Feed additive for the reduction of rumen methane emissions

Abstract

The current invention relates to a feed additive wherein said feed additive comprises an effective amount of one or more methanotrophic bacteria encapsulated into a gel or gel-like matrix.

Description

[0001] FEED ADDITIVE FOR THE REDUCTION OF RUMEN METHANE EMISSIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a feed additive for the reduction of methane emissions in ruminants.

[0004] BACKGROUND

[0005] Methane is the second most prevalent greenhouse gas emitted. Methane's lifetime in the atmosphere is much shorter than carbon dioxide, but methane is more efficient at trapping radiation than carbon dioxide. Pound for pound, the comparative impact of methane on climate change is more than 25 times greater than carbon dioxide over 100 years. As a particularly potent greenhouse gas, methane emissions are responsible for about twenty percent of planetary warming and thus represent a significant environmental concern. Accordingly, there have been numerous efforts in the past to remediate, control and / or otherwise treat methane emissions.

[0006] Domestic livestock, such as cattle, buffalo, sheep, goats and camels produce large amounts of methane as part of their normal digestive process. Ruminant animal methane emissions, or, more specifically, enteric fermentation methane emissions, originate in the four-stomach digestive tract common to all ruminant animals, which includes the rumen, a large forestomach connected to the four-stomach digestive tract. The rumen contains a host of digestive enzymes, fungi, bacteria and protozoa and the bulk of digestion, as well as methane production via enteric fermentation, takes place here. The organisms responsible for methane production are the methanogenic archaea species inhabiting the rumen.

[0007] Thus, there exists a need for a method for reducing the methane emission from livestock. Indirectly, the reduction of methane emission results in an increase in milk production in cattle due to more efficient energy expenditure.

[0008] Methane-utilizing, or methanotrophic, microorganisms are well-known in the microbiology art for their capacity to grow and reproduce using methane as a source of carbon and / or energy, particularly in a wide range of diverse methane availability conditions. Accordingly, methanotrophic microorganisms have been proposed in the past as a potential tool for the remediation of methane emissions. EP1924683 describes the process of treatment of methane emissions using methanotrophic bacteria. Methylococcus capsulatus was used in US11304427 and US2019082717, US2019271019 in feed compositions.

[0009] Methanotrophic bacteria are however aerobic organisms and thus their survival and methane metabolization capacity is limited in the anaerobic rumen environment. For this reason, they are not widely used.

[0010] KR20180053085 and Patel Sanjay K.S.et al. 2018 disclose encapsulation of Methanotrophic bacteria for the production of methanol and US2020368292 discloses the encapsulation of Methanotrophic bacteria in an enteric coating. None of these however provide a suitable solution for assuring the survival of Methanotrophic bacteria in the rumen of animals.

[0011] It remains thus a need for providing a feed or feed additive that reduces methane production in ruminant animals. The invention thereto aims to provide a solution that allows the administration of methanotrophic bacteria to livestock, in a fashion that ensures the survival and flourishing of said bacteria in the anaerobic environment of the rumen and thus causes a significant reduction of methane emissions and indirectly an increase in milk and meat production.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention and embodiments thereof serve to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present invention relates to a feed additive comprising an effective amount of one or more methanotrophic bacteria encapsulated into a gel or gel-like matrix, according to claim 1. Preferred embodiments of the feed additive are shown in any of claims 2 to 11.

[0014] In a second aspect, the present invention relates to a method of reducing methane emissions in a ruminant animal according to claim 12. More, in particular, the method as described herein provides the administration of a feed additive to said animals. Preferred embodiments of the method are shown in any of claims 13 to 16.

[0015] In a third aspect, the present invention relates to the use of the feed additive, according to claim 17. In a third aspect, the present invention relates to a method for the production of a feed additive for lowering methane emissions in ruminants, according to claim 18. The method comprises encapsulating methanotrophic bacteria in a gel or gel-like matrix.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention concerns a feed additive suitable for the reduction of methane emissions in ruminant animals. The feed additive comprises methanotrophic bacteria encapsulated in a gel or gel-like matrix.

[0018] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0019] As used herein, the following terms have the following meanings:

[0020] "A", "an", and "the" as used herein refer to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0021] "About" as used herein refers to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0022] "Comprise", "comprising", "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specify the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. Furthermore, the terms first, second, third and the like in the description and the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0023] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0024] The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0025] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0026] The term "methanotrophs" or "methanotrophic bacteria" as used in the present disclosure, relates to those microorganisms that use methane as their sole source of carbon and energy.

[0027] The term "methanogens" or "methanogenic archaea" as used herein refers to those microorganisms that produce methane as a metabolic byproduct in hypoxic conditions.

[0028] "Encapsulation" or "encapsulated" as used herein relates to bacteria that are enclosed or embedded into a protective or containment matrix. The bacteria may be completely or partially covered by said containment matrix.

[0029] "Gel" or "gel-like matrix" as used herein refers to a three-dimensional network or structure formed by a material. They may exhibit the properties of a solid, a liquid or both a solid and liquid.

[0030] "Gels" typically have a solid-like consistency and form a gel structure when dispersed in water or other appropriate solvents. They exhibit characteristics such as a semi-solid texture, the ability to hold their shape, and the formation of a distinct gel network.

[0031] The substances categorized as "gel-like" do not exhibit the same level of strength as gels but possess some gel-like properties. The gel-like matrix has thus a semisolid consistency, possesses some degree of elasticity or stiffness, and has the ability to immobilize or enclose the bacteria. It is characterized by a continuous phase containing a substantial amount of liquid or solvent trapped within the solid network. A "gel-like" is a material that exhibits similar properties to a gel, even if it does not strictly meet the criteria of a gel.

[0032] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0033] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0034] In a first aspect, the invention relates to a feed additive wherein said feed additive comprises an effective amount of one or more methanotrophic bacteria encapsulated into a gel or gel-like matrix. The inventors have unexpectedly observed that administration of a feed additive comprising methanotrophic bacteria embedded into a gel or gel-like matrix, to a ruminant animal, significantly decreases the methane emissions of said animal. The gel or gel-like matrix provides physical support and stability to the methanotrophic bacteria, while allowing for the transport of gases, nutrients and other substances. Overall, the gel or gel-like matrix provides a protective environment for the encapsulated methanotrophic bacteria, ensuring its stability, viability, and controlled interactions within the matrix.

[0035] Moreover, the encapsulation of the bacteria into the gel or gel-like matrix increases the resistance of aerobic methanotrophs to the anaerobic environment of the rumen, presumably due to the gasses and compounds dissolved into said gel or gel-like matrix. As a consequence, the methanotrophs metabolize the enteric methane produced in the rumen, releasing CO2 and significantly reducing the absolute methane emissions. Feedstuff fermentation by the microbial communities in the rumen results in H2release, which H2is usually used by methanogenic archaea. It follows that said methanogenic archaea are essential in maintaining a low redox potential in the environment of the rumen. As a consequence, regular supplements used in the art for reducing methane emissions by actively inhibiting methanogenic archaea, present the major drawback that are causing an increase in H2pressure in the rumen. They inhibit thus reoxidation of reduced enzymatic co-factors (NADH, NADPH and FADH) and cause the rate of rumen fermentation to cease. Unexpectedly, the feed additive as disclosed herein does not present these drawbacks, as it does not inhibit methanogenic archaea but rather provides an alternative conversion of the CH4produced by said methanogenic archaea. The methanotrophic bacteria provided by the feed additive of the invention disclosed herein, metabolize the CH4produced by methanogenic archaea.

[0036] Moreover, the inventors observed an increase in the amount of particular volatile fatty acids (VFAs) produced during rumen fermentation upon administration of a feed additive comprising methanotrophic bacteria encapsulated in a gel or gel-like matrix like a significant increase in the production of propionate. VFAs are the main energy source for ruminants, providing approximately 70% of the total energy requirements. They are used primarily by the microorganisms to support their activities and growth, with the excess production being used by the ruminant itself. The three main volatile fatty acids produced in ruminants are acetate, butyrate and propionate. Propionate provides energy via the conversion of blood glucose in the liver. It is used in lactose (milk sugar) synthesis. By administrating the feed additive of the invention to ruminant animals, an increase in lactation is obtained due to the effect of said feed additive on VFAs production and composition.

[0037] A gel or gel-like matrix typically comprises a gelling agent such as a polymer or a hydrocolloid that can form a cross-linked or entangled network. The polymers within the matrix can be natural, synthetic, or a combination of both. In an embodiment of the feed additive as disclosed herein the gel or gel-like matrix comprises a gelling agent selected from alginate, agar, agarose, carrageenan, gelatin, silica gel or cellulose derivatives. Alginate, agarose, gelatine and cellulose derivatives are polymers while agar and carrageenan are hydrocolloids. Silica gel belongs to neither polymers nor hydrocolloids. It will be obvious that any suitable gelling agent known in the art may be used with the feed additive as disclosed herein.

[0038] In some embodiments the gelling agent is cellulose and / or a cellulose-based compound.

[0039] In some embodiments of the feed additive, the gel or gel-like matrix comprises two or more gelling agents. In other embodiments said feed additive comprises a mixture of two, three, four, five, six, seven, eight, nine, or ten gelling agents.

[0040] In a further embodiment of the feed additive, as disclosed herein, the gelling agent is alginate. The alginate may be selected from sodium alginate, potassium alginate, calcium alginate, propylene glycol alginate (PGA) or ammonium alginate. Alginate is a naturally occurring polysaccharide extracted from brown seaweeds, and its chemical structure consists of blocks of guluronic acid (G-blocks) and blocks of mannuronic acid (M-blocks).

[0041] Sodium alginate is soluble in water and forms a viscous gel when it comes into contact with calcium ions.

[0042] Calcium alginate can be produced from a sodium alginate solution by the addition of a calcium salt such as calcium chloride. This forms insoluble calcium alginate salt which precipitates out of solution. The resulting calcium alginate gel is more rigid than sodium alginate gel. The calcium alginate may then be redissolved in various sodium carbonate solutions to produce alginate products containing specific ratios of sodium to calcium. This influences the alginate's physical and chemical properties. Similar to sodium alginate, potassium alginate is obtained by replacing sodium ions with potassium ions. It has properties similar to sodium alginate.

[0043] PGA is a modified form of alginate that has improved solubility in water and enhanced stability.

[0044] Ammonium alginate is prepared by replacing sodium ions in sodium alginate with ammonium ions and is soluble in water.

[0045] In an embodiment of the feed additive as disclosed herein, said feed additive is obtained by forming the gel or gel-like matrix and the methanotrophic bacteria into particles, preferably spherical particles, like beads.

[0046] Methods for particle formation include, but are not limited to extrusion, droplet formation, spray drying, coacervation, electrostatic encapsulation or co-extrusion.

[0047] Extrusion is the process of shaping or forming a material by forcing it through a specially designed opening such as an aperture or die. The gel or gel-like matrix and bacteria are mixed and formed into particles by force-passing the mixture to the opening of an aperture or die. As the material passes through the aperture or die, it takes on the shape and dimensions of the die opening. The aperture or die may have various configurations, such as round, square, or custom shapes, depending on the desired outcome.

[0048] Droplet formation is the process of forming liquid material into small, spherical droplets. This phenomenon occurs when a liquid is subjected to certain conditions or forces that cause it to overcome surface tension and take on a rounded shape. Droplets are formed when mechanical force or pressure is applied with a syringe, pipet, middle or nozzle to the mixture comprising the gel or gel-like matrix and bacteria.

[0049] Spray drying involves atomizing the gel or gel-like matrix and bacteria into small droplets. These droplets are then exposed to hot air, which evaporates the solvent and leaves behind solid particles or microcapsules.

[0050] Coacervation is a phase separation technique that involves the formation of a liquidrich droplet phase within a polymer solution. In the case of encapsulating bacteria, coacervation can be employed by mixing the bacteria with a suitable polymer solution, inducing phase separation, and forming a liquid droplet phase around the bacteria. This liquid phase can then be solidified to create the encapsulating matrix.

[0051] Electrostatic encapsulation involves applying an electric field to generate charged droplets or particles. By manipulating the charge of the bacteria biomass to be encapsulated and the surrounding gel or gel-like matrix, the bacteria is encapsulated within the generated charged droplets.

[0052] Emulsion techniques, such as emulsion polymerization or multiple emulsion methods (e.g., water-in-oil-in-water), can be employed to encapsulate bacteria. By creating an emulsion system with appropriate polymerization or cross-linking mechanisms, the bacteria can be dispersed within droplets and subsequently stabilized and solidified to form the encapsulation gel or gel-like matrix.

[0053] Co-extrusion involves combining multiple substances or components through separate channels of an extrusion system. In the context of encapsulating bacteria, co-extrusion can be used to create multi-layered structures, where different layers or materials are extruded simultaneously to form a complex encapsulation matrix around the bacteria.

[0054] In an embodiment of the feed additive, as disclosed herein, the particles have an average particle size between 1 and 4 mm. In some embodiments, the particles have a particle size between 1 and 3.5 mm, 1 and 3 mm, 1 and 2.5 mm, 1 and 2 mm, or 1 and 1.5 mm. In alternative embodiments, the particles have a particle size between 1.5 and 4 mm, between 2 and 4 mm, between 2.5 and 4 mm, between 3 and 4 mm, or between 3.5 and 4 mm.

[0055] The particle size of said particles may be determined by any method known in the art. Non-limiting examples of particle measuring techniques include microscopy using millimeter scale, laser diffraction, dynamic light scattering, or sedimentation techniques.

[0056] In some embodiments of the feed additive, as disclosed herein, the particles have a regular shape and are preferably spheric. In such an embodiment the particles have a diameter between 0.1 and 8 mm, preferably between 1 and 4 mm. In some embodiments, the particles have a diameter between 0.1 and 7.5 mm, 0.1 and 7 mm, 0.1 and 6.5 mm, 0.1 and 6 mm, 0.1 and 5.5 mm, 0.1 and 5 mm, 0.1 and 4.5 mm, 0.1 and 4 mm, 0.1 and 3.5 mm, 0.1 and 3 mm, 0.1 and 2.5 mm, 0.1 and 2 mm, 0.1 and 1.5 mm, 0.1 and 1 mm, or between 0.1 and 0.5 mm. In alternative embodiments, the particles have a diameter between 0.5 and 8 mm, 1 and 8 mm, 1.5 and 8 mm, 2 and 8 mm, 2.5 and 8 mm, 3 and 8 mm, 3.5 and 8 mm, 4 and 8 mm, 4.5 and 8 mm, 5 and 8 mm, 5.5 and 8 mm, 6 and 8 mm, 6.5 and 8 mm, 7 and 8 mm or 7.5 and 8 mm.

[0057] In other embodiments, the particles have an irregular shape. The particles may be formed irregularly or they may be formed by breaking a layer of gel or gel-like matrix in which bacteria biomass is encapsulated.

[0058] In an embodiment of the invention, the methanotrophic bacteria of said feed additive are selected from the genus of Methanomonas, Methylococcus, Methylomonas, Methylobacter, Methylosinus, Methylocystis, Methylothermus, Methanoperedens, Methylacidimicrobium, Methylobvolum, Methylacidiphilum,

[0059] Methylobvolum, Methylocaldum, Methylocaspa, Methylocella, Methyloferula, Methylohalobius, Methylomagnum, Methylomarinovum, Methylomarinum,

[0060] Methylomicrobium, Methylomirabilis Methyloparacoccus, Methyloprofundus,

[0061] Methylosarcina Methylosoma and mixtures thereof. Preferred genera include Methylomonas, Methylobacter, Methylococcus, Methylosinus and mixtures thereof.

[0062] In a preferred embodiment, said methanotrophic bacteria are of the genus Methyloccocus, preferably the species Methylococcus capsulatus. Alternatively said methanotrophic bacteria are Methylosinus trichosporium, Methanomonas methanica, Methanomonas methanooxidans, Methylomicrobium alcaliphilum, Methanoperedens manganicus, Methanoperedens manganireducens, Methanoperedens nitroreducens, Methylacidimicrobium sp., Methylobacter sp., Methylacidiphilum sp., Methylobvolum sp., Methylocaldum sp., Methylocaspa sp., Methylocella sp., Methylocystis sp., Methyloferula sp., Methylothermus sp., Methylogea sp., Methyloglobulus sp., Methylohalobius sp., Methylomagnum sp., Methylomarinovum sp., Methylomarinum sp., Methylomicrobium alcaliphilum, Methylomirabilis oxyfera, Methylomirabilis sp., Methylomonas agile, Methylomonas albus, Methylomonas methanica, Methylomonas methanolica, Methylomonas rubrum, Methyloparacoccus sp., Methyloprofundus sp., Methylosarcina sp., Methylosoma sp.

[0063] In an embodiment, the feed additive as disclosed herein comprises one bacteria species. In another embodiment said feed additive comprises a mixture of two, three, four, five, six, seven, eight, nine, or ten bacteria species. In yet another embodiment, the feed additive comprises bacteria from one, two, three, four, five, six, seven, eight, nine, or ten genera.

[0064] The methanotrophic bacteria may be added as an inoculum comprising essentially methanotrophic bacteria and / or an isolated methanotrophic bacteria or spore.

[0065] In an embodiment of the feed additive, as disclosed herein, the particles comprise between 90% (w / w) and 99.9% (w / w) gelling agent. In preferred embodiments, the particles comprise between 90% (w / w) and 99.8% (w / w), between 90% (w / w) and 99.7% (w / w), between 90% (w / w) and 99.6% (w / w), between 90% (w / w) and

[0066] 99.5% (w / w), between 90% (w / w) and 99.4% (w / w), between 90% (w / w) and

[0067] 99.3% (w / w), between 90% (w / w) and 99.2% (w / w), between 90% (w / w) and

[0068] 99.1% (w / w), between 90% (w / w) and 99% (w / w), between 90% (w / w) and 98.5%

[0069] (w / w), between 90% (w / w) and 98% (w / w), between 90% (w / w) and 97.5% (w / w), between 90% (w / w) and 97% (w / w), between 90% (w / w) and 96.5% (w / w), between 90% (w / w) and 96% (w / w), between 90% (w / w) and 95.5% (w / w), between 90% (w / w) and 95% (w / w), between 90% (w / w) and 94.5% (w / w), between 90% (w / w) and 94% (w / w), between 90% (w / w) and 93.5% (w / w), between 90% (w / w) and 93% (w / w), between 90% (w / w) and 92.5% (w / w), between 90% (w / w) and 92% (w / w), between 90% (w / w) and 91.5% (w / w), between 90% (w / w) and 91% (w / w), or between 90% (w / w) and 90.5% (w / w) gelling agent.

[0070] Alternatively said particles comprise between 90.5% (w / w) and 99.9% (w / w), between 91% (w / w) and 99.9% (w / w), between 91.5% (w / w) and 99.9% (w / w), between 92% (w / w) and 99.9% (w / w), between 92.5% (w / w) and 99.9% (w / w), between 93% (w / w) and 99.9% (w / w), between 93.5% (w / w) and 99.9% (w / w), between 94% (w / w) and 99.9% (w / w), between 94.5% (w / w) and 99.9% (w / w), between 95% (w / w) and 99.9% (w / w), between 95.5% (w / w) and 99.9% (w / w), between 96% (w / w) and 99.9% (w / w), between 96.5% (w / w) and 99.9% (w / w), between 97% (w / w) and 99.9% (w / w), between 97.5% (w / w) and 99.9% (w / w), between 98% (w / w) and 99.9% (w / w), between 98.5% (w / w) and 99.9% (w / w), between 99% (w / w) and 99.9% (w / w), between 99.1% (w / w) and 99.9% (w / w), between 99.2% (w / w) and 99.9% (w / w), between 99.3% (w / w) and 99.9% (w / w), between 99.4% (w / w) and 99.9% (w / w), between 99.5% (w / w) and 99.9% (w / w), between 99.6% (w / w) and 99.9% (w / w), between 99.7% (w / w) and 99.9% (w / w) or between 99.8% (w / w) and 99.9% (w / w) gelling agent. In yet another alternative embodiment, the particles comprise between 50% (w / w) and 99.9% (w / w), between 60% (w / w) and 99.9% (w / w), between 70% (w / w) and 99.9% (w / w), or between 80% (w / w) and 99.9% (w / w) gelling agent.

[0071] In yet another alternative embodiment, the particles comprise between 50% (w / w) and 90% (w / w), between 50% (w / w) and 80% (w / w), between 50% (w / w) and 70% (w / w) or between 50% (w / w) and 60% (w / w) gelling agent.

[0072] In a preferred embodiment, the particles comprise between 0.1% (w / w) and 10% (w / w) methanotrophic bacteria biomass.

[0073] In other embodiments, said particles comprise between 0.1% (w / w) and 9.5%

[0074] (w / w), between 0.1% (w / w) and 9% (w / w), between 0.1% (w / w) and 8.5% (w / w), between 0.1% (w / w) and 8% (w / w), between 0.1% (w / w) and 7.5% (w / w), between 0.1% (w / w) and 7% (w / w), between 0.1% (w / w) and 6.5% (w / w), between 0.1% (w / w) and 6% (w / w), between 0.1% (w / w) and 5.5% (w / w), between 0.1% (w / w) and 5% (w / w), between 0.1% (w / w) and 4.5% (w / w), between 0.1% (w / w) and 4% (w / w), between 0.1% (w / w) and 3.5% (w / w), between 0.1% (w / w) and 3% (w / w), between 0.1% (w / w) and 2.5% (w / w), between 0.1% (w / w) and 2% (w / w), between 0.1% (w / w) and 1.5% (w / w), between 0.1% (w / w) and 1% (w / w), or between 0.1% (w / w) and 0.5% (w / w) methanotrophic bacteria biomass.

[0075] Alternatively said particles comprise between 0.5% (w / w) and 10% (w / w), between 1% (w / w) and 10% (w / w), between 1.5% (w / w) and 10% (w / w), between 2% (w / w) and 10% (w / w), between 2.5% (w / w) and 10% (w / w), between 3% (w / w) and 10% (w / w), between 3.5% (w / w) and 10% (w / w), between 4% (w / w) and 10% (w / w), between 4.5% (w / w) and 10% (w / w), between 5% (w / w) and 10% (w / w), between 5.5% (w / w) and 10% (w / w), between 6% (w / w) and 10% (w / w), between 6.5% (w / w) and 10% (w / w), between 7% (w / w) and 10% (w / w), between 7.5% (w / w) and 10% (w / w), between 8% (w / w) and 10% (w / w), between 8.5% (w / w) and 10% (w / w), between 9% (w / w) and 10% (w / w), or between 9.5 (w / w) and 10% (w / w) methanotrophic bacteria biomass.

[0076] In yet another alternative embodiment, the particles comprise between 0.1% (w / w) and 50% (w / w), between 0.1% (w / w) and 40% (w / w), between 0.1% (w / w) and 30% (w / w), between 0.1% (w / w) and 20% (w / w) or between 0.1% (w / w) and 15% (w / w) methanotrophic bacteria biomass. In yet another alternative embodiment, the particles comprise between 10% (w / w) and 50% (w / w), between 15% (w / w) and 50% (w / w), between 20% (w / w) and 50% (w / w), between 30% (w / w) and 50% (w / w) or between 40% (w / w) and 50% (w / w) methanotrophic bacteria biomass.

[0077] In yet another alternative embodiment, the particles comprise at least 0.1% (w / w), 0.2% (w / w), 0.3% (w / w), 0.4% (w / w), 0.5% (w / w), 0.6% (w / w), 0.7% (w / w), 0.8% (w / w), 0.9% (w / w), 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 11% (w / w), 12% (w / w), 13% (w / w), 14% (w / w), 15% (w / w), 16% (w / w), 17% (w / w), 18% (w / w), 19% (w / w), 20% (w / w), 21% (w / w), 22% (w / w), 23% (w / w), 24% (w / w), 25% (w / w), 26% (w / w), 27% (w / w), 28% (w / w), 29% (w / w), 30% (w / w), 31% (w / w), 32% (w / w), 33% (w / w), 34% (w / w), 35% (w / w), 36% (w / w), 37% (w / w), 38% (w / w), 39% (w / w), 40% (w / w), 41% (w / w), 42% (w / w), 43% (w / w), 44% (w / w), 45% (w / w), 46% (w / w), 47% (w / w), 48% (w / w), 49% (w / w) or 50% (w / w) methanotrophic bacteria biomass.

[0078] In an embodiment of the feed additive, as disclosed herein, the particles comprise methanotrophic bacteria cells at an optical density, measured prior to mixing with the gelling agent at 600 nm, of between 0.2 and 0.3 , preferably between 0.24 and 0.25. In some embodiments, the OD of the methanotrophic bacteria cells measured prior to mixing with the gelling agent at 600 nm, is between 0.2 and 0.29, 0.2 and 0.28, 0.2 and 0.27, 0.2 and 0.26, 0.2 and 0.25, 0.2 and 0.24, 0.2 and 0.23, 0.2 and 0.22, or 0.2 and 0.21. In other embodiments, said OD is between 0.21 and 0.3, 0.22 and 0.3, 0.23 and 0.3, 0.24 and 0.3, 0.25 and 0.3, 0.26 and 0.3, 0.27 and 0.3, 0.28 and 0.3, or 0.29 and 0.3.

[0079] The optical density of the methanotrophic bacteria cells is measured using methods known in the art. In an embodiment, the OD is measured at 600 nm using a spectrophotometer. The spectrophotometer is calibrated using a standard curve generated from known concentrations of methanotrophic bacteria. The standard curve is prepared by culturing methanotrophic bacteria to various known concentrations, measuring the optical densities at 600 nm, and plotting the optical densities against the concentrations to generate a linear calibration curve. This ensures accurate and reproducible measurements of the bacterial cell density. In embodiments of the feed additive, as disclosed herein, said methanotrophic bacteria are alive.

[0080] In the context of the disclosure, "alive" or "living" methanotrophic bacteria are defined as those bacteria that are in an active physiological state capable of performing essential biological functions necessary for their growth, maintenance, and metabolic activities. These functions include metabolic activity, where the bacteria can uptake and process methane (CH4) as a carbon and energy source, indicating active metabolic pathways. The bacteria must also exhibit reproductive capability, meaning they can undergo cellular division and proliferation under suitable environmental conditions. Viability is another crucial aspect, wherein the bacteria maintain cell membrane integrity and functionality, which can be confirmed using viability assays such as staining techniques or by plating on appropriate growth media to form colonies. Additionally, the bacteria must show respiratory activity, whether aerobic or anaerobic, as part of their energy production processes. For species of methanotrophic bacteria that possess structures for movement, such as flagella, being alive includes their ability to exhibit motility towards or away from chemical stimuli (chemotaxis). Finally, the bacteria must be able to respond to environmental changes or stimuli, such as alterations in nutrient availability, temperature, or pH, which indicates active regulatory and adaptive mechanisms. To determine that the methanotrophic bacteria in the feed additive are alive, standard microbiological and biochemical assays can be performed, including monitoring growth curves, viability staining, metabolic assays, and colony-forming unit (CFU) assays.

[0081] In an embodiment of the feed additive disclosed herein, said feed additive comprises between 0.15*106CFU / ml and 0.35*106CFU / ml methanotrophic bacteria. In some embodiments, the feed additive comprises between 0.15*106CFU / ml and 0.30*106CFU / ml, between 0.15*106CFU / ml and 0.25*106CFU / ml, or between 0.15*106CFU / ml and 0.20*106CFU / ml, preferably between 0.15*106CFU / ml and 0.25*106CFU / ml methanotrophic bacteria.

[0082] Alternatively, said feed additive comprises between 0.2*106CFU / ml and 0.35*106CFU / ml, between 0.25*106CFU / ml and 0.35*106CFU / ml, or between 0.3*106CFU / ml and 0.35*106CFU / ml, preferably between 0.25*106CFU / ml and 0.35*106CFU / ml methanotrophic bacteria methanotrophic bacteria. In yet another alternative embodiment, said feed additive comprises between 0.16*106CFU / ml and 0.34*106CFU / ml, between 0.17*106CFU / ml and 0.33*106CFU / ml, between 0.18*106CFU / ml and 0.32*106CFU / ml, between 0.19*106CFU / ml and 0.31*106CFU / ml, between 0.20*106CFU / ml and 0.30*106CFU / ml, between 0.21*106CFU / ml and 0.29*106CFU / ml, preferably between 0.22*106CFU / ml and 0.28*106CFU / ml, more preferably between 0.23*106CFU / ml and 0.27*106CFU / ml, or even more preferably between 0.24*106CFU / ml and 0.26*106CFU / ml.

[0083] In some embodiments, the feed additive may further comprise one or more stabilizers and / or emulsifiers that improve the mixability of said feed additive in the animal feed or drinking water. Such stabilizers may comprise glycerol.

[0084] In further embodiments, the feed additive may further comprise vitamins, minerals, trace elements, or a combination thereof. Vitamins include but are not limited to Vitamin A, Vitamin D3, Vitamin E, Vitamin K3, Thiamin, Riboflavin, Pantothenic acid, Biotin, Folic acid, Vitamin B12, Niacin, Pyridoxine, Ascorbic acid, Inositol and Choline. Minerals and trace elements include but are not limited to magnesium, sodium, manganese, iron, zinc, copper, selenium, phosphorous, cobalt and iodine. The addition of these components further contributes to a complete and healthy diet of the animals and positively influences their zootechnical performance. The inclusion of these components in the feed additive simplifies the steps needed for feeding the animals as these components no longer need to be added separately to the feed, resulting in improved efficiency of the feeding process.

[0085] In further embodiments, the feed additive as disclosed herein is formulated as a liquid or as a solid form. The term "solid form" means a powder, a granule, or a pellet in particular. The term "liquid form", in particular, means a solution in water or means a solution in oil and includes a viscous paste and a non-viscous solution. In an embodiment, the feed additive is formulated as a gel. In particular, said feed additive is suitable for oral administration.

[0086] A feed comprising the feed additive according to an embodiment of the invention is produced or manufactured by means known to a person skilled in the art. In an embodiment, the feed comprising the feed additive as disclosed herein is provided as a dry extruded feed pellet. This formulation allows a relatively long shelf life and also permits the packaging and storage of large amounts of feed. In some embodiments, the feed may be supplemented with other well-known ingredients so as to provide a nutritionally balanced complete food, including, but not limited to, plant matter, e.g., flour, meal, starch or cracked or processed grain produced from a crop plant such as wheat or other cereals, alfalfa, corn, oats, potato, rice, soybeans or other legumes; cellulose in a form that may be obtained from wood pulp, grasses, plant leaves and waste plant matter such as rice or soy bean hulls, or corn cobs; animal matter, e.g., fish or crustacean meal, oil, protein or solubles and extracts, krill, meat meal, bone meal, feather meal, blood meal or cracklings; algal matter; yeast; bacteria; vitamins, minerals and amino acids; organic binders or adhesives; and chelating agents and preservatives.

[0087] In another embodiment, the feed additive as disclosed herein comprises metabolites and enzymes produced by the methanotrophs bacterial strains.

[0088] In a second aspect, the present disclosure relates to a method for reducing methane emissions in a ruminant animal, said method comprises administering an effective dose of the feed additive as disclosed herein. As used herein, an "effective amount" refers to an amount capable of providing bioavailable levels of methanotrophic bacteria and a reduction in methane emissions.

[0089] In an embodiment of the method as disclosed herein said effective dose comprises the feeding of 0.01% to 0.5% of said feed additive on animal weight basis per day. In some embodiments of the method as disclosed herein, said effective dose comprises feeding 0.01% to 0.45%, 0.01% to 0.4%, 0.01% to 0.35%, 0.01% to 0.3%, 0.01% to 0.25%, 0.01% to 2%, 0.01% to 0.15%, 0.01% to 1%, 0.01% to 0.09%, 0.01% to 0.08%, 0.01% to 0.07%, 0.01% to 0.06%, 0.01% to 0.05%, 0.01% to 0.04%, 0.01% to 0.03% or 0.01% to 0.02% of said feed additive on animal weight basis per day.

[0090] In other embodiments of the method as disclosed herein, said effective dose comprises feeding 0.02% to 0.5%, 0.03% to 0.5%, 0.04% to 0.5%, 0.06% to 0.5%, 0.07% to 0.5%, 0.08% to 0.5%, 0.09% to 0.5%, 0.1% to 0.5%, 0.15% to 0.5%, 0.2% to 0.5%, 0.25% to 0.5%, 0.3% to 0.5%, 0.35% to 0.5%, 0.4% to 0.5 or 0.45% to 0.5% of said feed additive on animal weight basis per day.

[0091] In an embodiment, the method of lowering the methane emissions in a ruminant animal comprises administrating said feed additive to an animal feed, feed ingredient, or drinking water of said animal. According to a further embodiment, said method comprises adding the feed additive as disclosed herein at a concentration of between 1% (w / w) and 10% (w / w) to said animal feed, feed ingredient, or drinking water.

[0092] In another embodiment said method comprises adding the feed additive as disclosed herein at a concentration of between 1% and 10%, between 1% (w / w) and 9.5% (w / w), between 1% (w / w) and 9% (w / w), between 1% (w / w) and 8.5% (w / w), between 1% (w / w) and 8% (w / w), between 1% (w / w) and 7.5% (w / w), between 1% (w / w) and 7% (w / w), between 1% (w / w) and 6.5% (w / w), between 1% (w / w) and 6% (w / w), between 1% (w / w) and 5.5% (w / w), between 1% (w / w) and 5% (w / w), between 1% (w / w) and 4.5% (w / w), between 1% (w / w) and 4% (w / w), between 1% (w / w) and 3.5% (w / w), between 1% (w / w) and 3% (w / w), between 1% (w / w) and 2.5% (w / w), between 1% (w / w) and 2% (w / w), or between 1% (w / w) and 1.5% (w / w) to a feed or an animal drinking water.

[0093] Alternatively, the method of the invention comprises adding to a feed or an animal drinking water between 1.5% (w / w) and 10% (w / w), between 2% (w / w) and 10% (w / w), between 2.5% (w / w) and 10% (w / w), between 3% (w / w) and 10% (w / w), between 3.5% (w / w) and 10% (w / w), between 4% (w / w) and 10% (w / w), between 4.5% (w / w) and 10% (w / w), between 5% (w / w) and 10% (w / w), between 5.5% (w / w) and 10% (w / w), between 6% (w / w) and 10% (w / w), between 6.5% (w / w) and 10% (w / w), between 7% (w / w) and 10% (w / w), between 7.5% (w / w) and 10% (w / w), between 8% (w / w) and 10% (w / w), between 8.5% (w / w) and 10% (w / w), between 9% (w / w) and 10% (w / w), or between 9.5% (w / w) and 10% (w / w) of the feed additive as disclosed herein.

[0094] In yet another embodiment, the method comprises adding to a feed or an animal drinking water at least 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 90% (w / w) or 95% (w / w) of the feed additive as disclosed herein.

[0095] Preferably, the animals are treated or fed at least once per day, more preferably two or more times per day such as, for example, 2-6 or 4-6 times per day. It is preferred that any excess feed for treatment by oral administration be removed after the feeding period, e.g., by flushing out of a raceway system, or through removal out of the feed trunks. By preference, the method comprises administrating said feed additive or feed comprising said additive to a ruminant animal. Said ruminant animal is selected from the members of the Antilocapridae, Bovidae, Cervidae, Girraffidae, Moschidae, or Tragulidae families. In a further embodiment, said ruminant animal is a cattle, goat, sheep, giraffe, bison, yak, water buffalo, deer, camel, alpaca, llama, wildebeest, antelope, pronghorn, nilgai, or moose. In a preferred embodiment, said ruminant animal is a cattle, goat, or sheep.

[0096] In another embodiment, the method disclosed herein may comprise administrating a feed additive or feed comprising a feed additive to a non-ruminant or monogastric animal. Non-limitative examples of monogastric animals include humans, poultry, pigs, horses, rabbits, dogs and cats. While monogastric animals emit significantly lower levels of methane when compared to ruminants, increases in methane emissions are reported in monogastric subjects affected by diseases or pathogens such as worms or bacteria.

[0097] Administrating the feed additive or a feed supplemented with the feed additive reduced methane emissions of ruminant animals by between 25% and 80% compared to an untreated animal. In another embodiment, said methane reduction is between 25% and 75%, between 25% and 70%, between 25% and 65%, between 25% and 60%, between 25% and 55%, between 25% and 50%, between 25% and 45%, between 25% and 40%, between 25% and 35%, or between 25% and 30% compared to an untreated animal.

[0098] Alternatively said methane reduction is between 30% and 80%, between 35% and 80%, between 40% and 80%, between 45% and 80%, between 50% and 80%, between 55% and 80%, between 60% and 80%, between 65% and 80%, between 70% and 80%, or between 75% and 80% compared to an untreated animal.

[0099] In yet another embodiment, administration of the feed additive of the invention reduces the methane emissions in ruminant animals with at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90% or 95% compared to an untreated animal.

[0100] In another embodiment of the method, the feed additive contains enzymes and metabolites derived from the bacterial strain. The methanotrophic bacteria in the feed additive, convert the methane to carbon dioxide by combining the methane from the rumen and the oxygen "trapped" into the gel or gel-like matrix that the bacteria are embedded in. The inventors observed an unexpected reduction in methane emissions, without increasing hydrogen pressure in the rumen. The hydrogen in the rumen is taken up by the methanogenic archaea that produce methane which is further metabolized by the methanotrophic bacteria provided in the feed additive.

[0101] Moreover, the feed additive of the invention indirectly enhances the zootechnical performance of animals, growth rate, milk quality and / or feed conversion efficiency by increasing VFAs production, especially propionate.

[0102] In a third aspect, the invention relates to the use of the feed additive as described in any of the previous embodiments, for reducing the methane emission in a ruminant animal wherein said animal is a cattle, goat, or sheep.

[0103] In an embodiment, said uses comprise administering an effective dose of the feed additive as disclosed herein. As used herein, an "effective amount" refers to an amount capable of providing bioavailable levels of methanotrophic bacteria and a reduction in methane emissions.

[0104] In an embodiment of the use as disclosed herein said effective dose comprises the feeding of 0.01 to 5 % of said feed additive on animal weight basis per day.

[0105] In some embodiments of the use as disclosed herein, said effective dose comprises feeding 0.01% to 0.45%, 0.01% to 0.4%, 0.01% to 0.35%, 0.01% to 0.3%, 0.01% to 0.25%, 0.01% to 2%, 0.01% to 0.15%, 0.01% to 1%, 0.01% to 0.09%, 0.01% to 0.08%, 0.01% to 0.07%, 0.01% to 0.06%, 0.01% to 0.05%, 0.01% to 0.04%, 0.01% to 0.03% or 0.01% to 0.02% of said feed additive on animal weight basis per day.

[0106] In other embodiments of the use as disclosed herein, said effective dose comprises feeding 0.02% to 0.5%, 0.03% to 0.5%, 0.04% to 0.5%, 0.06% to 0.5%, 0.07% to 0.5%, 0.08% to 0.5%, 0.09% to 0.5%, 0.1% to 0.5%, 0.15% to 0.5%, 0.2% to 0.5%, 0.25% to 0.5%, 0.3% to 0.5%, 0.35% to 0.5%, 0.4% to 0.5 or 0.45% to 0.5% of said feed additive on animal weight basis per day. In an embodiment, the use comprises administrating said feed additive to an animal feed, feed ingredient, or drinking water of said animal. According to a further embodiment, said use comprises adding the feed additive as disclosed herein at a concentration of between 1% (w / w) and 10% (w / w), preferably 6% (w / w) to said animal feed, feed ingredient, or drinking water.

[0107] In another embodiment said use comprises adding the feed additive as disclosed herein at a concentration of between 1% and 10%, between 1% (w / w) and 9.5% (w / w), between 1% (w / w) and 9% (w / w), between 1% (w / w) and 8.5% (w / w), between 1% (w / w) and 8% (w / w), between 1% (w / w) and 7.5% (w / w), between 1% (w / w) and 7% (w / w), between 1% (w / w) and 6.5% (w / w), between 1% (w / w) and 6% (w / w), between 1% (w / w) and 5.5% (w / w), between 1% (w / w) and 5% (w / w), between 1% (w / w) and 4.5% (w / w), between 1% (w / w) and 4% (w / w), between 1% (w / w) and 3.5% (w / w), between 1% (w / w) and 3% (w / w), between 1% (w / w) and 2.5% (w / w), between 1% (w / w) and 2% (w / w), or between 1% (w / w) and 1.5% (w / w) to a feed or an animal drinking water.

[0108] Alternatively, the use comprises adding to a feed or an animal drinking water between 1.5% (w / w) and 10% (w / w), between 2% (w / w) and 10% (w / w), between 2.5% (w / w) and 10% (w / w), between 3% (w / w) and 10% (w / w), between 3.5% (w / w) and 10% (w / w), between 4% (w / w) and 10% (w / w), between 4.5% (w / w) and 10% (w / w), between 5% (w / w) and 10% (w / w), between 5.5% (w / w) and 10% (w / w), between 6% (w / w) and 10% (w / w), between 6.5% (w / w) and 10% (w / w), between 7% (w / w) and 10% (w / w), between 7.5% (w / w) and 10% (w / w), between 8% (w / w) and 10% (w / w), between 8.5% (w / w) and 10% (w / w), between 9% (w / w) and 10% (w / w), or between 9.5% (w / w) and 10% (w / w) of the feed additive as disclosed herein.

[0109] In yet another embodiment, the use comprises adding to a feed or an animal drinking water at least 1% (w / w), 2% (w / w), 3% (w / w), 4% (w / w), 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 90% (w / w) or 95% (w / w) of the feed additive as disclosed herein.

[0110] Preferably, the animals are treated or fed at least once per day, more preferably two or more times per day such as, for example, 2-6 or 4-6 times per day. It is preferred that any excess feed for treatment by oral administration be removed after the feeding period, e.g., by flushing out of a raceway system, or through removal out of the feed trunks.

[0111] By preference, the use comprises administrating said feed additive or feed comprising said additive to a ruminant animal. Said ruminant animal is selected from the members of the Antilocapridae, Bovidae, Cervidae, Girraffidae, Moschidae, or Tragulidae families. In a further embodiment, said ruminant animal is a cattle, goat, sheep, giraffe, bison, yak, water buffalo, deer, camel, alpaca, llama, wildebeest, antelope, pronghorn, nilgai, or moose. In a preferred embodiment, said ruminant animal is a cattle, goat, or sheep.

[0112] In another embodiment, the use disclosed herein may comprise administrating a feed additive or feed comprising a feed additive to a non-ruminant or monogastric animal. Non-limitative examples of monogastric animals include humans, poultry, pigs, horses, rabbits, dogs and cats. While monogastric animals emit significantly lower levels of methane when compared to ruminants, increases in methane emissions are reported in monogastric subjects affected by diseases or pathogens such as worms or bacteria.

[0113] Administrating the feed additive or a feed supplemented with the feed additive reduced methane emission of ruminant animals by between 25% and 80%. In another embodiment, said methane reduction is between 25% and 75%, between 25% and 70%, between 25% and 65%, between 25% and 60%, between 25% and 55%, between 25% and 50%, between 25% and 45%, between 25% and 40%, between 25% and 35%, or between 25% and 30%.

[0114] Alternatively said methane reduction is between 30% and 80%, between 35% and 80%, between 40% and 80%, between 45% and 80%, between 50% and 80%, between 55% and 80%, between 60% and 80%, between 65% and 80%, between 70% and 80%, or between 75% and 80%.

[0115] In yet another embodiment, administration of the feed additive of the invention reduces the methane emissions in ruminant animals with at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90% or 95%.

[0116] In a final aspect, the invention relates to a method for the production of a feed additive according to any of the previous embodiments, wherein said feed additive reduces methane emissions in ruminants, characterized in that said method comprises the steps: a) providing a methanotrophic bacteria biomass; b) mixing the methanotrophic bacteria biomass with a gelling agent to encapsulate the methanotrophic bacteria biomass in a gel or gel-like matrix; and c) formation of particles from the gel or gel-like matrix.

[0117] The methanotrophic bacteria biomass may be obtained by any bacterial culture methodologies known in the art as suitable for growing methanotrophs. For example, bacteria may be grown by batch culture or continuous culture methodologies. In certain embodiments, the cultures are grown in a controlled culture unit, such as a fluidized bed reactor, a fermenter, a bioreactor, a hollow fiber membrane bioreactor, a packed bed bioreactor, or the like.

[0118] A classical batch culturing method is a closed system where the composition of the media is set at the beginning of the culture and not subject to external alterations during the culture process. Thus, at the beginning of the culturing process, the media is inoculated with the desired organism and growth or metabolic activity is permitted to occur without adding anything to the system. Typically, however, a "batch" culture is batch with respect to the addition of carbon source (e.g., methane, natural gas, unconventional natural gas, methanol, a methylamine, a methylthiol, or a methylhalogen) and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures, cells moderate through a static lag phase to a high-growth logarithmic phase and finally to a stationary phase where the growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in the logarithmic growth phase are often responsible for the bulk production of end products or intermediate in some systems. Stationary or postexponential phase production can be obtained in other systems.

[0119] The fed-batch system is a variation of the standard batch system. Fed-batch culture processes comprise a typical batch system with the modification that the substrate is added in increments as the culture progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors, such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2.

[0120] Continuous cultures are "open" systems where a defined culture media is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in logarithmic phase growth. Alternatively, continuous culture may be practised with immobilized cells where carbon and nutrients are continuously added and valuable products, byproducts and waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural or synthetic materials.

[0121] Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end-product concentration. For example, one method will maintain a limited nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to modulate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to media being drawn off must be balanced against the cell growth rate in the culture.

[0122] The methanotrophic bacteria are preferably grown in nitrate mineral salts (NMS) culture medium. However, it will be obvious to the skilled person that any culture medium suitable for growing methanotrophic bacteria, may be used with the method as disclosed herein. Non-limiting examples include nitrogen mineral salt, amino acid mineral 1 (AMI), yeast extract medium (Y-medium), or other mineral salts media (MSM).

[0123] Methyl- or methane-containing carbon substrates can be used to produce the bacteria biomass and to culture said bacteria biomass. Exemplary carbon substrates include natural gas, unconventional natural gas, syngas and methane. As used herein, "natural gas" refers to naturally occurring gas mixtures that have formed in porous reservoirs and can be accessed by conventional processes (e.g., drilling) and are primarily made up of methane, but may also have other components such as carbon dioxide, nitrogen or hydrogen sulfide. As used herein, "unconventional natural gas" refers to a naturally occurring gas mixture created in formations with low permeability that must be accessed by unconventional methods, such as hydraulic fracturing, horizontal drilling or directional drilling. Exemplary unconventional natural gas deposits include tight gas sands formed in sandstone or carbonate, coal bed methane formed in coal deposits and adsorbed in coal particles, shale gas formed in fine-grained shale rock and adsorbed in clay particles or held within small pores or microfractures, methane hydrates that are a crystalline combination of natural gas and water formed at low temperature and high pressure in places such as under the oceans and permafrost. Sources of methane include natural sources, such as natural gas fields, "unconventional natural gas" sources (such as shale gas or coal bed methane, wherein content will vary depending on the source) and biological sources where it is synthesized by, for example, methanogenic microorganisms, manure digest and industrial or laboratory synthesis. Methane includes pure methane, substantially purified compositions, such as "pipeline-quality natural gas" or "dry natural gas", which is 95-98% percent methane, and unpurified compositions, such as "biogas" and "wet natural gas" (comprising a higher percentage of liquid natural gases (such as ethane and butane) than dry natural gas, the latter being almost completely methane), wherein other hydrocarbons have not yet been removed, and methane comprises from about 50% to about 75% of the biogas composition and at least about 60% to 85% or less of the wet natural gas composition.

[0124] When the methanotrophic bacteria have grown sufficiently, the bacteria biomass is harvested from the culture. Without being bound to theory, methods of harvesting the bacteria include centrifugation, filtration, sedimentation or flocculation. In some embodiments, the bacteria biomass is washed after harvesting. In other embodiments, the bacteria biomass is not washed.

[0125] After harvesting, the methanotrophic bacteria biomass is mixed with a gelling agent to form a gel or gel-like matrix.

[0126] The gel or gel-like matrix may be characterized by any of the features disclosed in the previous embodiments. The gelling agent may be any of the gelling agents disclosed in the previous embodiments.

[0127] The gel or gel-like matrix comprising the encapsulated methanotrophic bacteria is then formed in particles by methods such as extrusion, droplet formation, spray drying, coacervation, electrostatic encapsulation or co-extrusion, as disclosed in previous embodiments. The formed particles may have any of the characteristics disclosed in the previous embodiments.

[0128] The feed additive is obtained by collecting the particles. Surprisingly, the feed additive comprising methanotrophic bacteria encapsulated in a gel or gel-like matrix reduces methane emissions in ruminant animals.

[0129] The particles may be stored at 4°C or preserved after harvesting, by lyophilization, freezing, or spray-drying.

[0130] The invention is not limited to this application. The method according to the invention can be applied in all sorts of instances where methane emissions are caused by bacterial fermentation. The disclosed additive and the treatment method may be applied to treating methane emissions in all sorts of monogastric animals including humans.

[0131] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0132] EXAMPLES

[0133] Example 1. Bacteria biomass production

[0134] Methylococcus capsulatus (Foster and Davis strain ACM 3302) bacterial cultures of preexisting 10 days old stock cultures were inoculated in fresh NMS medium without copper, at 10% bacteria concentration. The cultures were each feed with an atmosphere comprising 50% CH4and 50% air, at the start of the culture, after 72h in culture, and further on each 24 to 48h. The cultures were incubated at 37°C with shaking (150 rpm) for the first 3 days and without agitation on the 4th day.

[0135] The bacteria biomass production was done by quantifying the optical density (OD) of the cultures. Measurements were done with a spectrophotometer at 600nm.

[0136] The initial OD of the culture was 0.037. 4 days after the start of the culture, the bacteria reached the plateau phase at an OD between 0.24 and 0.25. Bacteria biomass at an OD between 0.24 and 0.25 were centrifuged at 5000 rpm for 10 minutes. The supernatant was removed and the collected sediment comprised the bacterial biomass.

[0137] Example 2. Bacteria biomass encapsulation

[0138] The bacterial biomass obtained according to Example 1 was briefly washed and mixed with a 5% (w / v) Na-alginate solution (1 ml bacteria biomass to 19 ml 5% Na-alginate).

[0139] The resulting mixture, comprising the bacteria biomass in Na-alginate solution, was extruded dropwise using a syringe into a CaCI? solution of 1.5 M for the preparation of encapsulated bacteria beads.

[0140] When parts of the sodium ions in the alginate solution were exchanged with calcium ions, a gel matrix formed. This gel matrix consisted of Na-alginate beads in which bacteria biomass is encapsulated.

[0141] Thereafter, Na-alginate beads in which bacteria biomass was encapsulated were washed with saline solution to remove any loosely bound cells.

[0142] Example 3. Beads size measurement

[0143] Optical microscopy was used for determining the diameter of the beads comprising the encapsulated bacterial biomass.

[0144] About 20 beads obtained according to Example 2 were randomly sampled and visualized under a light microscope equipped with a camera. The generated images were processed using a software and the diameter of the beads was calculated based on the 1 mm scale bar.

[0145] All the measured beads had a diameter of between 1 and 4 mm.

[0146] Example 4. The effect of the feed additive on methane and hydrogen emissions and VFAs composition in in vitro rumen bioassav

[0147] The encapsulated bacteria biomass obtained according to Example 2 was used in an in vitro rumen assay where the effect of said feed additives on methane and hydrogen emissions and total and individual volatile fatty acids (VFAs) composition was measured in a comparative test.

[0148] A batch incubation mimicking the rumen fermentation using sealed 250-ml flasks in triplicate and containing 50 mg of dairy cow ration substrate, consisting of grass silage, maize silage, and a balanced dairy cow concentrate (175 / 175 / 150, w / w / w) and CC>2-saturated bicarbonate / phosphate-buffered rumen fluid (buffer + water: rumen fluid 4: 1, v:v) to reach a total liquid volume of 50 ml was performed in the presence of either 1% and 10% encapsulated bacteria biomass according to Example 2, or in the presence of 1% and 10% Na-alginate beads and 1% and 10% methanotrophic bacteria liquid culture. Also, a negative control consisting of the dairy cow ration substrate alone and a positive control consisting of the dairy cow ration substrate and IpM Bromoform were used.

[0149] Gas and liquid samples were collected at the end of the incubation period to determine gas composition (CH4and H2) and VFAs. The gas-phase analysis was performed using a micro-GC equipped with two gas chromatographic modules and a thermal conductivity detector (3000 micro-GC, Agilent, USA). Ethane (C2H6; 2 ml / flask) was used as an internal standard.

[0150] For VFA analysis, 1 ml of incubation medium was acidified with 100 pl of formic acid containing the internal standard (10 mg 2-ethyl butyric acid / ml formic acid). After 15 min centrifugation at 4 °C and 22000 g, the supernatant was filtered and an aliquot was transferred into a 1.5-ml glass vial. Volatile fatty acids were analyzed through gas chromatography on an HP 7890A (Agilent Technologies, Diegem, Belgium) equipped with a Nukol column (30 m x 0.25 mm x 0.25 pm, Supelco) with a flame ionization detector.

[0151] The net production of VFA was calculated by subtracting the amounts in rumen fluid before the incubation from amounts measured after incubation.

[0152] The methane production for the encapsulated bacteria biomass added at 10% was on average 914 pmol / g DM (Table 1), which represents a 52% reduction in CH4production compared to the untreated cow ration substrate. The methane production for the bacteria biomass alone added at 10% was on average 1061 pmol / g DM (Table 1), which represents a 52% reduction in CH4production compared to the untreated cow ration substrate. For the Na-alginate beads alone, the methane reduction was up to 30% compared to the negative control. The encapsulated bacteria biomass added at 10% was the most effective in reducing the methane production compared to any other tested conditions.

[0153] There was no significant difference in the H2content between the encapsulated bacteria and the Na-alginate beads.

[0154] The propionate production for the encapsulated bacteria biomass added at 10% was on average 1527 pmol / g DM (Table 2), which represents a significant increase of 9% compared to the negative control condition. Other VFA were significantly reduced compared to the negative control condition.

[0155] Notably, the encapsulated bacteria biomass added at 10% outperformed the positive control, Bromoform, in both the reduction of CH4and the increase in propionate. Bromoform is a compound known for inhibiting methanogenesis.

[0156] Table 1. The effect of the encapsulated methanotrophic bacteria biomass on the production of methane (CH4) and hydrogen (H2) in a 24 h batch in vitro incubation.

[0157] Treatment CH4H2

[0158] Negative control 1730d1.90

[0159] Positive control 1370bc1.94

[0160] 1% Bacteria biomass 1730d1.68

[0161] 10% Bacteria biomass 1061ab60.44

[0162] 1% Na-alginate beads 1558cd1.39

[0163] 10% Na-alginate beads 1256bc2.19

[0164] 1% Encapsulated biomass 1420cd1.46

[0165] 10% Encapsulated biomass 914a3.21

[0166] SEM191.9 20.700

[0167] P-Value <0.0001 0.13

[0168] Standard Error of the Means,

[0169] Means with different superscripts within a column differ significantly (P < 0.05)

[0170] Table 2. The effect of the encapsulated methanotrophic bacteria biomass on the production of volatile fatty acids (pmol / g DM, unless stated otherwise) in a 24 h batch in vitro incubation.

[0171]

[0172] The incubation of proteinaceous feed material with methanotrophic bacteria followed by fermentation is described in US2019082717 and US2019271019. In the current experiment, 1% and 10% bacterial biomass were used without encapsulation. These conditions are similar to those outlined in the aforementioned documents, as the dairy cow ration substrate— comprising grass silage, maize silage, and a balanced dairy cow concentrate— serves as a proteinaceous feed material. However, the current results indicate that simple incubation of bacterial biomass with the proteinaceous feed material is not as effective in reducing methane as encapsulating the methanotrophs in a gel matrix. Example 5. The effect of the concentration of the encapsulated bacteria biomass on the methane reduction in in vitro rumen bioassav

[0173] The encapsulated bacteria biomass obtained according to Example 2 is used in an in vitro rumen assay as described in Example 4 at different concentrations between 1% and 10% total weight feed.

[0174] Example 6. The effect of the encapsulated bacteria biomass on methane and hydrogen emissions and VFAs composition in sheep

[0175] The encapsulated bacteria biomass obtained according to Example 2 is used to evaluate its effectiveness in inhibiting methane emissions in sheep.

[0176] Twenty adult non-pregnant sheep are used in a randomized block design in which five sheep are allocated to one of the following experimental treatments (n=5): control untreated feed, feed supplemented with encapsulated bacteria biomass, feed supplemented with Na-alginate beads and feed supplemented with liquid methanotrophic bacteria culture.

[0177] The diet is provided twice a day to each animal at a 1.2 energy maintenance level. The nutritional composition of the feed additive brings additional energy (per 100g = 3700kj / 900kcal) and is taken into account in the formulation of the diet. The animals are adapted to the diet 14 days prior to the CH4measurement.

[0178] After the adaptation period, the animals are moved for three days to the chambers for CH4and H2emissions measurements. All animals belonging to the same treatment group are measured at a time. If a significant (20-30% or more) drop in dry matter intake is observed in any animal while in the chamber as compared to the adaptation period, the measuring period is extended until a satisfactory intake is achieved.

[0179] The CH4and H2emissions and total and individual VFAs composition are measured according to the method described in Example 4.

[0180] After measurements, rumen fluids are collected 2 hours after feeding using the stomach tubing technique (Ramos-Morales et al., 2014) to determine the microbial fermentation profile (volatile fatty acids -VFA, NH3, lactate and pH). An additional aliquot is kept at -80 °C for further molecular analyses. It is expected that animals treated with the encapsulated bacteria biomass feed additive according to the invention, emit less methane than the animals treated with Na-alginate or liquid cultures of methanotrophic bacteria. No significant difference in H2emissions is expected in animals treated with the encapsulated bacteria biomass compared to the animals treated with the Na-alginate or liquid bacteria cultures. Total VFA in animals treated with encapsulate bacteria biomass is expected to significantly increase including propionate but not the other VFA compared to the animals treated with Na-alginate or methanotrophic bacteria liquid cultures.

[0181] It is clear that the method according to the invention, and its applications, are not limited to the presented examples. The experiments provided in the above examples may be reproduced with other methanotrophic bacteria. The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. A feed additive characterized in that said feed additive comprises an effective amount of one or more methanotrophic bacteria encapsulated into a gel or gel-like matrix.

2. Feed additive according to claim 1, wherein said methanotrophic bacteria are alive.

3. Feed additive according to any of the claims 1 or 2, wherein said feed additive comprises between 0.15*106CFU / ml and 0.35*106CFU / ml methanotrophic bacteria.

4. Feed additive according to any of the claims 1 to 3, wherein said gel or gellike matrix comprises a gelling agent selected from alginate, agar, agarose, carrageenan, gelatin, silica gel or cellulose derivatives.

5. Feed additive according to claim 4, wherein the gelling agent is alginate and wherein said alginate is selected from sodium alginate, potassium alginate, calcium alginate, propylene glycol alginate or ammonium alginate.

6. Feed additive according to any of the claims 1 to 5, wherein said feed additive is obtained by forming the gel or gel-like structure and the methanotrophic bacteria into particles, preferably spherical particles, like beads.

7. Feed additive according to claim 6 wherein said particles have an average particle size between 1 and 4 mm.

8. Feed additive according to any of the claims 1 to 7, wherein said methanotrophic bacteria are selected from the genus of Methylomonas, Methylobacter, Methylococcus, Methylosinus and mixtures thereof.

9. Feed additive according to any of the claims 1 to 8, wherein said methanotrophic bacteria are of the genus Methylococcus, preferably the species Methylococcus capsulatus.

10. Feed additive according to any of the claims 6 to 9, wherein the particles comprise between 90% (w / w) and 99.9% (w / w) gelling agent.

11. Feed additive according to any of the claims 1 to 10, wherein the particles comprise at least 0.1% (w / w) methanotrophic bacteria biomass.

12. Feed additive according to any of the claims 1 to 11, wherein the particles comprise between 0.1% (w / w) and 10% (w / w) methanotrophic bacteria biomass.

13. Feed additive according to any of the claims 1 to 12, wherein the particles comprise methanotrophic bacteria cells at an optical density, measured prior to mixing with the gelling agent at 600 nm calibrated with a standard curve of known bacterial concentrations, of between 0.2 and 0.3.

14. A method for reducing methane emissions in a ruminant animal said method comprises administering an effective dose of a feed additive according to claims 1 to 13.

15. The method according to claim 14, wherein said feed additive is administered to an animal feed, feed ingredient, or drinking water of said animal.

16. The method according to any of the claims 14 or 15, wherein said animal feed, feed ingredient, or drinking water comprises between 1% (w / w) and 10% (w / w) of said feed additive.

17. The method according to any of the claims 14 to 16, wherein said effective dose comprises the feeding of 0.01% to 0.5% of said feed additive on animal weight basis per day.

18. The method according to any of the claims 14 to 17, wherein said ruminant animal is chosen from the group comprising cattle, goat, or sheep.

19. Use of a feed additive according to any of the claims 1 to 13 for reducing the methane emission in a ruminant animal wherein said animal is a cattle, goat, or sheep.

20. A method for production of a feed additive according to any of the claims 1 to 9, wherein said feed additive reduces methane emissions in ruminants, characterized in that said method comprises the steps: a) providing a methanotrophic bacteria biomass; b) mixing the methanotrophic bacteria biomass with a gelling agent to encapsulate the methanotrophic bacteria biomass in a gel or gel-like matrix; and c) formation of particles from the gel or gel-like matrix.

Images