 0.39 1.80 + 0.11 20d. 27 d.

[0351] BG1 M 5.25 + 0.85 1.49 + 0.24 22j. 29 j.

[0352] F 5.94 + 0.28 1.69 + 0.08 21 h- 26 h.

[0353] E 7.99 + 0.60 2.63 + 0.63 22j. 26 j.

[0354] M 6.43 + 2.71 1.38 + 0.03 21 h- 26 h.

[0355] BG6

[0356] F 6.87 + 1.09 1.70.15 + 23 h. 28 j.

[0357] TF 10.36 + 0.78 2.94 + 0.22 24h. 29 j.

[0358] E 5.72 + 1.16 1.62 + 0.32 19j- 27 y.

[0359] PI M 5.55 + 1.06 1.76 + 0.32 20j. 25 h.

[0360] F 6.18 + 1.57 1.76 + 0.45 20h. 25 h.

[0361] P3 12.18 + 1.49 3.46 + 0.42 23 h. 27 j.

[0362] BG6 + TAA2025 9.45 + 0.62 2.48 + 0.24 24h. 29 j.

[0363] BG6 + PI 6.36 + 0.03 1.73 + 0.14 23 h. 28 j.

[0364] BG6 + BG1 13.12 + 1.63 3.73 + 0.47 25 h. 29 j.

[0365]

[0366] The results in Table 5 show that each of the microbial products tested alone (Trichoderma atroviride TAA2025, live yeast Saccharomyces cerevisiae BG1, and Bacillus / Peribacillus strain mixture) increases the initial rate of substrate (cattle manure) degradation. In particular, an increase of 211.8% was observed for Trichoderma atroviride TAA2025 at a concentration of 0.000000001% by weight of cattle manure; 206.5% for the Bacillus / Peribacillus strain mixture at a concentration of 0.0000002% by weight of cattle manure; and 87.8% for the live yeast Saccharomyces cerevisiae BG1 at a concentration of 0.00200% by weight of cattle manure. The results in Table 5 also show that each of the microbial products tested alone reduces the duration of the degradation of bovine manure by anaerobic digestion.In particular, 90% substrate degradation is achieved in 5 to 6 days less with Trichoderma atroviride; in 1 to 4 days less with the live yeast Saccharomyces cerevisiae BG1; and in 1 to 4 days less with the Bacillus / Peribacillus strain mixture compared to manure alone. Regarding the compositions including the Bacillus / Peribacillus strain mixture, Table 5 shows that they all increase the initial rate of substrate degradation: an 88.2% increase for the BG6 + PI composition; a 179.6% increase for the BG6 + TAA2025 composition; and a 288.2% increase for the BG6 + BG1 composition, compared to manure alone. These compositions also decrease the manure degradation time by one or two days compared to manure alone.

[0367] It was also observed that the composition (%CO4, %COi) of the biogas produced varies only very slightly depending on the presence or absence of microbial products, and on the nature of the microbial product(s) used (data not shown). The methanogenic potential values ​​of the manure calculated in the preliminary assessment are presented in Table 6 below.

[0368] Table 6. Methanogenic potential of cattle manure in the absence or presence of various microbial products or fermented molasses at high (E, according to Table 4), medium (M, according to Table 4), low (F, according to Table 4), or very low (TF, according to Table 4) levels, or in the presence of different compositions including the Bacillus / Peribacillus strain mixture, the respective levels of which are presented in Table 4. Methanogenic potential (m²) 3 / T MB) (m W MO) none 28.10 99.00

[0369] E 46.37 65.02% 163.22 TAA2025 M 65.39 132.70% 230.16

[0370] F 65.06 131.53% 228.99 E 37.89 34.84% 133.36 BG1 M 26.46 -5.84% 93.14

[0371] F 32.50 15.66% 114.41 E 56.22 100.07% 197.89 M 40.20 43.06% 139.30 BG6

[0372] F 42.09 49.79% 148.15 TF 67.05 138.61% 235.99 E 39.75 41.46% 139.93 PI M 43.34 54.23% 152.56

[0373] F 39.47 40.46% 138.92 P3 65.83 134.27% 231.72

[0374] BG6 + TAA2025 49.20 75.09% 173.18

[0375] BG6 + PI 41.59 48.01% 146.40

[0376] BG6 + BG1 73.84 162.78% 259.91

[0377]

[0378] The results in Table 6 show that each of the microbial products tested (Trichoderma atroviride TAA2025, live yeast Saccharomyces cerevisiae BG1, and Bacillus / Peribacillus strain mixture) increases the methanogenic potential of cattle manure. The best results were obtained for the medium and low levels (0.000001% and 0.000000001% by weight of cattle manure) of Trichoderma atroviride TAA2025; the very low level (0.0000002% by weight of cattle manure) of BG6; and the high level (0.024%) of BGL. Regarding the compositions including the Bacillus / Peribacillus strain mixture, Table 6 shows that at the respective levels tested, they all increase the methanogenic potential of cattle manure: by 48.0% for the BG6 + PI composition; of 75.09% for the BG6 + TAA2025 composition; and of 162.78% for the BG6 + BGLC composition.Measurement of the Effects of Compositions including the Mixture of Bacillus / Peribacillus Strains on the Methane-Producing Potential of Bovine Manure.

[0379] The products tested in this study are:

[0380] a mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 and the live yeast Saccharomyces cerevisiae BG1 (referred to as BG6 + BG1 hereafter); a mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 and the fungal strain Trichoderma atroviride TAA2025 (referred to as TAA2025 + BG6); and a competition product as defined in the preceding example (referred to as P3).

[0381] The methanogenic potential of cattle manure was determined through anaerobic digestion at 37.5°C, which continued for 37 days. Anaerobic digestions were conducted both with and without each of the following compositions. The respective contents of the components in the different compositions used in the anaerobic digestion reactions are presented in Table 7.

[0382] Table 7. Contents of the compositions tested in the anaerobic digestion of bovine manure. Each composition was tested at medium (M), low (F), and very low (TF) content. The respective contents of the components in each composition containing the Bacillus / Peribacillus strain mixture are provided.

[0383] Microbial Products Concentration Content (expressed as a % by weight of manure)

[0384] P3 Recommended content 0.0010%

[0385] TAA2025 0.000005%

[0386] BG6 M

[0387] BG6 0,000200%

[0388] TAA2025 0,000005%

[0389] BG6 F

[0390] BG6 0,000020%

[0391] TAA2025 0,000005%

[0392] BG6 TF

[0393] BG6 0,000002%

[0394] TAA2025 + BG6

[0395] TAA2025 0,000005%

[0396] TAA2025 M

[0397] BG6 0,000020%

[0398] TAA2025 0,000001%

[0399] TAA2025 F

[0400] BG6 0,000020%

[0401] TAA2025 0,000000001% TAA2025 TF

[0402] BG6 0,000020%

[0403]

[0404] BG1 0,01%BG6

[0405] M 0,00020%

[0406] BG1 0,01%

[0407] F BG1 + BG6 BG6 0,00002%

[0408] BG1 0,01%

[0409] TF BG6 0,000002%

[0410]

[0411] The kinetics of the anaerobic digestion reaction were monitored for each sample, and the important parameters were determined (see Figure 1). The kinetic data and the biogas composition are presented in Table 8 below.

[0412] Table 8. Kinetic data for the anaerobic digestion of bovine manure in the absence or presence of different compositions with medium (M, according to Table 7), low (F, according to Table 7), or very low (TF, according to Table 7) content. The respective contents of the components in the compositions including the Bacillus / Peribacillus strain mixture are shown in Table 7.

[0413] V max V max Degradation. Degradation. Microbial products (m 3 of CHV (m 3 of CH4 / at 80% to 90% day / T MO) day / T MB) (days) (days) none 8.25 + 0.33 1.39 + 0.06 29 days 31 days

[0414] BG6 M 8.01 + 0.97 1.35 + 0.16 22j. 29 j. BG6 F 8.75 + 1.47 1.51 + 0.20 22j. 29 j. BG6 F 8.05 + 1.80 1.33 + 0.28 22j. 29 j. TAA2025 + BG6

[0415] TAA2025 M 8.67 + 0.72 1.47 + 0.12 22j. 29j. TAA2025 F 5.65 0.76 + 0.89 + 0.15 23j. 30j. TAA2025 F 6.01 + 0.78 1.02 + 0.13 23j. 29j. M 12.93 + 0.71 2.08 + 0.21 28j. 31j. BG6 + BG1 F 8.55 + 1.24 + 1.44 + 0.21 27j. 31j.

[0416] TF 4.00 + 1.01 1.03 + 0.17 27 j. 32 j. P3 5.16 + 0.81 0.87 + 0.13 25 j. 31 j.

[0417]

[0418] The results in Table 8 show that the BG6 + BG1 composition (particularly the M content) increases the initial rate of bovine manure degradation. A reduction of 1 or 2 days in the substrate degradation time is also observed for the TAA2025 + BG6 composition. It was also observed that the composition (%CEU, %COi) of the biogas formed varies only very slightly depending on the presence or absence of microbial product compositions, and on the nature of the microbial product(s) used (data not shown).

[0419] The values ​​of the methanogenic potential of the manure calculated in the preliminary assessment are presented in the following Table 9.

[0420] Table 9. Methane potential of cattle manure in the absence or presence of different compositions including the mixture of Bacillus / Peribacillus strains at medium (M, according to Table 7), low (F, according to Table 7), or very low (TF, according to Table 7) levels. The respective levels of the components contained in the compositions are presented in Table 7.

[0421] Potential Methanogen Potential Methanogen Gain (%) Methanogen (m 3 / T MB) (m W MO)

[0422] none 19.69 116.44

[0423] BG6 M 26.08 32.45% 153.63 BG6 + TAA2025 BG6 F 31.55 60.23% 179.88

[0424] BG6 TF 23.00 16.81% 136.00 TAA2025 M 24.86 26.26% 147.03 BG6 + TAA2025 TAA2025 F 18.36 -6.75% 106.97

[0425] TAA2025 TF 18.73 -4.88% 110.79 M 32.13 63.18% 190.01 BG6 + BG1 F 23.20 17.83% 137.20

[0426] TF 13.95 -29.15% 82.50

[0427] P3 18.08 -8.18% 106.90

[0428]

[0429] The results in Table 9 show that compositions including the Bacillus / Peribacillus strain mixture increase the methanogenic potential of cattle manure. The best results were obtained for the average content of the BG6 + BG1 composition (i.e., 0.01% of the live yeast strain Saccharomyces cerevisiae BG1 by weight of manure and 0.0002% of the Bacillus / Peribacillus strain mixture); and the low BG6 content of the BG6 + TAA2025 composition (i.e., 0.000005% of the Trichoderma atroviride TAA2025 strain by weight of manure and 0.000002% of the Bacillus / Peribacillus strain mixture by weight of manure). Example 2: Effects of the Bacillus / Peribacillus BG6 Strain Mixture and Its Compositions on the Kinetics and Production Yield of Biogas by Anaerobic Digestion of Different Methanogenic Substrates

[0430] Materials and Methods

[0431] Measurement of Biochemical Methane Potential (BMP) See Example 1.

[0432] Methane-producing substrates studied

[0433] Four different methanogenic substrates were studied:

[0434] Substrate I: substrate consisting of 50% by weight of cattle manure, 15% by weight of corn, and 35% by weight of triticale;

[0435] Substrate II: substrate consisting of 50% by weight of maize, 25% by weight of triticale, and 25% of pig slurry;

[0436] Substrate III: substrate consisting of 15% by weight of maize, 35% by weight of beet pulp, and 50% triticale; and

[0437] Substrate IV: substrate consisting of 15% by weight of maize, 35% by weight of cattle manure, and 50% of triticale.

[0438] Contents of the Microbial Products Studied

[0439] The microbial products tested in the evaluations of this Example are studied at the concentrations indicated below and expressed as a percentage of the weight of methanogenic substrate used:

[0440] the mixture of bacterial strains of the genus Bacillus / Peribacillus (designated BG6) at a content of 0.00002%;

[0441] the strain of the fungus Trichoderma atroviride TAA2025 (referred to as TAA2025 hereafter) at a content of 0.000005%;

[0442] the live yeast Saccharomyces cerevisiae BG1 (designated BG1) at a content of 0.012% when used alone and 0.01% when used in combination with another microbial product;

[0443] fermented molasses as defined in Example 1 supplemented with downgraded yeast extracts (approximately 10% crude) (designated VO2) at a content of 1%;

[0444] a competitor product defined above (designated P3) with a content of 0.001%; ​​a mixture designated “TAA2025 + BG6” containing the Trichoderma atroviride strain TAA2025 (content of 0.000005%) and the mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 (0.00002%);

[0445] a mixture designated “BG1 + BG6” containing the live yeast strain Saccharomyces cerevisiae BG1 (0.01%) and the mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 (0.00002%);

[0446] a mixture designated “TAA2025 + VO2 + BG1 + BG6” containing the Trichoderma atroviride TAA2025 strain (content of 0.000005%), fermented molasses (1%), the live yeast strain Saccharomyces cerevisiae BG1 (0.01%) and the mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 (0.00002%).

[0447] Results

[0448] A. Evaluation of the Effects of the Bacillus / Peribacillus BG6 Strain Mixture and Its Composition on the BMP of Substrate I

[0449] As a reminder, substrate I is a substrate made up of 50% by weight of cattle manure, 15% by weight of corn, and 35% by weight of triticale (i.e. a substrate made up of cattle manure and plant fibers).

[0450] The methanogenic potential of substrate I was determined by anaerobic digestion carried out at 37.5°C and continued for 36 days. The anaerobic digestions were conducted in the absence or presence of each of the different products to be tested at the concentrations indicated above.

[0451] Table 10. Kinetic data of anaerobic digestion of substrate I in the absence or in the presence of the different microbial products and compositions.

[0452] V max V max Degradation. Degradation. Microbial products (m 3 of CHV (m 3of CH4 / at 80% to 90% day / T MO) day / T MB) (days) (days) none 36.45 + 3.51 9.48 + 0.91 14d. 21 d- TAA2025 0.45 + 0.03 1.69 + 0.08 14d. 21 days - VO2 35.57 + 2.14 9.26 + 0.55 14 days - 21 days - BG1 32.52 + 3.16 9.18 + 0.78 14 days - 21 days - BG6 1.85 + 0.09 2.22 + 0.17 14 days - 21 days - BG1 + BG6 37.44 + 4.86 8.43 + 1.52 14 days - 21 days - TAA2025 + BG6 35.98 + 4.08 9.53 + 0.80 14 days - 21 days

[0453]

[0454] TAA2025 + VO2 +BG1 + BG6 33.74 + 3.87 8.89 + 1.00 14d. 21 days - P3 38.25 + 2.95 9.93 + 0.76 14 days. 21 d-

[0455]

[0456] The results show that the microbial products BG6 and TAA2025, used alone, significantly reduce the initial rate of substrate I degradation, while all other products and combinations studied, including the competing product P3, do not significantly alter this rate. No reduction in the time required to achieve 80% or 90% degradation of substrate I was observed for any of the products studied, including P3.

[0457] It was also observed that the composition (%CÜ4, %COi) of the biogas formed varies only very slightly depending on the presence or absence of microbial product compositions, and the nature of the microbial product(s) used (data not shown).

[0458] The values ​​of the methanogenic potential of substrate I calculated in the evaluation are presented in the following Table 11.

[0459] Table 11. Methanogenic potential of substrate I in the absence or presence of different microbial products and compositions.

[0460] Potential Methanogen Potential Methanogen Gain (%) Methanogen (m a / T MB) (m 3 / T MO) none 69.74 267.95 TAA2025 62.69 -10.11% 235.00 VO2 65.44 -6.16% 251.44 BG1 72.41 +3.83% 278.19 BG6 68.39 -1.94% 262.77 BG1 + BG6 74.26 +6.48% 269.47 TAA2025 + BG6 70.41 +2.39% 270.53 TAA2025 + VO2 + BG1 + BG6 67.48 -3.24% 246.80

[0461] P3 72.46 +3.90% 275.49

[0462]

[0463] The results in Table 11 show that the BG6 product, used alone or in combination with all the other products (TAA2025 + VO2 + BG1 + BG6 mixture), decreases the methanogenic potential of substrate I. A slight increase in the methanogenic potential of substrate I is observed for the TAA2025 + BG6 mixture. This slight increase is of the same order of magnitude as that observed for the competing product P3. A larger increase is observed for the BG1 + BG6 combination.

[0464] B. Evaluation of the Effects of the Bacillus / Peribacillus BG6 Strain Mixture and Its Composition on the BMP of Substrate II

[0465] As a reminder, substrate II is a substrate consisting of 50% by weight of maize, 25% by weight of triticale, and 25% of pig slurry (i.e. a substrate consisting of pig slurry and plant fibers).

[0466] The methanogenic potential of substrate II was determined by anaerobic digestion carried out at 37.5°C and continued for 36 days. The anaerobic digestions were conducted in the absence or presence of each of the different products to be tested at the concentrations indicated above.

[0467] Table 12. Kinetic data of anaerobic digestion of substrate II in the absence or in the presence of the different microbial products and compositions.

[0468] V max V max Degradation. Degradation. Microbial products (m 3 of CHV (m 3of CH4 / at 80% to 90% day / T MO) day / T MB) (days) (days) none 48.36 + 3.32 13.27 + 0.89 10d. 16d. TAA2025 1.21 + 0.10 1.56 + 0.02 10d. 16d. VO2 58.10 + 2.60 15.69 + 1.15 14d. 21d- BG6 1.46 + 0.66 2.81 + 0.10 16d. 22d. BG1 56.74 + 2.41 15.60 + 0.65 9d- 10d. BG1 + BG6 54.50 + 1.15 14.94 + 0.29 16d. 22d. TAA2025 + BG6 59.06 + 4.08 15.96 + 0.69 10d. 16d. TAA2025 + VO2 + BG1 + BG6 51.37 + 4.49 13.87 + 1.21 16d. 22d. P3 66.22 + 0.73 17.68 + 0.36 16d. 22d.

[0469]

[0470] The results show that each of the microbial products BG6 and TAA2025, when used alone, leads to a significant decrease in the initial rate of substrate II degradation, while all other products and combinations studied significantly increase this rate. With the exception of strain BG1, all other products studied require more time to induce 80% or 90% degradation of substrate II than when no product is used.

[0471] It has also been observed that the composition (%CÜ4, %COi) of the biogas formed varies only very slightly depending on the presence or absence of microbial product compositions, and the nature of the microbial product(s) used (data not shown).

[0472] The values ​​of the methanogenic potential of substrate II calculated in the evaluation are presented in the following Table 13.

[0473] Table 13. Methanogenic potential of substrate II in the absence or presence of different microbial products and compositions.

[0474] Potential Methanogen Potential Methanogen Gain (%) Methanogen (m a / T MB) (m a / T MO)

[0475] none 88.27 326.75 TAA2025 86.98 -1.46% 321.97

[0476] VO2 111.54 +26.36% 412.79

[0477] BG1 114 +29.15% 422.34

[0478] BG6 89.31 -1.18% 330.60

[0479] BG1 + BG6 107.68 +21.99% 398.60 TAA2025 + BG6 103.98 +17.80% 384.90 TAA2025 + VO2 + BG1 + BG6 93.07 +5.44% 348.56

[0480] P3 127.96 +44.96% 476.49

[0481]

[0482] The results in Table 11 show that the BG6 product used alone decreases the methanogenic potential of substrate II. A slight increase in the methanogenic potential of substrate II (a gain of approximately 5%) is observed when BG6 is combined with all other products. Combining BG6 with strain TAA2025 or with strain BG1 results in a greater increase in the methanogenic potential of substrate II (a gain of approximately 20%), but this increase is less than that observed in the presence of product P3 (a gain of approximately 45% in the methanogenic potential of substrate II). C. Evaluation of the Effects of the Bacillus / Peribacillus BG6 Strain Mixture and Its Composition on the BMP of Substrate III

[0483] As a reminder, substrate III is a substrate consisting of 15% by weight of maize, 35% by weight of beet pulp, and 50% of triticale (i.e. a substrate consisting solely of plant fibers).

[0484] The methanogenic potential of substrate III was determined by anaerobic digestion carried out at 37.5°C and continued for 36 days. The anaerobic digestions were conducted in the absence or presence of each of the different products to be tested at the concentrations indicated above.

[0485] Table 14. Kinetic data of anaerobic digestion of substrate III in the absence or in the presence of the different microbial products and compositions.

[0486] V max V max Degradation. Degradation. Microbial products (m 3 of CHV (m 3of CH4 / at 80% to 90% day / T MO) day / T MB) (days) (days) none 75.12 + 4.09 14.36 + 0.87 15 days 22 days. TAA2025 0.88 + 0.04 15.72 + 0.99 10 days 16 days. VO2 88.44 + 2.65 16.91 +0.50 10 days 15 days. BG6 2.18 + 0.07 20.00 + 1.28 15 days 22 days. BG1 86.18 + 5.68 15.93 + 1.60 9 days - 15 days. BG1 + BG6 72.71 + 3.51 14.26 + 0.70 16d. 22d. TAA2025 + BG6 73.94 + 5.13 14.05 + 0.89 16d. 22d. TAA2025 + VO2 + BG1 + BG6 87.60 + 6.00 16.75 + 1.14 10d. 15 days. P3 77.97 + 2.60 14.64 + 0.11 7d-10d.

[0487]

[0488] The results obtained show that the BG6 product and the TAA2025 strain, used alone, each lead to a significant decrease in the initial degradation rate of substrate III, expressed in m 3 of CH4 / day / T MO, while all other products and combinations do not affect or only very slightly increase this rate. The initial rate of substrate degradation, expressed in m 3The CH4 / day / T MB, on the other hand, remains practically unchanged in the presence of the different products and compositions tested. A degradation of 80% or 90% of substrate III is obtained more rapidly for TAA2025, VO2, BG1 and the combination of all products. It was also observed that the composition (%CEU, %COi) of the biogas formed varies only very slightly depending on the presence or absence of microbial product compositions, and on the nature of the microbial product(s) used (data not shown).

[0489] The values ​​of the methanogenic potential of substrate III calculated in the evaluation are presented in the following Table 15.

[0490] Table 15. Methanogenic potential of substrate III in the absence or presence of the different microbial products and compositions.

[0491] Potential Methanogen Potential Methanogen Gain (%) Methanogen (m a / T MB) (m a / T MO)

[0492] none 105.21 379.66 TAA2025 105.48 -0.26% 378.10

[0493] VO2 112.42 +6.85% 405.57

[0494] BG1 109.43 +4.01% 399.30

[0495] BG6 115.67 +9.94% 417.19

[0496] BG1 + BG6 103.61 -1.52% 373.87 TAA2025 + BG6 102.29 -2.78% 369.11 TAA2025 + VO2 +BG1 + BG6 112.22 +6.67% 404.97

[0497] P3 110.92 +5.43% 400.27

[0498]

[0499] The results in Table 15 show that the BG6 product, used alone or in combination with all the other products (TAA2025 + VO2 + BG1 + BG6 mixture), increases the methanogenic potential of substrate III, and this increase is greater than that observed with the competing product P3. A slight decrease in the methanogenic potential of substrate III is observed for the BG1 + BG6 combination and the TAA2025 + BG6 combination.

[0500] D. Evaluation of the Effects of the Bacillus / Peribacillus BG6 Strain Mixture and Its Composition on the BMP of Substrate IV

[0501] As a reminder, substrate IV is a substrate consisting of 15% by weight of maize, 35% by weight of cattle manure, and 50% of triticale (i.e. a substrate consisting of cattle manure and plant fibers, but with a lower proportion of cattle manure than substrate I).

[0502] The methanogenic potential of substrate IV was determined by anaerobic digestion carried out at 37.5°C and continued for 38 days. The anaerobic digestions were conducted in the absence or presence of each of the different products to be tested at the concentrations indicated above.

[0503] Table 16. Kinetic data of anaerobic digestion of substrate III in the absence or in the presence of the different microbial products and compositions.

[0504] V max V max Degradation. Degradation. Microbial products (m 3 of CHV (m 3 of CHV at 80% to 90% day / T MO) day / T MB) (days) (days) none 29.8 + 2.2 8.9 + 0.6 9d- 22d. TAA2025 28.9 + 2.0 8.4 + 0.3 12d- 19d. VO2 29.5 + 4.4 8.8 + 1.31 12d- 19d. BG6 29.3 + 1.1 10.1 + 1.0 13d. 20d. BG1 32.2 + 2.8 9.6 + 1.0 11d- 17d- BG1 + BG6 29.23 + 0.8 8.7 + 0.3 14d. 20d. TAA2025 + BG6 28.1 + 1.0 8.4 + 0.3 12d. 18 days TAA2025 + VO2 +BG1 + BG6 28.4 + 4.0 8.3 + 1.3 12d-20d. P3 22.3 + 6.9 14.64 + 0.11 12d-13d.

[0505]

[0506] The results obtained show that the initial degradation rate of substrate IV is not significantly altered by the presence of the different products and combinations studied. A 90% degradation of substrate IV is achieved during a shorter digestion time (1 to 5 days) in the presence of the different products and combinations than in their absence. By comparison, in the presence of the competing product P3, a shorter reaction time of 9 days is required to achieve 90% degradation of substrate IV.

[0507] It was also observed that the composition (%CÜ4, %COi) of the biogas formed varies only very slightly depending on the presence or absence of microbial product compositions, and on the nature of the microbial product(s) used (data not shown). The values ​​of the methanogenic potential of substrate IV calculated in the evaluation are presented in Table 17 below.

[0508] Table 17. Methanogenic potential of substrate IV in the absence or presence of different microbial products and compositions.

[0509] Potential Methanogen Potential Methanogen Gain (%) Methanogen (m 3 / T MB) (m 3 / T MO)

[0510] None 74.17 + 4.09 248.17 + 13.70 TAA2025 80.67 + 4.09 +8.76% 270.07 + 13.69 VO2 80.98 + 4.32 +9.18% 271.11 + 14.45 BG1 81.19 + 3.59 +9.46% 271.78 + 12.05 BG6 80.15 + 0.59 +8.06% 268.32 + 1.97 BG1 + BG6 72.45 + 2.15 -2.32% 242.55 + 7.19 TAA2025 + BG6 71.14 + 4.82 -4.09% 241.52 + 16.13 TAA2025 + VO2 + BG1 + BG6 75.22 + 6.38 -1.42% 251.82 + 21.35 P3 73.20 + 9.37 -1.31% 245.06 + 31.36

[0511]

[0512] The results in Table 17 show that the BG6 product used alone increases the methanogenic potential of substrate IV (gain of approximately 9%) more effectively than the competing product P3. A slight decrease in the methanogenic potential of substrate IV is observed for the BG1 + BG6 combination, and for the combination of BG6 with all other products (TAA2025 + VO2 + BG1 + BG6 mixture). This slight decrease (approximately 1.5%) is of the same order of magnitude as that observed for the competing product P3. A greater decrease in the methanogenic potential of substrate IV (approximately 4%) is obtained with the TAA2025 + BG6 mixture.

[0513] Conclusions

[0514] The results concerning the effects of the mixture of Bacillus! Peribacillus BG6 strains and its compositions on the methanogenic potential of the different substrates studied are summarized in the following Table 18.

[0515] Table 18. Effects of BG6 and its compositions on the methanogenic potential of substrates I, II, III, and IV, compared to the effects of the competing product, P3. Gain calculated relative to the methanogenic potential expressed in m 3 / T MB in the absence of Substrate product

[0516] I II III IV BG6 -1.94% -1.18% +9.94% +8.06% TAA2025 + BG6 +2.39% +17.80% -2.78% -4.09% BG1 + BG6 +6.48% +21.99% -1.52% -2.32% TAA2025 + VO2 + BG1 + BG6 -3.24% +5.44% +6.67% -1.42%

[0517]

[0518] P3 +3.90% +44.96% +5.43% -1.31% The results obtained show that product BG6 is the most effective (i.e., it causes an increase in the methanogenic potential of the substrate, and this increase is greater than that observed for the competing product P3) on substrate III (substrate composed solely of fibers) and on substrate IV (substrate composed of cattle manure and plant fibers in a 35:65 ratio). The mixtures TAA2025 + BG6 and BG1 + BG6 are each effective in increasing the potential of substrate II (substrate composed of pig slurry and plant fibers), but less so than P3. The gain provided by the mixture TAA2025 + BG6 on the methanogenic potential of substrate I (the substrate composed of cattle manure and plant fibers in a 50:50 ratio) is of the same order of magnitude as that observed for P3.However, a higher increase in the methanogenic potential of substrate I is observed in the presence of the BG1 + BG6 mixture. The effects of the mixture of all products (TAA2025 + VO2 + BG1 + BG6) on the methanogenic potential of substrate III are of the same order of magnitude as product P3.

[0519] Example 3: Effects of the two mixtures “TAA2025 + BG6” and “BG1 + BG6” on the continuous methanization of livestock effluents

[0520] The aim of the study presented in this Example was to evaluate the effect of two combinations according to the invention, the mixture “TAA2025 + BG6” and the mixture “BG1 + BG6”, on the continuous methanation of a slurry. BMP tests were previously carried out to determine the optimal concentration of the solutions to be added to the slurry.

[0521] Materials and Methods

[0522] Microbial Products, Slurry, and Digesters: The microbial product solutions (TAA2025, BG1, and BG6) were stored protected from moisture, and the TAA2025 solution was stored at 4°C. Regarding the inputs used to feed the 10 L digesters, hereafter referred to as chemostats, manure was collected, then freeze-dried and ground. Cattle slurry was also collected and sieved through a 5 mm screen to limit the presence of large particles. Continuous Digestion

[0523] The following microbial products were tested in the evaluations of this Example at the levels indicated below and expressed as a percentage of the dry weight of substrate used:

[0524] the mixture designated “TAA2025 + BG6” containing the Trichoderma atroviride strain TAA2025 (content of 0.000005%) and the mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 (0.00002%); and

[0525] the mixture designated “BG1 + BG6” containing the live yeast strain Saccharomyces cerevisiae BG1 (0.01%) and the mixture of bacterial strains of the genus Bacillus / Peribacillus BG6 (0.00002%).

[0526] Three chemostats were used: a control chemostat, providing a reference production of biogas / biomethane, and two experimental chemostats, one for each of the two mixtures studied. The digestion temperature was set at 40°C (mesophilic regime), and digestion was carried out with continuous agitation and an average residence time of 40 days. The chemostats were fed daily with the rations indicated in Table 19 below.

[0527] Table 19. Rations used to feed the different chemostats.

[0528] Initial rations

[0529] Chemostat 1 Chemostat 2 Chemostat 3

[0530] 8 g freeze-dried manure and 8 g freeze-dried manure and 8 g freeze-dried and ground manure + 242 g ground water + 242 g ground water + 242 g ground water + 242 g ground water

[0531] BG1 + BG6 mix, TAA2025 + BG6 mix. Rations starting February 20, 2025.

[0532] Chemostat 1 Chemostat 2 Chemostat 3

[0533] 8 g freeze-dried manure and 8 g freeze-dried manure and 8 g freeze-dried and ground manure + 50 g ground slurry + 50 g ground slurry + 50 g sifted cattle slurry + 192 g sifted cattle water + 192 g sifted cattle water + 192 g water + BG1 + BG6 mixture

[0534]

[0535] The rations were modified during the trial due to low biogas production in one of the cropping systems. Adding cattle slurry to the ration stimulated biogas production, as the mixture had difficulty degrading the freeze-dried manure on its own.

[0536] The quantity of microbial products was calculated from the analysis of the dry matter (DM) content of the manure sample, which was 25% DM. Eight grams of freeze-dried manure is therefore equivalent to 24 g of dry matter (DM) of fresh manure. Despite a change in the ration, with the addition of raw material, the quantity of microbial products remained the same throughout the trial, based on 24 g of DM.

[0537] The microbial product solutions were prepared daily to ensure the quality of the mixtures. The protocol used for the three solutions was as follows:

[0538] For the BG1 solution: preparation of a solution containing 10 mg / l of BG1; taking 356 ml of this solution and adding the sample to the ration;

[0539] For the BG6 solution: preparation of a stock solution containing 10 mg / l of BG6; dilution of 5 ml of stock solution in 1 liter; taking 142 ml of the diluted solution and adding the sample to the ration;

[0540] For the TAA2025 solution: preparation of a stock solution containing 10 mg / l of TAA2025; dilution of 100 ml of the stock solution in 1 liter; taking 1.78 ml of the diluted solution and adding the sample to the ration.

[0541] The quality of the biogas was monitored to determine whether the addition of the mixtures would result in a significant change in its composition. The gas analysis focused on methane and CO2 levels.

[0542] Microbiological Analyses

[0543] To ensure that the microbial products had no impact on the pathogens and that there was no health risk, samples of digestate were taken and analyzed. Samples were also taken from the three chemostats before the start of the trial to check for any microbiological changes after the addition of the microbial product combinations according to the invention. The species tested are as follows:

[0544] Escherichia coli (standard: NF ISO 16649-2)

[0545] Salmonella (standard: BKR 23 / 07 - 10 / 11) Clostridium perfringens (standard: NF EN ISO 15213-2)

[0546] Presumed Enterobacteriaceae at 37°C (standard: NF V 08-054)

[0547] Listeria monocytogenes (standard: AES 10 / 309 / 00).

[0548] Further analyses were carried out to obtain a more detailed composition of the digestate: (1) agronomic analyses, and (2) ISMO (Indicator of Organic Matter Stability) analyses. Analyses of the composition of digestate from chemostats (dried samples) and freeze-dried ground manure were also performed.

[0549] Results and Discussion

[0550] The freeze-dried manure introduced during the trial was characterized: a biogas production potential (BMP) test was performed, and its organic matter content was determined. The freeze-dried manure contained 81.68% organic matter and had a methanogenic capacity of 114.5 m³. 3 / TüM, or 26.6 m a / Tï \i.

[0551] A. Daily Production Method

[0552] The daily methane production of the three chemostats throughout the trial is shown in Figure 2(A). A stabilization period was observed to ensure identical production for each digester. In this configuration, all digesters received the same feed ration, namely the ration for chemostat 1 (control). The introduction of the combinations according to the invention began on January 27, 2025.

[0553] From the outset of the experiment, biomethane production from the TAA2025 + BG6 mixture ceased. The cause of this cessation, which contradicts the results obtained in batch mode, and its origin have not been clearly identified, but several hypotheses can be put forward: (a) difficulty in digesting a ration composed solely of freeze-dried manure, (b) accidental overdosing of TAA2025, (c) negative impact of the combination in continuous mode.

[0554] To address these hypotheses, it was decided to add 50 g of liquid slurry to the mixture starting February 20, 2025, to ensure a minimum organic input in case the combination's biogas production from freeze-dried slurry was limited. This resulted in a two-stage resumption of biomethane production. For the other two chemostats, production was increased thanks to the additional organic matter from the slurry. After these production variations, the yield of each chemostat stabilized. Figure 2(B) presents an overview of the last 30 days of the trial. The data shown no longer correspond to the daily biomethane production but to the methanogenic potential of the ration as expressed in the digester.

[0555] Over the study period, the methanogenic potential of the ration in chemostat 2 (BG1 + BG6 mixture) was 82.56 ± 10.19 m 3 / TDM. On average, this production was 50% higher than that of the control (54.35 ± 4.95 m 3 / ToM). The difference between the two productions is significant and confirms the beneficial effect of the BG1 + BG6 mixture on biomethane production in the digester.

[0556] The methane production of chemostat 3 was equivalent to that of the control (50.66 ± 9.53 m 3 (ToM). The addition of the TAA2025 + BG6 mixture is therefore neutral on biomethane production. This conclusion should be interpreted in light of the negative effect observed at the beginning of the experiment, which does not reflect the positive effects initially obtained during the batch BMP trials. It would be worthwhile to confirm these results in a second trial.

[0557] Table 20 below shows the composition of the biogas produced in the different chemostats. The addition of solution mixtures does not have a significant impact on the composition of the biogas produced.

[0558] Table 20. Composition of biogas produced in the different chemostats.

[0559] Before the Chemostat 1 test Chemostat 2 Chemostat 3 % CH45 3% 53% 52% %CO2 38% 39% 37% Before the Chemostat 1 test Chemostat 2 Chemostat 3 % CH45 4% 53% 53%

[0560]

[0561] %CO2 37% 38% 37%

[0562] B. Microbiological Quality of Digestates

[0563] Table 21 below shows the results of the microbiological analyses of the digestates from the 10-litre reactors at the beginning of the experiment and after stabilization. Table 21. Composition of the biogas produced in the different chemostats at time T0 (January 26, 2025) and at time Tfinal (March 30, 2025).

[0564] Pathogen Reference BG1 + BG6 TAA2025 + BG6 T0 Tfinal T0 Tfinal T0 Tfinal Enterococcus ( / g) >15000 >15000 >15000 >15000 >15000 >15000 E. coli ( / g) 90 540 190 130 <40 presence 190 of germs Salmonella ( / g) ND ND ND ND ND ND Clostridium <100 <100 1600< <10 <10 <10 perfringens ( / g)

[0565]

[0566] Listeria ( / g) ND ND ND ND ND ND ND = not detected.

[0567] Microbiological analysis of the digestates from the chemostats shows that, for all pathogens except enterococci, the addition of a combination according to the invention does not significantly affect the presence of these pathogens in the digestates. Therefore, the addition of a combination according to the invention does not promote the growth of these pathogens. In the specific case of enterococci, the measurement of this pathogen is imprecise, making it impossible to draw conclusions about its evolution. Further experiments are necessary to guarantee the safety of the mixtures with respect to this pathogen.

[0568] C. Constitutive Analysis of the Dry Matter of Manure and Digestate at Tfînal The results obtained are presented in the following Table 22.

[0569] Table 22. Results of the constitutive analysis of the dry matter of the manure and digestate at Tfinal for the three chemostats.

[0570] % g / kgDM g / kgDM g / kgDM Origin MS NDF ADF ADL Chopped manure 95.7 574 336 90 Chemostat 1 97.8 604 402 178 Chemostat 2 98.1 596 390 168 Chemostat 3

[0571]

[0572] 97.5 558 368 160

[0573] The results of these analyses show that the action of the two combinations influences the dry matter composition of the digestates, with the NDF / ADF / ADL values ​​(where NDF = Neutral Detergent Fiber, ADF = Acid Detergent Fiber, and ADL = Acid Lignin Fiber) being lower for the digestates of the tested combinations compared to the control digestate. The effect of the TAA2025 + BG6 mixture is more significant than that of the BG1 + BG6 mixture for ADL. This observation raises the question of the measured production for the TAA2025 + BG6 combination, which, in light of these results, should have been higher than that of the control.

[0574] D. Agronomic Analysis of Digestates

[0575] The digestates were analyzed between times TO and Tfinal. The main points to remember are presented in Table 23 below.

[0576] Table 23. Results of agronomic analyses of digestates in the absence and in the presence of the mixture “BG1 + BG6” and the mixture “TAA2025 + BG6”.

[0577] Reference BG1 + BG6 TAA2025 + BG6 T0 Tfinal T0 Tfinal T0 Tfinal % DM 1.46 2.68 1.71 0.917 0.789 1.96 % OM (% of the DM) 66.5 73.4 66.6 39.8 42.3 69.3 PH 8.0 7.6 7.9 7.8 8.1 7.7 Total Nitrogen (% of the DM) 4.9 2.4 4.3 7.5 5.3 4.0 Total Organic Carbon 43.9 40.6 49.6 46.7 44.4 39 (% of the DM)

[0578] C / N 9.0 17.26 10.95 6.20 8.35 7.70

[0579] 54 64

[0580]

[0581] ISMO (% OM DM) 59 59 55 58 The evolution of digestates between the beginning and the end of the experiment presents characteristics specific to each modality.

[0582] Regarding the dry matter (DM) and organic matter (OM) content of the digestates, it should be noted that the DM levels are very low compared to those of a digester from a biogas plant (approximately 10% DM versus 1 to 2% in this case). Measurement uncertainties are therefore greater. In particular, the initial digestate contents differ from one reactor to another. This may be due to the low DM content of the digestate and the homogenization of the sample just before collection. Nevertheless, an increase in DM and OM content is observed in the reference digester, reflecting the transition from a ration composed solely of manure to a mixture of manure and slurry. The same trend was observed for the TAA2025 + BG6 mixture, but the increase was smaller.In contrast, for the BG1 + BG6 mixture, the DM and MO contents decrease quite markedly, probably indicating increased degradation of the substrate introduced into the digester.

[0583] Regarding pH, the observed values ​​are perfectly consistent with field observations. A decrease in pH was observed in all chemostats between the beginning and end of the experiment, indicating a change in digestion. In particular, the increased organic load in the ration can induce this acidification, visible in the reference chemostat. The more moderate decrease in pH in the two chemostats containing a combination of microbial products may indicate improved digestion of volatile fatty acids, limiting their acidifying effect.

[0584] The C / N (organic carbon / nitrogen) ratios are slightly below normal (optimal values ​​between 20 and 30). The control chemostat shows an increase in this ratio, which could be induced by the introduction of slurry, while the other two chemostats show a decrease in this ratio.

[0585] The ISMO (Indicator of Organic Matter Stability) decreased in all chemostats, similarly between the control chemostat and the chemostat containing the BG1 + BG6 mixture, and more markedly in the chemostat containing the TAA2025 + BG6 mixture, which had a higher initial ISMO. The three digestates obtained at the end of the experiment had equivalent ISMO values, although the evaluation of this parameter varied depending on the individual chemostat.

[0586] Conclusion

[0587] The “BG1 + BG6” mixture showed a significant 50% increase in biomethane production compared to the control. This result confirms the positive impact of this mixture on methane production during batch trials.

[0588] The second mixture, “TAA2025 + BG6”, showed no difference in biomethane production compared to the control. However, this conclusion appears to contradict the results obtained during batch trials and the quality of the digestates produced. The experiment needs to be repeated to confirm these findings.

[0589] The presence of these two mixtures had no negative impact on pathogen proliferation in the digestate, with the exception of enterococci. For enterococci, quantitative measurements did not yield precise values, thus preventing any conclusions from being drawn regarding the evolution of their populations. Agronomic analyses performed on the digestates from the chemostats revealed certain trends concerning the parameters generally evaluated in digestates. An impact on digestate quality (%DM / %OM, C / N ratio (total organic carbon / nitrogen), ISMO (Indicator of Organic Matter Stability)) was observed, but it is important to note that the digestates are very low in dry matter, indicating that these results should be interpreted with caution. They provide initial trends that need to be confirmed on a larger scale.

Claims

Demands 1. A process for producing biogas from a methanogenic substrate, characterized in that it comprises an anaerobic digestion step of said methanogenic substrate in the presence of: a mixture of Bacillus / Peribacillus BG6 strains consisting of: the Bacillus licheniformis strain deposited with the NRRL on November 20, 2024, under accession number NRRL B-68474; the Bacillus licheniformis strain deposited with the NRRL on November 20, 2024, under accession number NRRL B-68477; the Bacillus megaterium or Priestia megaterium strain deposited with the NRRL on November 20, 2024, under accession number NRRL B-68473; the Bacillus pumilus strain deposited at the NRRL on November 20, 2024, under accession number NRRL B-68475; the strain of Peribacillus butanolivorans or Bacillus butanolivorans deposited with the NRRL on November 20, 2024, under accession number NRRL B-68478; and the Bacillus velezensis strain deposited at the NRRL on November 20, 2024, under accession number NRRL B-68476; or in the presence of a combination or composition: of the mixture of Bacillus / Peribacillus BG6 strains; and of the Trichoderma atroviride TAA2025 strain deposited at the CNCM on February 5, 2025, under accession number CNCM 1-6171; or in the presence of a combination or composition: of the mixture of Bacillus / Peribacillus BG6 strains; and of the live yeast strain of Saccharomyces cerevisiae BG1 deposited at the CNCM, on January 21, 2025, under accession number CNCM I- 6169; or in the presence of a combination or composition: of the mixture of Bacillus / Peribacillus BG6 strains; and fermented molasses.

2. A process according to claim 1, characterized in that the anaerobic digestion of the methanogenic substrate is carried out in the presence of the Bacillus / Peribacillus BG6 mixture at a content between 0.0000002% and 0.002% or between 0.0000002% and 0.00002% of the weight of the methanogenic substrate, in particular a content of about 0.00002% of the weight of the methanogenic substrate.

3. A process according to claim 1, characterized in that the anaerobic digestion of the methanogenic substrate is carried out in the presence of the mixture of Bacillus / Peribacillus BG6 strains and the live yeast strain Saccharomyces cerevisiae BG1, wherein the content of the Saccharomyces cerevisiae BG1 strain is between 0.001% and 0.1%, in particular is about 0.010% of the weight of the methanogenic substrate, and the content of the mixture of Bacillus / Peribacillus BG6 strains is between 0.000002% and 0.0010% or between 0.00002% and 0.0005% of the weight of the methanogenic substrate, in particular is about 0.00002% of the weight of the methanogenic substrate.

4. A process according to claim 1, characterized in that the anaerobic digestion of the methanogenic substrate is carried out in the presence of the mixture of Bacillus / Peribacillus BG6 strains and the Trichoderma atroviride TAA2025 strain, wherein the content of the Bacillus / Peribacillus BG6 strain mixture is between 0.000002% and 0.0002% or between 0.00002% and 0.0002% by weight of the methanogenic substrate, in particular 0.00002% by weight of the methanogenic substrate, and the content of the Trichoderma atroviride TAA2025 strain is between 0.000000001% and 0.000001% or between 0.000000001% and 0.000005% by weight of the methanogenic substrate, in particular approximately 0.000005% of the weight of the methanogenic substrate.

5. A process according to claim 1, characterized in that the anaerobic digestion of the methanogenic substrate is carried out in the presence of the mixture of Bacillus / Peribacillus BG6 strains and fermented molasses, where the fermented molasses is fermented sugar beet molasses, fermented sugar cane molasses, or a mixture thereof.

6. A process according to claim 5, characterized in that the content of fermented molasses is between 0.01% and 5% or between 0.05% and 2% of the weight of methanogenic substrate, in particular about 1% of the weight of methanogenic substrate, and the content of mixture of Bacillus / Peribacillus BG6 strains is between 0.000002% and 0.0002% of the weight of methanogenic substrate, in particular about 0.00002% of the weight of methanogenic substrate.

7. A method according to any one of claims 1 to 6, characterized in that the anaerobic digestion of the methanogenic substrate is further carried out in the presence of an inoculum.

8. Composition or combination for improving biogas production by anaerobic digestion of a methanogenic substrate, characterized in that it comprises, or consists of: the mixture of Bacillus / Peribacillus BG6 strains; and / or in that it comprises, or consists of: the mixture of Bacillus / Peribacillus BG6 strains; and the Trichoderma atroviride strain TAA2025; or in what it includes, or consists of: the mixture of Bacillus / Peribacillus BG6 strains; and the live yeast strain Saccharomyces cerevisiae BG1; or in what it includes, or consists of: the mixture of Bacillus / Peribacillus BG6 strains; and fermented molasses.

9. Composition or combination according to claim 8, characterized in that it comprises, or consists of, the Trichoderma atroviride TAA2025 strain and the Bacillus / Peribacillus BG6 strain mixture in relative weight amounts of between 1:0.2 and 1:40000, in particular between 1:0.4 and 1:20000 or between 1:0.4 and 1:40 or between 1:0.4 and 1:20, more particularly are about 1:

4.

10. Composition or combination according to claim 8, characterized in that it comprises, or consists of, the mixture of Bacillus / Peribacillus BG6 strains and the live yeast strain Saccharomyces cerevisiae BG1, wherein the relative amounts by weight of the mixture of Bacillus / Peribacillus BG6 strains and the BG1 strain are between 1:10 and 1:10000 or between 1:100 and 1:5000, in particular between 1:100 and 1:1000, more particularly are about 1:500.

11. Composition or combination according to claim 8, characterized in that it comprises, or consists of, the mixture of Bacillus / Peribacillus BG6 strains and fermented molasses, wherein the fermented molasses is fermented sugar beet molasses, fermented sugar cane molasses, or a mixture thereof.

12. Composition or combination according to claim 11, characterized in that it comprises, or consists of, the mixture of Bacillus / Peribacillus BG6 strains and fermented molasses in relative weight amounts between 1:500 and 1:500000 or between 1:5000 and 1:50000, in particular are about 1:50000.

13. A method for preparing a composition defined in any one of claims 8 to 12, comprising a mixing step: of the mixture of Bacillus / Peribacillus BG6 strains and the Trichoderma atroviride TAA2025 strain; Or : of the mixture of Bacillus / Peribacillus BG6 strains and the live yeast strain Saccharomyces cerevisiae BG1; Or : of the mixture of Bacillus / Peribacillus BG6 strains and fermented molasses.

14. Use of a composition or combination defined in any one of claims 8 to 13 in a process for the production of biogas by anaerobic digestion of a methanogenic substrate.

15. Use according to claim 14 to increase biogas production yield.

16. Use of claim 14 or claim 15 to increase the rate of biogas production.

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