Delivery method for a volatile feed additive for mitigation of methane production

Abstract

A composition and a method for delivery are provided for reducing methane production in a ruminant, said composition comprising one or more dihalomethanes adsorbed onto particular solid carriers.

Description

[0001] DELIVERY METHOD FOR A VOLATILE FEED ADDITIVE FOR MITIGATION OF METHANE

[0002] PRODUCTION

[0003] TECHNICAL FIELD

[0004] A composition and a method are provided for reducing methane production in a ruminant, said composition comprising one or more dihalomethanes adsorbed onto a solid carrier selected from activated carbon, lignin or sepiolite. The composition has improved storage stability, while allowing rapid and effective release into the digestive system of a ruminant. A process for formation of the composition is also described, said process comprising the steps of: a. adsorption of dihalomethane onto solid carrier selected from activated carbon, lignin or sepiolite, b. optionally, adding a liquid stabiliser component to said composition, c. optionally, granulating or pelletising the composition from step a. or the stabilised composition of step b., d. optionally, coating the granules or pellets from step c. with a coating material, e. optionally, producing a bolus from the coated granules or pellets from step d..

[0005] BACKGROUND

[0006] Ruminants account for as much as 30% of global anthropogenic methane emissions or 44% of total greenhouse gas (GHG) emissions from livestock (Gerber et al. (2013); Tackling Climate Change Through Livestock - A global assessment of emissions and mitigation opportunities. Rome: Food and Agriculture Organization of the United Nations (FAO), 2013). Methane is a GHG 25 times more powerful than CO2. There is a substantial pressure to reduce climate footprint from milk and beef production.

[0007] Methane (CH4) is synthesised as follows: CO2+4H2-> CH4+2H2O. Rumen archaea are the microorganisms responsible for this synthesis (Patra et al. (2017).

[0008] Related publications include US3660562 and US11529310. Other related publications include W02010 / 036752, WO 2021 / 116395A1, WO 2023 / 212773A1, US2015 / 132432 Al, and CN 110 771 742 A. There is still a need for compositions and methods for reducing methane production in a ruminant, which provides an efficient reduction in methane formation without deleterious side-effects. Certain methane-reducing components are liquids at room temperatures and conditions, which often require additional processing if they are to be fed to animals. There is furthermore a need for such compositions to be storage stable, with reduced volatility of the components, while allowing rapid and effective release of active agent(s) into the digestive system of a ruminant. Suitably, milk production of the ruminant should not be compromised.

[0009] SUMMARY

[0010] It has been found by the present inventor(s) that storage stability and effective release of active agents can be provided by adsorbing active agents onto a solid substrate, being selected from activated carbon, lignin or sepiolite.

[0011] A composition for reducing methane production in a ruminant is thus provided, said composition comprising one or more dihalomethanes adsorbed onto a solid carrier selected from activated carbon, lignin or sepiolite, said one or more dihalomethanes being selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane.

[0012] Also provided is a feed premix for a ruminant, said feed premix comprising the composition described herein. A method for reducing methane production in a ruminant, is also provided, said method comprising the step of administering the composition described herein, or the feed premix described herein, to said ruminant.

[0013] Further details of the present technology are presented in the following description, examples and claims.

[0014] LEGENDS TO THE FIGURES

[0015] Figure 1 shows a TGA curve of a sample of activated carbon.

[0016] Figure 2 shows a TGA curve of a sample of activated carbon with DBM.

[0017] Figure 3 shows a TGA curve of a sample of sepiolite.

[0018] Figure 4 shows a TGA curve of a sample of sepiolite with DBM. Figure 5 shows a TGA curve of a sample of lignin.

[0019] Figure 6 shows a TGA curve of a sample of lignin with DBM.

[0020] DETAILED DISCLOSURE

[0021] Definitions

[0022] The term "methanogenesis", "methane production" and "methane emission" is used interchangeably in the present context to describe the production of methane emanating from the digestive activities of ruminants. Methane emission by ruminants can easily be measured in individual animals in metabolic chambers by methods known in the art, see e.g. Ding et al 2006: Chapter 10: Emissions from Livestock and Manure Management. 2006 IPCC Guidelines for National Greenhouse Gas Inventories. Volume 4: Agriculture, Forestry and Other Land Use. https: / / www.ipcc-nggip.iges.or.jP / public / 2006gl / pdf / 4.

[0023] The term "ruminant" refers to any of a subfamily of bovinae including cattle and cows, sheep, goats, buffaloes, bison, and other antelopes, preferably cattle and cows.

[0024] The term "feed dry matter" or "feed DM" is used in the present context to refer to the material remaining after all of the water is evaporated out of a feed.

[0025] The overall goal of this technology is to formulate a physical carrier including the active component dihalomethane for mitigating the enteric methane emission in ruminants.

[0026] A composition is provided for reducing methane production in a ruminant, said composition comprising one or more dihalomethanes adsorbed onto a solid carrier selected from activated carbon, lignin or sepiolite, said one or more dihalomethanes being selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane.

[0027] Of the dihalomethanes, dibromomethane, diiodomethane and bromoiodomethane are preferred, dibromomethane and diiodomethane are more preferred, wherein dibromomethane is most preferred. Optionally, a mixture of two or more dihalomethanes may be included in the composition.

[0028] The dihalomethanes are adsorbed onto a solid carrier selected from activated carbon, lignin or sepiolite, whereby the storage stability is improved. At the same time, rapid and effective release of the dihalomethane(s) into the digestive system of a ruminant is maintained. By administering one or more dihalomethanes to a ruminant in this manner, it is possible to achieve a significant reduction in methane production in said ruminant without significantly influencing milk production and / or feed intake. While a significant reduction in methanogenesis is achieved through the administration of one or more dihalomethanes accompanied by some or a substantial reduction in feed intake a much less pronounced reduction in milk yield is observed, and hence an improved feed conversion rate - measured as amount of milk produced per unit of ingested feed - is obtained and thereby an improved feed efficiency.

[0029] Activated carbon is classified as safe for human and animal consumption in the USA and in Europe. Activated carbon, also called activated charcoal or active carbon, is a form of carbon commonly used to filter contaminants from water and air, among many other uses. It is processed (activated) to have small, low-volume pores that greatly increase the surface area available for adsorption or chemical reactions.

[0030] Activated carbon is produced from carbon-rich materials such as wood, coal, coconut shells, and peat through processes that involve carbonization and activation. The activation process can be physical or chemical, creating a network of micro, meso, and macropores, which are crucial for adsorption applications.

[0031] Types of Activated Carbon

[0032] 1. Based on Raw Material:

[0033] - Wood-Based : Known for their high microporosity and low density, suitable for vapor phase adsorption.

[0034] - Coal-Based : Typically have a balanced pore structure, offering versatility for both liquid and vapor phase applications.

[0035] - Coconut Shell-Based : Characterized by high hardness and high micropore volume, ideal for gas phase adsorption and decolorization processes.

[0036] - Peat-Based : Offer unique adsorption properties due to their origin, often used in water treatment.

[0037] 2. Based on Production Process: - Physical Activation: Involves carbonization at high temperatures followed by activation with steam or carbon dioxide, resulting in a wide range of pore sizes. This include biochar, fine-carbon, fine-grained residues that are produced via pyrolysis.

[0038] - Chemical Activation: Involves impregnation with activating agents (e.g., phosphoric acid, potassium hydroxide) followed by carbonization, typically yielding a highly porous structure with a significant amount of mesopores.

[0039] 3. Based on Final Structure:

[0040] - Powdered Activated Carbon (PAC) : Fine particles with high surface area, primarily used in liquid phase applications for rapid adsorption.

[0041] - Granular Activated Carbon (GAC) : Larger particles suitable for both liquid and gas phase applications, often used in fixed-bed systems.

[0042] - Pelletized Activated Carbon: Compressed form of GAC, offering uniform particle size and low dust content, ideal for gas phase applications and ease of handling.

[0043] A particularly preferred activated carbon has the following analysis parameters:

[0044] Moisture < 11%

[0045] Carbon on dry matter (NF B55-101) > 82%

[0046] Ash (NF EN1860-2) < 5%

[0047] Crude fiber (Regulation EC 152 / 2009) < 70%

[0048] Apparent density: 0.25 - 0.40 kg / L (or g / ml).

[0049] Granulometry (sieves) : > 90% below 1600 pm. or

[0050] Average specific area (BET) : 150-300 m2 / g. Lignins

[0051] Lignins are a class of naturally-occuring polymers obtained from plants, typically woody plants. Lignin comprises highly heterogeneous, highly crosslinked polymers containing phenol functionalities and phenol derivatives.

[0052] Sepiolite

[0053] Sepiolite is a white, soft clay mineral. Sepiolite complex magnesium silicate with a typical chemical formula: Mg4Si5Oi5(OH)2-6H2O.

[0054] Dihalomethanes, such as dibromomethane, interact with solid carriers primarily through physisorption. This process involves van der Waals forces and does not alter the chemical structure of the adsorbate or the adsorbent. The large surface area and the pore structure of the solid carrier facilitate the trapping of dihalomethane molecules within its pores. The efficiency of adsorption depends on factors such as pore size distribution, surface area, and the nature of the solid carrier.

[0055] Loading Dihalomethane onto solid carriers e.g. activated carbon.

[0056] 1. Impregnation Method (Liquid-Solid Interaction) :

[0057] - Process: Activated carbon is spray with a solution containing the dihalomethane.

[0058] - Advantages: Allows for uniform distribution of dihalomethane within the carbon structure and is suitable for precise control of loading levels.

[0059] - Disadvantages: Requires handling and disposal of liquid solvents.

[0060] 2. Gas Phase Loading (Vapor-Solid Interaction) :

[0061] - Process: Dihalomethane vapors are passed over activated carbon until the desired loading is achieved.

[0062] - Advantages: Cleaner process with no need for solvents, and typically faster than liquid impregnation. It is also more suitable for large-scale applications. - Disadvantages: May result in less uniform loading compared to liquid impregnation, and requires careful control of vapor flow and concentration.

[0063] The composition may further comprise a liquid stabiliser component for further improving stability. The liquid stabiliser component is suitably also adsorbed onto said activated carbon. The liquid stabiliser component may be selected from the group consisting of glycerin, polyethylene glycol, glycerol, monopropylene glycol (MPG), glyceryl polyethylene glycol ricinoleate, mono-, di- and triglycerides of C6-C20 fatty acids, vegetable oils such as sunflower oil, and mixtures thereof. Of these, polyethylene glycol (of various molecular weights), propylene glycol, glycerin and glycerol are preferred. More preferable is a mixture of glycerin and glyceryl polyethyleneglycol ricinoleate.

[0064] Liquid stabiliser components are particularly useful when the solid carrier is selected from lignin or sepiolite. In other words, a composition is provided for reducing methane production in a ruminant, said composition comprising one or more dihalomethanes adsorbed onto a solid carrier selected from lignin or sepiolite, said one or more dihalomethanes being selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane, and wherein said composition further comprises a liquid stabiliser component.

[0065] The total dihalomethane content in the composition is suitably 12 wt% or less, preferably 10 wt% or less, preferably between 0.1 and 5 wt %, preferably between 0.5 and 2 wt%, preferably between 1.5 and 5 wt%, preferably between 3 and 5 wt%. For example, in one aspect, the dibromomethane content in the composition is 3-5 wt%.

[0066] After incubation of a homogenized sample of the composition for a period of 2h in demineralized water at 23°C, the concentration of said dihalomethanes in the demineralized water is suitably at least 100 pg / L, preferably at least 500 pg / L. After incubation of a homogenized sample of the composition for a period of 24h in demineralized water at 23°C - the concentration of said dihalomethanes in the demineralized water is suitably at least 500 pg / L, preferably at least 750 pg / L. These tests demonstrates that high release of dihalomethanes can be achieved in an in vitro test.

[0067] After incubation of a homogenized sample of the composition for a period of 30 minutes at 30°C - the concentration of said dihalomethanes in the headspace above said sample is suitably less than 100 pg / dm3, preferably less than 50 pg / dm3, more preferably less than 20 pg / dm3. This test demonstrates that the volatility of the dihalomethanes can be controlled, and thereby storage stability can be increased. The dihalomethane(s) should be released in rumen ingesta with typically value of pH 5.5 to 7.5 within a relatively short time, preferably within 4 hours.

[0068] The composition may be formed by adsorbing the dihalomethane(s) of interest onto solid carrier. Liquid stabiliser component(s) may be added as required. Adsorption can result from one or more steps of mixing, spray drying, fluid bed drying, and spray cooling, as known to a person skilled in the art. Suitably, the composition is provided in a one-step process in which dihalomethane(s) of interest are mixed with solid carrier, optionally in the presence of liquid stabiliser component(s). The composition is suitably in powder form.

[0069] The composition should preferably be stable in sealed bags for a period of at least 24 months.

[0070] The composition should be used supplemented to animals through a feed premix for a ruminant, said feed premix comprising the composition described herein.

[0071] The composition should be stable in combination with a feed premix - including micro- and microminerals and other components - for at least 6 months. The feed premix is preferably a ruminant base mix, such as a mineral premix or a vitamin premix comprising vitamins and minerals. Vitamin and mineral premixes are designed to provide ruminants with all their nutritional needs. Non-limiting commercially available examples thereof include fat-soluble vitamins as A, D and E, trace minerals such as manganese, zinc, cobalt, iron, iodine and selenium, macro-minerals as calcium, phosphorous and sodium.

[0072] In an embodiment, the composition is admixed to a total mixed ration (TMR) or partial mixed ration (PMR). In a TMR all dietary components, e.g. forage, silage and concentrate, are mixed before serving. Forage is a plant material, silage is grass or other green fodder made from green foliage crops which have been preserved such as by acidification, or achieved through fermentation. Concentrate refers to a product mainly consisting of cereals, such as, but not limited to, barley, maize, wheat, but may also include protein-rich feed ingredients such as soybean, rapeseed, and sunflower. The composition should be stable in combination with other feed components, eg. a total mixed ration for around 24 hours. The TMR feed will have dry matter percent of typically 40 to 60 percent. In a PMR ration, the main part of the ration, is mixed before serving. Additional feed, typically the concentrates, are feed beside the main ground ration.

[0073] The composition could be included in a feed bolus. A feed bolus will release a small amount of a dihalomethane over a period. This solution is especially relevant in pastoral farming systems. In an embodiment, the composition reduces methane emission by at least 5%, preferably by at least 8%, more preferably by at least 12%, more preferably by at least 15%, more preferably by at least 18%, more preferably by at least 20%, more preferably by at least 21% per ruminant per day.

[0074] The in vivo effect of adding dibromomethane to the TMR - in terms of methane production reduction - and a method of testing this effect are described in Example 4 further below.

[0075] The invention provides the following process: a. adsorption of dihalomethane onto solid carrier selected from activated carbon, lignin or sepiolite to provide a composition according to the invention, b. optionally, stabilising the composition by adding a liquid stabiliser component to said composition, c. optionally, changing the physical form of the composition from step a. or the stabilised composition of step b., e.g. by granulation or pelletisation, d. optionally, coating the composition (e.g. the granules or pellets) from step c. with a coating material, e. optionally, producing a bolus from the coated granules or pellets from step d..

[0076] The liquid stabilizer components which may be used in step b. may be those listed above.

[0077] Changing the physical form of the composition by e.g. granulation or pelletisation (step 3 above) increases the composition volume while decreasing the surface area, which can lead to increased stability and reduced volatility of the dihalomethane(s).

[0078] The purpose of coating the granules or pellets is to reduce the evaporation of the dihalomethane (DHM) and increase the shelf life of the product. The coating applied must be formulated and created so that the DHM is released into the rumen where it plays its role in inhibiting methanation. The following materials can be applied alone or in mixtures to achieve the indicated objectives.

[0079] The application of the coating can take place in different ways: in pans, by spray, in extrusion, in fluidized beds; hot or cold; in batch systems or in continuous systems; through the use of solutions or dry coating.

[0080] Usable materials, by categories:

[0081] Oils or fats: o Triglycerides o Monoglycerides (preferred as they dissolve well in the rumen) o Free fatty acids; both saturated and mono- or polyunsaturated considering the softening and melting point o Carbohydrates:

[0082] ■ Monosaccharides or disaccharides (by application of solutions and syrups);

[0083] ■ Oligosaccharides (for example, maltodextrins and cyclodextrins)

[0084] ■ Starch polysaccharides

[0085] ■ Non-starch polysaccharides

[0086] ■ Cellulosic: Hydroxy Propyl Methyl Cellulose "HPMC"

[0087] • Hydroxy Propyl Cellulose "HPC"

[0088] • Methyl Hydroxy Ethyl Cellulose "MHEC"

[0089] • Methyl Cellulose

[0090] Ethyl Cellulose

[0091] Sodium Carboxy Methyl Cellulose "CMC Sodium • Cellulose Acetate

[0092] Waxes: o Castor wax o Bees wax o Parafin wax o Carnauba wax

[0093] Pectine:

[0094] Chitosan

[0095] Alginates

[0096] Synthetic polymers: o Vinyls

[0097] ■ PVP: polyvinylpyrrolidone o Glycols

[0098] ■ Polyethylene Glycols o Acrylates

[0099] ■ Dimethylaminoethyl Methacrylate (Eduragit E)

[0100] One goal of the coating material is to reduce the solubility of the composition in the rumen.

[0101] A method for reducing methane production in a ruminant is also provided, said method comprising the step of administering the composition, or the feed premix, as described herein, to said ruminant. In one aspect, said method comprises the step of administering the composition such that said one or more dihalomethanes are provided to said ruminant in an amount of 5-50 mg / kg feed DM, such as 10-50 mg / kg feed DM, such as 10-40 mg / kg feed DM, such as 10-30 mg / kg feed DM, such as 10-25 mg / kg feed DM, such as 10-20 mg / kg feed DM, such as 10- 15 mg / kg feed DM, such as 12-15 mg / kg feed DM, such as an amount in the range of 8-15 mg / kg feed DM.

[0102] In one aspect, said method comprises the step of administering the composition such that said one or more dihalomethanes are provided to said ruminant in an amount of 15-25 mg / kg feed DM, such as 17-23 mg / kg feed DM, such as about 20 mg / kg feed DM.

[0103] In one aspect, said method comprises the step of administering the composition such that dibromomethane is provided to said ruminant in an amount of 1-32 mg / kg feed DM, e.g. 3- 32 mg / kg feed DM, such as 6-32 mg / kg feed DM, such as 6-26 mg / kg feed DM, such as 6-19 mg / kg feed DM, such as 6-16 mg / kg feed DM, such as 6-13 mg / kg feed DM, such as 6-10 mg / kg feed DM, such as 8-10 mg / kg feed DM, such as an amount in the range of 5-10 mg / kg feed DM.

[0104] In one aspect, said method comprises the step of administering the composition such that dibromomethane is provided to said ruminant in an amount of 15-25 mg / kg feed DM, such as 17-23 mg / kg feed DM, such as about 20 mg / kg feed DM.

[0105] In one aspect, said method comprises the step of administering the composition such that diiodomethane is provided to said ruminant in an amount of 2-50 mg / kg feed DM, e.g. 5-50 mg / kg feed DM, such as 10-50 mg / kg feed DM, such as 10-40 mg / kg feed DM, such as 10- 30 mg / kg feed DM, such as 10-25 mg / kg feed DM, such as 10-20 mg / kg feed DM, such as 10-15 mg / kg feed DM, such as 12-15 mg / kg feed DM, such as an amount in the range of 8- 15 mg / kg feed DM.

[0106] In one aspect, said method comprises the step of administering the composition such that bromoiodomethane is provided to said ruminant in an amount of 2-41 mg / kg feed DM, e.g. 4-41 mg / kg feed DM, such as 8-41 mg / kg feed DM, such as 8-33 mg / kg feed DM, such as 8- 25 mg / kg feed DM, such as 8-21 mg / kg feed DM, such as 8-16 mg / kg feed DM, such as 8-12 mg / kg feed DM, such as 10-12 mg / kg feed DM, such as an amount in the range of 7-12 mg / kg feed DM.

[0107] In one aspect, said method comprises the step of administering the composition such that chloroiodomethane is provided to said ruminant in an amount of 1-33 mg / kg feed DM, e.g. 3- 33 mg / kg feed DM, such as 7-33 mg / kg feed DM, such as 7-26 mg / kg feed DM, such as 7-20 mg / kg feed DM, such as 7-16 mg / kg feed DM, such as 7-13 mg / kg feed DM, such as 7-10 mg / kg feed DM, such as 8-10 mg / kg feed DM, such as an amount in the range of 5-10 mg / kg feed DM.

[0108] The total amount of the composition to be supplemented to the ruminant is preferably around 10 to 20 grams pr day pr animal as this is practically manageable and a typical volume for a feed additive.

[0109] The daily amount of the active dihalomethane to be supplemented the ruminants is likely to be 7 mg pr kg dm intake. Assuming a cow eats around 20 kg dm feed pr day, the total daily amount of dihalomethane would be around 150 mg.

[0110] In one aspect, the daily amount of dihalomethane, e.g. dibromomethane, to be supplemented to the ruminant is 10-25 mg / kg DM intake, such as 17-23 mg / kg DM intake, such as around 20 mg / kg DM intake.

[0111] EXAMPLES

[0112] In the process of developing the composition, several solids and liquids and their combinations were scanned to study properties of evaporation, absorption and release pattern. The methods of evaluating include Thermogravimetric Analysis (TGA) to estimate absorption and release pattern of dihalomethane. GC-MS analysis will be used to determine evaporation properties (Head Space Analysis) and release pattern of dihalomethane in neutral solution mimicking rumen digesta.

[0113] Example 1. Thermogravimetric analysis release of dihalomethane in liquid and evaporation head space analysis

[0114] Technique: TGA measures the changes in the weight of a specimen measured while its temperature is increased. Moisture and volatile contents of a sample can be measured by TGA.

[0115] Conditions: All the analyses have been carried out from room temperature to 350°C, with a ramp of 10°C / min, under a constant flow of gaseous nitrogen (to avoid combustion of the components).

[0116] Instruments: Perkin-Elmer TGA800 equipped with a Pt-crucible (inert material). Nitrogen flux: 80mL / min.

[0117] Sampling : The free adsorbent and a combination of 90 % absorbent-i- 10 % DBM was tested for each adsorbent. From each sampling a trace showing the relationship between temperature and weight was compiled into a plot. The first derivative of the plot indicates inflection point (T-max) with biggest loss in weight (w-loss). For each adsorbent and 90 % adsorbent + 10 % DBM combination the T-max and w-loss value were used for summarizing the results The tested adsorbents were silica, zeolite, bentonite, sepiolite, activated carbon, corncob and lignin.

[0118] Dibromomethane (DBM) has a boiling point of 96.95°C. The main interest is therefore to see the weight change above this temperature.

[0119] Table 1. Weight change and corresponding highest maximum in weight change (T-max) for potential adsorbents with and without 10 % DBM.

[0120] It can be observed in Table 1 that for the adsorbent silica, zeolite and bentonite the w-loss and T-max are very similar for free adsorbent compared the adsorbent + DBM combination.

[0121] This suggests that the DBM not is absorbed or released in these compositions. For the sepiolite, there is absorption of DBM as there is a higher weight change when the DBM is included. This weight change is - however - happening at low temperature (T-max = 80 Celcius).

[0122] The activated carbon shows a release of 8.4 % at T-max 162 degree celcius. This demonstrates that the DBM has been absorbed and can be released in activated carbon.

[0123] The ligning including DBM shows a local weight decrease of 4.8 % at 131 degrees Celsius and another local decrease of 11.6 % at 183 degrees Celsius, suggesting a further release of DBM.

[0124] The TGA analysis demonstrated that the adsorbents carbon, sepolite and lignin comprising adsorbed DBM show a considerable weight change in the temperature range 100-200 degrees. This is not observed in the free adsorbent, suggesting a release of DBM, indicating their suitability as adsorbents.

[0125] Figures 1-6 show TGA curves of samples of activated carbon, sepiolite, and lignin as adsorbents, with and without DBM. The results (weight (w) loss in % and Tmax in °C) are summarised in the Table 1 above. The weight loss is the percentage of mass lost by the sample as a function of temperature. Tmax is the temperature at which the rate of weight loss is maximum.

[0126] Figure 1 shows a TGA curve of a sample of activated carbon.

[0127] Figure 2 shows a TGA curve of a sample of activated carbon with 10 % DBM.

[0128] Figure 3 shows a TGA curve of a sample of sepiolite.

[0129] Figure 4 shows a TGA curve of a sample of sepiolite with 10 % DBM.

[0130] Figure 5 shows a TGA curve of a sample of lignin.

[0131] Figure 6 shows a TGA curve of a sample of lignin with 10 % DBM.

[0132] Example 2. Release pattern and head space analysis of various solid adsorbents using GC-MS analysis.

[0133] A. Release pattern of dibromomethane in water 4 adsorbents (silica, activated carbon, clinoptilolite and sepiolite) were tested for their ability to release dibromomethane (DBM) in neutral water. Four bottles were prepared including 98.5 % adsorbent + 1.5 % dibromomethane. A homogeneous sample of the composition was placed in a container containing milliQ water. The incubation time was 2 and 24 hours. Each combination was repeated 3 times. Thus, a total of 24 bottles were prepared. Gas Chromatography-Mass Spectrometry (GC-MS) was performed to measure the concentration of dibromomethane, and the results are provided in Table 2.

[0134] DBM

[0135] Concentration(ug / L) release, %

[0136] Mean St. dev.

[0137] Silica 2 17.8 0.3 0.8

[0138] 24 89 4 4.2

[0139] Activated Carbon 2 578 6 27.5

[0140] 24 900 10 42.9

[0141] Clinoptilolite 2 32.2 0.2 1.5

[0142] 24 55 2 2.6

[0143] Sepiolite 2 52 3 2.5

[0144] 24 236.5 0.7 11.3

[0145] Table 2. Concentration of DBM after 2 and 24 hours in liquid for combination of 98,5 % of silica, activated carbon, clinoptilolite and sepiolite combined with 1.5 % DBM.

[0146] The results in Table 2 show that the concentration of DBM was significantly higher for the activated carbon compared to all other solid adsorbents. For example, at 2 hours the release was 27.5 percent for activated carbon and below 2.5 percent for all other adsorbents. At 24 hours the DBM release was 42.9 percent for activated carbon but lower than 12 percent for all other treatments. This demonstrates a higher release of DBM in neutral liquid.

[0147] As appears from the results in Table 2 as well, the concentration of DBM was higher for sepiolite compared to all other solid absorbents apart from activated carbon. For example, at 2 hours, the release was 2.5 percent for sepiolite and 1.5 percent or less for all other adsorbents (excluding activated carbon). At 24 hours, the DBM release was 11.3 percent for sepiolite and 4.2 percent or less for all other treatments (excluding activated carbon).

[0148] B. Head space analysis Gas Chromatography-Mass Spectrometry (GC-MS) with headspace injection is abbreviated as HS / GC-MS. It is a method that allows first to extract compounds present in the surrounding air. Then, the extracted compounds are analyzed by gas chromatography coupled to mass spectrometry. The compounds are thus identified and analyzed in a qualitative, quantitative and semi-quantitative way.

[0149] This method is particularly adapted for the analysis of volatile compounds, such DBM.

[0150] SAMPLE PREPARATION : A homogenized sample of a composition comprising DBM adsorbed onto activated carbon is placed in an empty vial; heated at 30°C for 15 minutes and then the air in the vial is sampled and injected into the gas chromatograph for GC-MS analysis.

[0151] DBM release,

[0152] Concentration(ug / L) %

[0153] Mean St. dev.

[0154] Silica 3.3 0.1 0.2

[0155] Activated Carbon 10 2 0.7

[0156] Clinoptilolite 22 5 1.5

[0157] Sepiolite 552 20 36.8

[0158] Table 3. Head space concentrations of DBM

[0159] The results in table 3 show low concentrations of DBM indicating low evaporation of DBM for silica, activated carbon, clinoptilolite but high (36.8 percent) concentrations of DBM for sepiolite. Low head space values can be obtained if either the adsorbent has not absorbed the DBM or a high interaction between adsorbent and DBM has been reached. As the release of DBM in water and head space values were low for clinoptilolite and silica, this suggests that DBM only was absorbed in a very small amount. The high head space value for sepiolite demonstrate that sepiolite has a high capacity for DBM absorption.

[0160] Example 3. Head space analysis of liquid stabiliser components combined with DBM and solid carriers

[0161] A solid carrier (activated carbon and sepiolite) mixed with liquid stabiliser component (propylene glycol, polyethylene glycol, glyceryl polyethylene glycol ricinoleate, sunflower oil and glycerol) were tested for their for evaporation properties. Head space analysis was used for evaluating evaporation. The tested carrier material was mixed in the ratio of 90 % solid carrier + 8.5 % liquid material + 1.5 % dibromomethane. A homogenized sample was placed in an empty vial; heated at 30°C for 15 minutes and then the air in the vial is sampled and injected into the gas chromatograph for GC-MS analysis.

[0162] Concentration DBM release

[0163] (ug / L) %

[0164] Liquid stabiliser

[0165] Solid component Mean St. dev.

[0166] Activated Carbon 14.7 0.2 1

[0167] Activated Carbon Polyethylene glycol 4 18.1 0.3 1.2

[0168] Activated Carbon Polyethylene glycol 15 9.99 0.05 0.7 polyethylene glycol

[0169] Activated Carbon ricinoleate 9.1 0.3 0.6

[0170] Activated Carbon Sunflower oil 12.8 0.4 0.9

[0171] Sepiolite No liquid 520 10 34.8

[0172] Sepiolite Propylene glycol 360 10 24

[0173] Sepiolite Polyethylene glycol 4 510 10 34.3 polyethylene glycol

[0174] Sepiolite ricinoleate 466 8 31.1

[0175] Sepiolite Sunflower oil 249 8 16.6

[0176] Sepiolite Glycerol 199 4 13.1 Table 4. Head space concentration of DBM

[0177] Table 4 shows overall a significant lower DBM head space concentrations for the activated carbon compared to sepiolite. Head space values for activated carbon and DBM without liquid agent did have low head space concentrations. Adding a liquid stabiliser component to the adsorbent and DBM mixture showed that the evaporation was still low but differences between liquid stabilisers could not be concluded due to low variation in the analyzed concentrations. A higher variation of the evaporation reducing agent were found for the sepiolite data which enable a clearer insight into the effect of the liquid agents. The evaporation profiles for the sepiolite demonstrates that propylene glycol, glycerol, polyethylene glycol ricinoleate and sunflower oil can reduce evaporation of DBM. Example 4. In vivo demonstration of efficacy of a composition The efficacy of a composition was tested in vivo in a 4x4 latin square design. Four periods of 14 days (d) using four Danish Holstein cows. In the first period, no dibromomethane was added to the TMR, while either 8, 16, or 20 mg dibromomethane / kg feed DM were added to the TMR in the three remaining periods in a change-over design. The treatment consisted of 4 different levels (CON, LOW, MED, and HIGH) of dibromomethane (0, 7, 14 and 21 mg / kg feed DM). The control diet (0 mg / kg feed DM) consisted of corn silage, grass / clover silage, dried sugar beet pulp, barley, rapeseed cake, soybean meal, and minerals. Dibromomethane was added to the remaining treatments by use of a composition consisting of 1.5 % dibromomethane, 87.5 % activated carbon, 1.5 % glyceryl polyethyleneglycol ricinoleate, and 9.5 % glycerin. All diets were fed on an ad libitum basis. Gas was measured in individual respiratory chambers.

[0178] A statistical analysis was performed on the experimental results. The methodology employed for the analysis was substantially similar to the statistical approach described by Thorsteinsson et al., 2023 in Journal of Dairy Science 106:6921-6937. In brief, in the statistical analysis, observations of all variables were averaged within cow and period. The effect of diet on the various animal responses was analysed with a linear mixed model:

[0179] Ytpc=p "F Gd T Yp T Ac T E pc, where Ypcis the dependent response variable, p is the overall mean, a is the fixed effect of diet (d = CON, LOW, MID, or HIGH), y is the fixed effect of period (p = 1 to 4), A is the random effect of cow (c = 1 to 4), and Epcis the random residual error assumed to be independent with constant variance and normally distributed.

[0180] The experimental results and the results of the statistical analysis are shown in Table 5 below. As appears, the composition significantly reduced the methane emissions with up to 23 % per cow per day. CH4yield per kg DMI and per kg ECM was likewise significantly reduced. No significant effects were observed on DMI and ECM.

[0181] Treatments P-value

[0182] Item CON LOW MED HIGH SEM Treatment Linear Quadratic

[0183] DMI, kg / d 21.6 22.4 21.7 20.2 1.33 0.40 0.24 0.23

[0184] ECM, kg / d 29.3 29.4 28.4 28.0 2.69 0.30 0.09 0.71

[0185] Gas exchange, g / d

[0186] CH4357a370a337ab274b19.4 0.01 <0.01 0.03

[0187] CO214486 15133 14566 14236 870 0.41 0.44 0.22

[0188] O210766 11139 10649 10558 694 0.42 0.35 0.38

[0189] H20.753b1.70b4.00ab9.42a1.36 <0.01 <0.01 0.10

[0190] Respiration 0.983 0.991 1.00 0.983 0.0143 0.42 0.76 0.16 coefficient

[0191] Gas yield, g / kg DMI

[0192] CH416.5 16.6 15.6 13.8 1.01 0.08 0.02 0.22

[0193] CO2670 675 671 711 16.5 0.31 0.15 0.32

[0194] O2498 497 490 528 14.6 0.26 0.20 0.18

[0195] H20.0353b0.0756b0.1928ab0.4976a0.0777 0.01 <0.01 0.10

[0196] Gas yield, g / kg ECM

[0197] CH412.3 13.1 12.0 10.4 1.31 0.06 0.03 0.08

[0198] CO2502 534 521 528 51.5 0.47 0.35 0.42

[0199] O2373 394 381 393 41.1 0.52 0.38 0.69

[0200] H20.0266b0.0619b0.155ab0.328a0.0539 <0.01 <0.01 0.15

[0201] Table 5. Abbreviations: Dry matter intake ("DMI"); energy corrected milk ("ECM"); day ("d"). DMI, ECM and Gas yield from lactating cows supplemented a dibromomethane containing feed additive. Cows were supplemented (CON; 0 mg / kg feed DM, LOW; 7 mg / kg feed DM, MED; 14 mg / kg feed DM or HIGH; 21 mg / kg feed DM).a bValues within the same row with different superscripts differ significantly (P < 0.05).

Claims

CLAIMS1. A composition for reducing methane production in a ruminant, said composition comprising one or more dihalomethanes adsorbed onto a solid carrier selected from activated carbon, lignin or sepiolite, said one or more dihalomethanes being selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane.

2. The composition according to claim 1, wherein the solid carrier is activated carbon, preferably wood-based activated carbon.

3. The composition according to any one of the preceding claims, wherein said composition is in powder form.

4. The composition according to any one of the preceding claims, wherein the total dihalomethane content in the composition is 12 wt% or less, preferably between 0.1 and 5 wt %, preferably between 0.5 and 2 wt%, preferably between 1.5 and 5 wt%, preferably between 3 and 5 wt%.

5. The composition according to any one of the preceding claims, wherein said composition further comprises a liquid stabiliser component, preferably wherein said liquid stabiliser component is also adsorbed onto said solid carrier.

6. The composition according to claim 5, wherein the liquid stabiliser component is selected from the group consisting of glycerin, polyethylene glycol, glycerol, monopropylene glycol (MPG), glyceryl polyethylene glycol ricinoleate, mono-, di- and triglycerides of C6-C20 fatty acids, vegetable oils such as sunflower oil, and mixtures thereof.

7. The composition according to any one of claims 5-6, wherein said composition, including said liquid stabiliser component, is in granulate or pellet form.

8. The composition according to any one of the preceding claims, being further coated in a coating material, e.g. a polymeric material.

9. The composition according to any one of the preceding claims, wherein - after incubation of a homogenized sample of the composition for a period of 2h in demineralized water at 23°C - the concentration of said dihalomethanes in the demineralized water is at least 100 pg / L, preferably at least 500 pg / L.

10. The composition according to any one of the preceding claims, wherein - after incubation of a homogenized sample of the composition for a period of 24h in demineralized water at 23°C - the concentration of said dihalomethanes in the demineralized water is at least 500 pg / L, preferably at least 750 pg / L.

11. The composition according to any one of the preceding claims, wherein - after incubation of a homogenized sample of the composition for a period of 30 minutes at 30°C - the concentration of said dihalomethanes in the headspace above said sample is less than 100 pg / dm3, preferably less than 50 pg / dm3, more preferably less than 20 pg / dm3.

12. The composition according to any one of the preceding claims, wherein the composition reduces methane emission by at least 5%, preferably by at least 8%, more preferably by at least 12%, more preferably by at least 15%, more preferably by at least 18%, more preferably by at least 20%, more preferably by at least 21% per ruminant per day.

13. A feed premix for a ruminant, said feed premix comprising the composition according to any one of claims 1-12.

14. A method for reducing methane production in a ruminant, said method comprising the step of administering the composition according to any one of claims 1-12, or the feed premix according to claim 13, to said ruminant.

15. The method according to claim 14, said method comprising the step of administering the composition such that said one or more dihalomethanes are provided to said ruminant in an amount of 15-25 mg / kg feed DM, such as 17-23 mg / kg feed DM, such as about 20 mg / kg feed DM.

16. A process for formation of the composition according to any one of claims 1-12, said process comprising the steps of: a. adsorption of dihalomethane onto solid carrier selected from activated carbon, lignin or sepiolite, b. optionally, adding a liquid stabiliser component to said composition, c. optionally, granulating or pelletising the composition from step a. or the stabilised composition of step b., d. optionally, coating the granules or pellets from step c. with a coating material, e. optionally, producing a bolus from the coated granules or pellets from step d..

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