Compositions, systems, and methods of reducing methane emissions

Abstract

The present disclosure provides for compositions, for example, anti-methanogenic compositions comprising a: i) a haloform; and ii) a high surface area material. The present disclosure further provides a process for preparing compositions, for example, anti-methanogenic compositions and associated methods for reducing methane emission.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit ofU.S. Provisional Patent Application No. 63 / 615,422, filed on December 28, 2023, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD The present disclosure relates to the use of high surface area materials as a platform for stabilizing bromoform formulations to inhibit methanogenesis in ruminants. Compositions, methods of preparing compositions, and systems are further provided. BACKGROUND In the realm of livestock management, one of the significant challenges is the control of methane emissions. Ruminants, such as cows, sheep, and goats, are known to produce methane as a byproduct of their digestive process. This methane is released into the atmosphere, contributing to greenhouse gas emissions and global warming. Furthermore, the production of methane also represents a loss of energy for the animal, as the energy contained in the methane could have been used for growth or milk production. Bromoform is being commercialized as a chemical agent to reduce methane emissions arising from ruminant livestock. Unfortunately, aqueous solutions of bromoform are unstable due to a combination of having an unfavorable air / water partition coefficient (due to weak water / bromoform intramolecular forces) and a relatively low boiling point leading to significant volatility. Asparagopsis seaweed represents a natural source of bromoform, and extracts into food oils (e.g., canola oil) have been used to improve the shelflife of bromoform as a feed supplement (Algal Research, Volume 51, October 2020, 102065, the contents of which are incorporated by reference in their entirety). The intermolecular forces between bromoform and hydrophobic molecules comprising the food oil are comparatively much stronger leading to markedly reduced volatility. Unlike 3-Nitrooxypropanol (3-NOP) which can undergo a hydrolysis reaction that impacts its shelflife ((EFSA Journal 2021; 19( 11):6905, the contents of which are incorporated by reference in their entirety), the shelflife of these bromoform-containing supplements is generally less impacted by chemical reactions but instead influenced predominantly by their volatility. However, bromoform is reactive under strongly alkaline or in the presence of certain strongly nucleophilic amines (Journal of Pharmaceutical Sciences, Volume 54, Issue 12, December 1965, 1736-1739, the contents of which are incorporated by reference in their entirety). Given the strong dependence of the rate of volatilization on the air / liquid surface area and convective air flow (whether laminar or turbulent) (Water Research, 32, 7, July 1998, 2106-2112, the contents of which are incorporated by reference in their entirety), food oil solutions of haloforms are not a robust methodology for stabilizing bromoform in a solid-based feed supplement likely to be used in a ruminant diet. Furthermore, canola oil has a very high energy value; it is often an ingredient in solid-based feed but has minimal applications on its own. Furthermore, it is often used in cattle feed manufacturing for dust suppression, improved moisture retention, and feed / equipment friction reduction. Importantly, bromoform-infused oils are relatively stable compared to aqueous solutions; however, dispersion of these oils onto a solid will greatly increase the air / liquid interfacial area which leads to a dramatic increase in volatilization. Various materials are being explored for their potential to stabilize bromoform in a manner suitable for administration to ruminants. Ideally, the material would bind bromoform sufficiently to stabilize it long enough to be fed to the animal and then allow for its ready release in the rumen where it can then inhibit methanogenesis. While zeolites are common sorbents used for VOCs (i.e., bromoform), they are typically hydrophilic and favor adsorption of water over VOCs (Microporous and Mesoporous Materials, 294, 109869, the contents of which are incorporated by reference in their entirety). As such, there is a need to identify suitable materials to stabilize bromoform prior to administration to an animal and then allow its release within the animal’s rumen. SUMMARY Activated carbon (or activated charcoal) exhibits significant VOC sorption capacity with bromoform in aqueous media (Environmental Science and Technology, 2019, 53, 5, 2647-2659, the contents of which are incorporated by reference in their entirety). Within the context of bromoform, activated carbon is currently used in water treatment applications where bromoform is removed from water through a sorption process (Toxicological and Environmental Chemistry, 63, 1997, 227-231, the contents of which are incorporated by reference in their entirety). This approach can typically remove bromoform to low ppb levels. Activated carbon is also used in gas sampling systems for environmental monitoring applications. In this application, bromoform can be sampled by then allowing it to sorb onto a quantity of activated carbon (Sandia Report, SAND2006-2447). Elevated temperatures (i.e., approximately 400 °C) are then used to quickly desorb the contents for gas chromatography analysis. Other high surface area carbon-based sorbent resin materials such as highly or hyper-crosslinked aromatic polymers can also be similarly used (Macromolecules 2017, 50, 4132-4143, the contents of which are incorporated by reference in their entirety). With both types of materials, the strong van der Waals surface interactions between bromoform and the carbon surface initially exhibit Langmuir isotherm behavior at low levels and more closely follow Freundlich or mixed isotherm behavior at higher concentrations. The present disclosure addresses a practical need for stabilized forms of bromoform for use in reducing methane production in ruminants without negatively affecting their health or productivity. As bromoform has as a relatively narrow therapeutic window (i.e., a sufficiently large dose to impart a significant methane reduction and small enough to eliminate or minimize the toxicity), it is important that a stabilized form of bromoform is used in practice in order to accurately dose bromoform in a practical setting. The advantage of using these materials is that they can be coupled with various bromoform synthesis strategies (either a synthetic or biologically mediated pathway) using either a solid-liquid or solid-vapor extraction technique; with this approach, the health and safety concerns with handling bromoform as a liquid solvent can be minimized. Furthermore, by reducing bromoform’s volatility during ruminant ingestion, the amount of bromoform inhaled (which results in an undesired general anesthetic effect) is reduced; the release of bromoform can be re-directed into the rumen where it can efficiently inhibit methanogenesis. In some aspects, the present disclosure relates to an approach where a haloform, such as, bromoform is thermally stabilized onto a high surface area carbon-based sorbent material through strong physical adsorption (i.e. van der Waals interactions) and / or absorption (i.e., swelling). As predicted by the shape of any isotherm, the equilibrium release characteristics will be favored with a higher concentration. For example, 10 grams of 10 wt.% loaded material will have a different methane reduction response compared to 100 gram of a 1 wt.% loaded material despite the same amount of bromoform being present in both scenarios. These materials will strike a balance between reducing volatility in the air while still providing sufficient release in an aqueous environment; this strategy is supported by literature finding showing that water vapor will cause a reduction in sorption capacity (Advances in Environmental Research, 4, 4, November 2000, 357362, the contents of which are incorporated by reference in their entirety). In some embodiments, a composition is provided, comprising, consisting of, or consisting essentially of: i)     a haloform; and ii)     a high surface area material; wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, ion exchange resins based on styrene and / or divinylbenzene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-based polymer, silica-based polymer, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof; and the haloform is physically sorbed onto the high surface area material. In some embodiments, a composition is provided, comprising, consisting of, or consisting essentially of: i)     a haloform; and ii)     a high surface area material; wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, ion exchange resins based on styrene and / or divinylbenzene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-based polymer, silica-based polymer, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof; if the high surface area material is a polymer then the polymer is cross-linked; and the haloform is physically sorbed onto the high surface area material. In an aspect, the compositions described herein are anti-methanogenic compositions. In some embodiments, the high surface area material is a carbonaceous material selected from the group consisting of activated carbon (also known as activated charcoal), graphene, graphene oxide, and derivatives thereof. In some embodiments, the carbonaceous material is an activated carbon selected from the group consisting of powdered activated carbon (PAC), granular activated carbon (GAC), extruded activated carbon (EAC), bead activated carbon (BAC), impregnated carbon, polymer coated carbon, woven carbon, and combinations thereof. In some embodiments, the activated carbon is granular activated carbon (GAC). In some embodiments, the activated carbon is derived from coconut shells, peat, coal, rice husks, wood, sawdust, corn cobs, or fruit pits. In some embodiments, the activated carbon is pharmaceutical-grade activated carbon, food-grade activated carbon, industrial activated carbon, or environmental activated carbon. In other embodiments, the composition further comprises a cyclodextrin, wherein the cyclodextrin has been sorbed onto the activated carbon prior to addition of the haloform to the composition and the haloform adsorbs preferentially to the cyclodextrin. In some aspects, the cyclodextrin is an alpha, beta, or gamma-cyclodextrin. In other aspects, the cyclodextrin is a water insoluble highly crosslinked cyclodextrin based polymer. In one aspect, the polymer is an epichlorohydrin-cyclodextrin polymer. In another aspect, the polymer is an epichlorohydrin and / or trimethylolpropane glycidyl ether crosslinked-cyclodextrin polymer In some embodiments, the cyclodextrin is sorbed onto the activated carbon through a water deposition method where the activated carbon is exposed to a saturated water solution of the cyclodextrin and then dried leaving the activated carbon coated in the cyclodextrin. In certain aspects, the cyclodextrin is sorbed onto the activated carbon at a concentration of about 100 mg / g to about 700 mg / g. In some embodiments, the high surface area material is selected from the group consisting of Amberlite (Rohm and Haas) XAD-2, XAD-4, XAD-7, XAD-16, XAD-18, XAD-1180, XAD-1600, XAD-2000, XAD-2010; Amberchrom (Toso Haas) CG-71m, CG-71c, CG-161m, CG161c; Diaion Sepabeads (Mitsubishi Chemicals) HP20, SP206, SP207, SP850, HP2MG, HP20SS, SP20MS; Dowex (Dow Chemical) XUS-40285, XUS-40323, XUS-43493 (also referred to as Optipore V493 (dry form) or Optipore L493 (hydrated form)), Optipore V503, Optipore SD-2; Hypersol Macronet (Purolite) MN-100, MN-102, MN-150, MN-152, MN-170, MN-200, MN-202, MN-250, MN-252, MN-270, MN-300, MN-400, MN-500, MN-502, Purosorb (Purolite) PAD 350, PAD 400, PAD 428, PAD 500, PAD 550, PAD 600, PAD 700, PAD 900, PAD 950, and combinations thereof. In some aspects, the high surface area material is PAD 600. In some embodiments, the haloform is selected from the group consisting of bromoform, chloroform, iodoform, and combinations thereof. In other embodiments, the haloform is bromoform. In some embodiments, the polymer is an organic polymer, an inorganic polymer, or a hybrid polymer, or combinations thereof. In some embodiments, the high surface area material has a surface area of about 1 to about 5000 m2 / g, about 100 to about 5000 m2 / g, about 200 to about 5000 m2 / g, about 300 to about 5000 m2 / g, about 500 to about 5000 m2 / g, about 500 to about 4000 m2 / g, about 500 to about 3000 m2 / g, about 750 to about 3000 m2 / g, about 900 to about 3000 m2 / g, about 1000 to about 3000 m2 / g, or about 500 to about 1500 m2 / g. In other embodiments, the high surface area material has a surface area of 50 to 5000 m2 / g, 100 to 5000 m2 / g, 200 to 5000 m2 / g, 300 to 5000 m2 / g, 500 to 5000 m2 / g, 500 to 4000 m2 / g, 500 to 3000 m2 / g, 750 to 3000 m2 / g, 900 to 3000 m2 / g, 1000 to 3000 m2 / g, or 500 to 1500 m2 / g. In some embodiments, an animal feed or feedstuff comprising, consisting essentially of, or consisting of compositions described herein is provided. In some embodiments, the animal feed or feedstuff further comprises a cereal, starch, vegetable waste, vitamin, mineral, trace element, emulsifier, aromatizing product, binder, colorant, odorant, thickening agent, or a combination thereof. In some embodiments, a method for reducing methane emission is provided, the method comprising administering compositions described herein or the animal feed or feedstuff to an animal in need thereof to elicit the desired response. In other aspects, the disclosure provides for methods of reducing methane emission by administering compositions described herein or the animal feed or feedstuff to a ruminant in an effective amount to reduce methane emission from the ruminant. In some embodiments, the ruminant is a cow, bull, steer, ox, heifer, goat, sheep, giraffe, yak, deer, or antelope. In some embodiments, compositions described herein or / or animal feed or feedstuffs is administered in an amount to deliver about 1 to about 100 mg, about 5 to about 50 mg, or about 10 to about 40 mg of bromoform per kg dry matter intake. In some embodiments, a process for preparing a composition, for example, an anti-methanogenic composition is provided, wherein the process comprises, consists essentially of, or consists of: i) combining an aqueous solution comprising a haloform with a high surface area material to extract the haloform onto the high surface area material; and ii) separating the high surface area material with the extracted haloform from the aqueous solution to prepare the anti-methanogenic composition; wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-based polymer, silica-based polymer, ion exchange resin, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof; and if the high surface area material is a polymer then the polymer is cross-linked. In some embodiments, a process for preparing compositions, for example, an anti-methanogenic composition, is provided, wherein the process comprises: i) bubbling air into an aqueous solution comprising a haloform to form a vapor phase with the haloform; ii) passing the vapor phase with the haloform over a stirred bed of a high surface area material to extract the haloform onto the high surface area material; and iii) collecting the high surface area material with the extracted haloform to prepare the anti-methanogenic composition; wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-based polymer, silica-based polymer, ion exchange resin, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof; and if the high surface area material is a polymer then the polymer is cross-linked. In some embodiments, the stirred bed of high surface area material is continuously mixed to ensure deposition of the haloform onto the high surface area material is substantially uniform. The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and processes that are consistent with the disclosed subject matter as claimed herein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 compares the bromoform retention of the following bromoform formulations: 1) a 2.5 x 10'3 wt.% aqueous solution of bromoform at 20°C; 2) a hyper-crosslinked poly( divinylbenzene) resin bead material (Purolite PAD600) at 10 wt.% loading and temperature of 40°C; 3) an activated charcoal (AC) material (C9154; Sigma-Aldrich) with bromoform 15 wt.% and temperature of 100°C; and 4) an activated charcoal (AC) material (C9154; Sigma-Aldrich) with bromoform 10 wt.% and temperature of 100°C. Despite a higher loading and temperature (both of which would favor a more rapid relative reduction in bromoform content), the PAD600 and AC samples had markedly higher retention even over several weeks while the water solution contained virtually no bromoform after only a few hours. FIG. 2 shows the thermogravimetric analysis results when a 10 wt.% bromoform loaded activated charcoal (C9157, Sigma-Aldrich) is ramped to 400 °C. This material was produced by introducing the activated charcoal into an aqueous solution of bromoform. The weight loss below 100°C corresponds to vaporization of water. The strong retention of bromoform with this material is evident with the incomplete release of bromoform over the short ramping period of less than 2 hours to 400 °C temperature. FIG. 3 shows isotherm curves for bromoform physically sorbed onto activated charcoal powder - C9157 (Sigma-Aldrich) and 161551 (Sigma-Aldrich); activated charcoal granules -C3014 (Sigma-Aldrich); carbon black - Vulcan XC72 (Cabot); or polydivinylbenzene resin beads - PUROSORB™ PAD600 (Purolite). FIGs. 4A-4C depict the daily (mean ± SE) total gas production (mL) (FIG. 4A), methane concentration (mL / 100 mL gas) (FIG. 4B), and methane yield (mL / g dry matter incubated) (FIG. 4C). Day 8 represents the pre-treatment period. PR-L dosage was increased by 50% on Day 16, AC-H was not added from Day 16 onwards. Note in FIG. 4A total gas is similar to control (p>0.05) for all treatments and timepoints apart from treatment AC-H on Day 11 (p<0.05). In FIG. 4B and FIG. 4C all treatments are significantly different to control (p<0.05) unless marked with “+” which indicates that some treatments are not significantly different (p>0.05) to the control (Table 7). FIG. 5 presents the pattern of methane emissions over 24 hours post feeding for steers fed canola meal without an activated carbon (AC) loaded with fungal derived bromoform (control). Group A and B each represent two steers measured over two consecutive days. FIG. 6 presents the pattern of methane emissions over 24 hours post feeding for steers fed canola meal with C9 carrier containing bromoform at nominal 10% (w / w) (i, ii) then bromoform at nominal 20% (w / w) (iii, iv). Group A and B each represent one steer measured over two consecutive days. FIG. 7 presents the pattern of methane emissions over 24 hours post feeding for steers fed canola meal with 16 carrier containing bromoform at nominal 10% (w / w) (i, ii) then bromoform at nominal 20% (w / w) (iii, iv). Group A and B each represent one steer measured over two consecutive days. FIG. 8 presents the pattern of methane emissions over 24 hours post feeding for steers fed canola meal with RP3 carrier containing bromoform at nominal 10% (w / w) (i, ii) then bromoform at nominal 20% (w / w) (iii, iv). Group A and B each represent one steer measured over two consecutive days. DETAILED DESCRIPTION Preferred features, embodiments and variations of the invention may be discerned from the following detailed description which provides sufficient information for those skilled in the art to perform the invention. The detailed description is not to be regarded as limiting the scope of the preceding summary of the invention in any way. In compliance with the statute, the invention has been described in language more or less specific to structural or methodical features. The term “comprises” and its variations, such as “comprising” and “comprised of’ is used throughout in an inclusive sense and not to the exclusion of any additional features. It is to be understood that the invention is not limited to specific features shown or described since the means herein described comprises preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted by those skilled in the art. Throughout the specification and claims (if present), unless the context requires otherwise, the term “substantially” or “about” will be understood to not be limited to the value for the range qualified by the terms. It is to be understood that unless specifically stated otherwise, references to “a,” “an,” and / or “the” may include one or more than one and that reference to an item in the singular may also include the item in the plural. Reference to an element by the indefinite article “a,” “an” and / or “the” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. As used herein, the term “comprise,” and conjugations or any other variation thereof, are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. As used herein, “activated charcoal” is synonymous with “activated carbon” and refers to a form of carbon that is processed (activated) to have small, low-volume pores that increase the surface area available for adsorption or chemical reactions. Activated charcoal may be derived from waste products such as coconut husks and waste from paper mills. These bulk sources are converted into charcoal before being “activated”. A “ruminant” is a mammal of the order Artiodactyla that digests plant-based food by initially softening and partially fermenting it within the animal's first stomach chambers, then regurgitating the semi-digested mass, now known as cud, and chewing it again. The process of rechewing the cud to further break down plant matter and stimulate digestion is called “ruminating”. Ruminants have a digestive tract with four chambers, namely the rumen, reticulum, omasum and abomasum. In the first two chambers, the rumen and the reticulum, the food is mixed with saliva and separates into layers of solid and liquid material. Solids clump together to form the cud, or bolus. The cud is then regurgitated, chewed slowly to completely mix it with saliva, which further breaks down fibers. Fiber, especially cellulose, is broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa and fungi. The broken-down fiber, which is now in the liquid part of the contents, then passes through the rumen into the next stomach chamber, the omasum. The food in the abomasum is digested much like it would be in the monogastric stomach. Digested gut contents are finally sent to the small intestine, where the absorption of the nutrients occurs. Almost all the glucose produced by the breaking down of cellulose is used by the symbiotic bacteria. Ruminants get their energy from the volatile short chain fatty acids (VFAs) produced by the bacteria, namely acetate, propionate, butyrate, valerate, and isovalerate. Ruminants include cattle, goats, sheep, giraffes, yaks, deer, antelope, and others. As used herein, the term “reducing” includes the reduction of amount of substance in comparison with a reference. For example, the reduction in the amount of total gas and / or methane produced by a ruminant animal or animals administered a composition according to the present invention, relative to an animal or animals not administered a composition of the present invention. The reduction can be measured in vitro with an artificial rumen system that simulates anaerobic fermentation, or in vivo with animals confined in respiration chambers. It is within the knowledge and skill of those trained in the art to assess enteric methanogenesis by a ruminant animal. As used herein, the term “reducing methane production” refers to the reduction of methane produced in the gastro-intestinal tract. The term includes the specific volume of methane generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of methane. The present invention aims to reduce this process, such as to reduce the total amount of methane produced in the gastrointestinal tract. It is within the knowledge and skill of those trained in the art to assess methane production by a ruminant animal. High Surface Area Materials High surface area materials are materials with high surface area. Typically, high surface area materials have a surface area of at least 1 m2 / g, as measured by the BET method (Brunauer-Emmett-Teller adsorption method). The high surface area materials applied in the present invention typically have a surface area of 1-5000 m2 / g, optionally 100-5000 m2 / g, optionally 2005000 m2 / g, optionally 300-5000 m2 / g, optionally 500-5000 m2 / g, optionally 500-4000 m2 / g, optionally 500-3000 m2 / g, optionally 750-3000 m2 / g, optionally 900-3000 m2 / g, optionally 10003000 m2 / g, optionally 500-1500 m2 / g. Details of each species of the high surface area materials are further described below. Activated Carbon Activated carbons are complex products which are difficult to classify on the basis of their behaviour, surface characteristics and other fundamental criteria such as pore size (i.e., a combination of microporosity, mesoporosity and microporosity). However, a broad classification is made for general purposes based on their size, preparation methods, and industrial applications. In certain aspects, the activated carbon of the present invention comprises one or more of the following categories. The categories are not mutually exclusive and can be combined (e.g., a pharmaceutical-grade or acid-washed activated carbon derived from wood or coconut shells). Powdered Activated Carbon (PAC) Normally, activated carbons (RI) are made in particulate form as powders or fine granules less than 1.0 mm in size with an average diameter between 0.15 and 0.25 mm. Thus, they present a large surface to volume ratio with a small diffusion distance. Activated carbon (RI) is defined as the activated carbon particles retained on a 50-mesh sieve (0.297 mm). Powdered activated carbon (PAC) material is a finer material. PAC is made up of crushed or ground carbon particles, 95-100% of which will pass through a designated mesh sieve. The ASTM classifies particles passing through an 80-mesh sieve (0.177 mm) and smaller as PAC. It is not common to use PAC in a dedicated vessel, due to the high head loss that would occur. Instead, PAC is generally added directly to other process units, such as raw water intakes, rapid mix basins, clarifiers, and gravity filters. Granular Activated Carbon (GAC) Granular activated carbon (GAC) has a relatively larger particle size compared to powdered activated carbon and consequently presents a smaller external surface. Diffusion of the haloform sorbate is thus an important factor. Granular activated carbons are suitable for adsorption of gases and vapours because gaseous substances diffuse rapidly through the pore structure. They are also suitable for sorption of dissolved low molecular weight organic species which rapidly diffuse through the pore structure as well. Granulated activated carbons are used for air filtration and water treatment, as well as for general deodorization and separation of components in flow systems and in rapid mix basins. GAC can be obtained in either granular or extruded form. GAC is designated by sizes such as 8><20, 12x40, 20x40, or 8x30 for liquid phase applications and 4x6, 4x8 or 4x10 for vapor phase applications. A 20x40 carbon is made of particles that will pass through a U.S. Standard Mesh Size No. 20 sieve (0.84 mm) (generally specified as 85% passing) but be retained on a U.S. Standard Mesh Size No. 40 sieve (0.42 mm) (generally specified as 95% retained). AWWA (1992) B604 uses the 50-mesh sieve (0.297 mm) as the minimum GAC size. The most popular aqueous-phase carbons are the 12x40 and 8x30 sizes because they have a good balance of size, surface area, and head loss characteristics. As opposed to powders, granules may have a lower propensity to produce dust during processing (e.g., feed manufacturing and delivery). Extruded Activated Carbon (EAC) Extruded activated carbon (EAC) combines powdered activated carbon with a binder, which are fused together and extruded into a cylindrical shaped activated carbon block with diameters from 0.8 to 130 mm. These are mainly used for gas phase applications because of their low pressure drop, high mechanical strength and low dust content. Bead Activated Carbon (BAC) Bead activated carbon (BAC) is made from petroleum pitch and supplied in diameters from approximately 0.35 to 0.80 mm. Like EAC, it is also noted for its low pressure drop, high mechanical strength and low dust content, but with a smaller grain size. Its spherical shape makes it preferred for fluidized bed applications such as water filtration. Impregnated Carbon Porous carbons containing several types of inorganic impregnates such as iodine and silver are impregnated carbon. Cations such as aluminium, manganese, zinc, iron, lithium, and calcium have also been prepared for specific applications. Due to its antimicrobial and antiseptic properties, silver loaded activated carbon is used as an sorbent for purification of domestic water. Drinking water can be obtained from natural water by treating the natural water with a mixture of activated carbon and aluminium hydroxide (A1(OH)3), a flocculating agent. Impregnated carbons are also used for the adsorption of hydrogen sulphide (H2S) and thiols. Polymer Coated Carbon This is a process by which a porous carbon can be coated with a biocompatible polymer to give a smooth and permeable coat without blocking the pores. The resulting carbon is useful for various applications including hemoperfusion. Woven Carbon There is a technology of processing technical rayon fiber into activated carbon cloth for carbon filtering. Adsorption capacity of activated cloth is greater than that of activated charcoal (BET theory) surface area: 500-1500 m2 / g, pore volume: 0.3-0.8 cm3 / g). Owing to the different forms of activated material, it can be used in a wide range of applications. Carbon Nanotubes (CNTs) CNTs are cylindrical structures made of carbon atoms arranged in a hexagonal lattice. They have an extremely high surface area and exceptional mechanical, thermal, and electrical properties. CNTs find applications in fields like electronics, materials science, and nanotechnology. Graphene Graphene is a single layer of carbon atoms arranged in a hexagonal lattice. It has an extremely high surface area and remarkable electronic and thermal conductivity. Graphene is used in various industries, including electronics, energy storage, and composite materials. Carbon Aerogels Carbon aerogels are lightweight, highly porous materials with a three-dimensional network structure. They are used in applications such as energy storage devices (supercapacitors), catalysis, and as lightweight structural materials. Carbon Black Carbon black is a fine powder produced by the incomplete combustion of hydrocarbons. It is widely used as a reinforcing filler in rubber products, such as tires, and as a pigment in inks and coatings. These materials generally have a much lower oxygen / nitrogen content compared to biologically derived activated carbons (or charcoals). Carbon Fibers Carbon fibers are composed of carbon atoms bonded together in a crystalline structure. They are known for their high tensile strength, low weight, and resistance to heat. Carbon fibers are used in aerospace, automotive, and sporting goods industries to make lightweight and strong composite materials. Carbon Nano fibers (CNFs) Similar to carbon nanotubes, carbon nanofibers are cylindrical structures with diameters in the nanometer range. They are used in various applications, including reinforcement in composites, energy storage devices, and catalyst supports. Carbon Cloth and Carbon Paper These materials are often made from carbon fibers and are used as electrodes in fuel cells and other energy storage devices. They provide a high surface area for electrochemical reactions. Carbon Foam Carbon foam is a three-dimensional, open-cell structure made from carbon. It is lightweight and possesses good thermal and electrical conductivity. Carbon foam finds applications in heat exchangers, thermal management systems, and as an electrode material. Coal-Based Activated Carbon Coal-based activated carbon is derived from bituminous coal, sub-bituminous coal, or lignite. It has a balanced micropore and mesopore structure; high density, mechanical strength, and hardness; and is versatile and cost-effective for bulk applications. Compared to wood-based, coconut-shell based or peat-based activated carbons, these carbons will generally have a much lower oxygen / nitrogen content. Wood-Based Activated Carbon Wood-based activated carbon is derived from hardwood, sawdust, or other plant-based biomass. It has a high mesopore volume and is ideal for sorbing large molecules. It is also lightweight, low density, and easily powdered. Coconut Shell-Based Activated Carbon Coconut shell-based activated carbon is derived from coconut shells, a renewable and sustainable resource. It is predominantly microporous with high adsorption capacity for small molecules. It has a high hardness and resistance to abrasion, allowing extended use. Peat-Based Activated Carbon Peat-based activated carbon is derived from peat, a partially decomposed plant material. It has a moderate surface area and adsorption capacity. Petroleum-Based Activated Carbon Petroleum-based activated carbon is derived from petroleum pitch or similar hydrocarbon residues. It has a tailored pore size distribution with high mesoporosity and is durable and chemically resistant. Steam-Activated Carbon Steam-activated carbon is produced with a high-temperature treatment (800-1000°C) in a controlled steam atmosphere. This process creates a microporous structure by removing volatile organic compounds and is an environmentally friendly process. Chemically Activated Carbon Chemically activated carbon is produced when taw material is treated with chemical agents like phosphoric acid or zinc chloride before heating (450-700°C). This results in an enhanced mesopore volume and larger surface area compared to steam activation. This process is suitable for processing softer raw materials like wood and peat. Microporous Activated Carbon Microporous activated carbon has a pore size of less than 2 nm. It has a high surface area and strong adsorption capacity for small molecules like gases and VOCs and is suitable for applications requiring precise adsorption. Mesoporous Activated Carbon Mesoporous activated carbon has a pore size of between 2 and 50 nm. Its larger pores accommodate medium-to-large molecules. Mesoporous activated carbon balances adsorption and desorption rates. Macroporous Activated Carbon Macroporous activated carbon has a pore size of greater than 50 nm. It has a lower surface area but allows rapid diffusion of large molecules. Acid-Washed or Impregnated Activated Carbon Acid-washed or impregnated activated carbon is produced when carbon is treated with acids or impregnated with specific chemicals (e.g., silver, sulfur). It has enhanced chemical selectivity and adsorption capacity. Acid-washed activated carbon is treated to remove impurities and reduce leachable ions like heavy metals. Catalytic Activated Carbon Catalytic activated carbon is produced with a modified process to enhance catalytic activity (e.g., surface-treated with metals). It performs chemical reactions while sorbing chemical agents (e.g., a haloform). Activated Carbon Composites Activated carbon composites are Blended with polymers or other materials for specialized functionality. They have enhanced mechanical or chemical properties for specific applications. Pharmaceutical-Grade Activated Carbon Pharmaceutical grade activated carbon is highly purified and processed to meet pharmaceutical and biotechnological standards. It has a low ash content to minimize ion leaching, is free from heavy metals and microbial contaminants, and has a high adsorption capacity for organic impurities and metabolic by-products. It is typically made from wood, coconut shells, or other high-purity raw materials and is generally activated through steam or chemical processes to achieve a fine pore structure. Food-Grade Activated Carbon Food-grade activated carbon has a high mesoporous structure for efficient adsorption of large organic molecules such as pigments and tannins. It is chemically neutral to avoid altering the flavor or nutritional content of food products and is free of harmful residues or contaminants that could compromise food safety. It is often derived from wood or coconut shells to achieve the required porosity and purity and can be steam-activated or chemically activated, depending on the intended application. Industrial Activated Carbon Industrial activated carbon has a high surface area and tailored pore structure for specific industrial needs. It has robust mechanical strength for durability in dynamic systems like fluidized beds and is often chemically or physically modified for enhanced performance in demanding applications. It is often made from coal, coconut shells, or petroleum pitch to provide the necessary hardness and chemical resilience and is generally subject to activation and post-treatment processes tailored to specific industrial applications. Environmental Activated Carbon Environmental activated carbon has tailored pore structures for broad-spectrum chemical absorption. It is resistant to fouling and capable of regenerating performance after cleaning or reactivation and is made with environmentally friendly production processes to align with sustainability goals. It is often derived from coal or coconut shells due to their high hardness and balanced porosity and can be impregnated or treated with chemicals (e.g., silver, potassium) for specialized applications. Cyclodextrin Activated Carbon Composite Materials Cyclodextrin is a well-known carrier for drug delivery applications (Advanced Drug Delivery Reviews, 26, 1999, 3-16 and Drug Development Research, 2018, 79, 201-217). Alpha, beta and gamma cyclodextrins are host-guest type molecules with varying ring sizes of 6, 7 and 8 glucose units, respectively. Intermolecular interactions act to stabilize the bromoform within the complex thus reducing its volatility. For various substrates, the binding affinity will be different; unlike activated carbon, cyclodextrin is a well-defined compound and thus its binding characteristics will not vary from sample to sample. Cyclodextrin is known to sorb onto various activated carbons (Journal of Colloid and Interface Science, 371, 2012, 89-100 and Journal of Colloid and Interface Science, 229, 2000, 615, 619). In these papers, adsorption capacity values ranging from approximately 100 to 700 mg / g are reported. Cyclodextrin binds to haloforms through intermolecular interactions with the cavity in the middle of cyclodextrin ring; this cavity will remain empty after adsorption onto activated carbon and as such it will be free to bind a haloform (e.g., bromoform). Unliked activated carbon which has considerable structural variation within a sample as well as between samples, there will be much more uniformity in the binding characteristics of cyclodextrin to a haloform. This will potentially lead to more predictable and accurate delivery of the haloform in the rumen. Activated carbon is a preferred carrier for cyclodextrin (as opposed to cyclodextrin on its own) as it is easier to incorporate into feed as activated carbon and this composite carrier does not directly dissolve in water. Furthermore, the activated carbon coated in cyclodextrin (as opposed to bromoform directly on activated carbon) will have better dispersibility in water and the release characteristics will be more uniform. In certain aspects, the cyclodextrin activated carbon composite material comprises a water insoluble highly crosslinked cyclodextrin based polymer such as epichlorohydrin-cyclodextrin polymer (see Gidwani, B., & Vyas, A. (2014). Synthesis, characterization and application of epichlorohydrin-P-cyclodextrin polymer. Colloids and Surfaces B: Biointerfaces, 114, 130-137). Various types of cyclodextrin-based materials are suitable for the compositions and processes disclosed herein. Native cyclodextrins include a-cyclodextrin, P-cyclodextrin, and y-cyclodextrin, which differ in the number of glucose units and cavity sizes, enabling inclusion complexes with small, medium, and large guest molecules, respectively. Chemically modified cyclodextrins expand their utility through structural alterations. Hydroxypropylated derivatives, such as hydroxypropyl-P-cyclodextrin (HP-P-CD) and hydroxypropyl-y-cyclodextrin (HP-y-CD), improve solubility and biocompatibility. Methylated cyclodextrins, including randomly methylated-P-cyclodextrin (RAMEB) and highly methylated-P-cyclodextrin, enhance hydrophobicity and guest-host interactions. Sulfated variants, such as sulfobutylether-P-cyclodextrin (SBE-P-CD), offer improved charge properties for applications in drug delivery, while carboxymethylated and phosphorylated cyclodextrins provide tailored solubility and biomedical functionality. Amine-modified and thiolated cyclodextrins enhance interactions with specific molecular targets, offering potential in controlled drug release and bioadhesion. Cyclodextrin-based polymers, such as cross-linked P-cyclodextrin polymers and hydrogels, find applications in controlled drug delivery. Cyclodextrin-polymer conjugates, particularly with polyethylene glycol (PEG), improve solubility and therapeutic compatibility. Additionally, cyclodextrin-metal-organic frameworks (MOFs) and inclusion complexes are integral to advanced separation techniques and stabilization of sensitive compounds. Supramolecular systems, such as cyclodextrin nanoparticles, micelles, and dendrimers, enable targeted therapeutic delivery. Branched cyclodextrins, including glucosylated and maltosylated forms, improve water solubility and reduce cytotoxicity, while chiral cyclodextrins are essential for enantiomeric separations in chromatography. Cyclodextrin-functionalized surfaces and grafted nanoparticles, such as those based on gold, silver, or silica, can also be used. Preparation of Bromoform Encapsulated Cyclodextrin / Activated Carbon Material Cyclodextrin can be sorbed onto activated carbon through a water deposition method where the activated carbon is exposed to a saturated water solution of the desired cyclodextrin. Cyclodextrin will sorb onto the activated carbon with a concentration dependent on the characteristics of the activated carbon and the residual cyclodextrin concentration (i.e., the concentration after sorption). Langmuir isotherm curves describe the sorption of cyclodextrin onto activated carbon better than Freudlich isotherms. More simply, in a general sense, a residual cyclodextrin concentration of 5 g / L will ensure that the sorption capacity has been reached. Following water-based deposition of cyclodextrin onto activated carbon, the sample can then be dried leaving an activated carbon nearly completely coated in cyclodextrin. A haloform (e.g., bromoform) can then be deposited onto this composite material through a vapor deposition process where the bromoform molecule fills the cyclodextrin cavity preferentially as the activated carbon is nearly completely coated. Crosslinked Polymers Polymeric Resins with Aromatic Rings Polymeric sorbents containing aromatic rings, such as those based on polystyrenedivinylbenzene (PS / DVB) copolymers, may be used for the adsorption of haloforms. The aromatic nature of these resins enhances their affinity for these compounds. Polydimethylsiloxane (PDMS) and Silica-Based Polymers PDMS is a silicone-based polymer with high affinity for haloforms. PDMS-coated fibers or particles, as well as other silica-based polymers, may be used in solid-phase microextraction (SPME) for the extraction haloforms. Polyacrylate Resins Polyacrylate resins are compatible with haloform compounds. They can be tailored to have specific functional groups that enhance their affinity for haloforms. Functional groups of interest include groups containing halides, groups with amide-containing ligands, groups with polar moieties such as hydroxyl and carbonyl groups, and ion exchange groups such as quaternary ammonium and sulfonic groups. Each of these functional groups can enhance the formation of favorable interactions with haloforms. Graphene Oxide and Modified Graphene-Based Materials Graphene oxide and modified graphene materials may be used for haloform adsorption due to their high surface area and unique properties. Functionalization of graphene can be done to improve selectivity for haloforms. Graphene oxide (GO) and reduced graphene oxide (rGO) can be functionalized with oxygen-containing groups (e.g., hydroxyl, carboxyl) on the basal plane and edges. These groups can enhance interactions with haloforms through hydrogen bonding. Introduction of halide-containing functional groups (e.g., chloromethyl or chlorophenyl groups) onto the graphene surface can enhance haloform binding. Nitrogen-doping of graphene introduces basic nitrogen functionalities, which interact favorably with haloforms. Nitrogen-containing groups can enhance the overall adsorption capacity of the graphene. Optimization of the graphene structure can maximizes 71-71 stacking interactions with haloforms. This involves controlling the number of graphene layers or introducing aromatic moieties. Incorporation of functionalized carbon nanotubes into graphene-based materials can increase their affinity for haloforms. The tubular structure and functional groups on carbon nanotubes can contribute to improved haloform adsorption. Poly(N-vinyl-2-pyrrolidone) (PVP) PVP and PVP-based materials are known for their compatibility with a variety of organic compounds. These materials can be used in various forms, such as films or particles, for adsorption applications with haloforms. Styrene-Divinylbenzene Polymers Styrene-divinylbenzene (Styrene-DVB) copolymers with a macroporous structure can also be used for the adsorption of haloforms. These macroporous structures are characterized by pores with diameters typically in the range of 50 nm to several micrometers. Amberchrom resins, such as Amberchrom CGI6IC, are macroporous Styrene-DVB copolymers that are commonly used for chromatographic separations. These resins have a high surface area and porosity. Chromabond resins, including Chromabond HR-X, are macroporous copolymers of styrene and divinylbenzene. They are utilized in solid-phase extraction (SPE) for sample preparation and purification. XAD resins, such as XAD-4 and XAD-16, are macroporous Styrene-DVB copolymers widely used for adsorption and extraction of organic compounds from air and water samples. Diaion HP (Hypercrosslinked Porous) resins are macroporous Styrene-DVB copolymers manufactured by Mitsubishi Chemical. They are designed for applications such as chromatography and separation processes. NeviPure offers macroporous hyper-cross-linked polystyrene-divinylbenzene resins for various applications, including the removal of impurities from pharmaceutical intermediates. MN200 is a macroporous Styrene-DVB copolymer resin used for adsorption applications, including solid-phase extraction (SPE) and chromatography. In certain aspects, the hyper-crosslinked polymer comprises a styrene-divinylbenzene group with a macroporous structure. In one aspect, the hyper-crosslinked polymer is selected from the group consisting of PUROSORB® PAD400, PAD500, PAD600, PAD900, PAD1200, PAD350, PAD610, PAD910, PAD950, PAD950C, Amberlite® FPX66, FPX68, Amberlite® XAD2, XAD4, XAD 16, XAD1 180, XAD200, XAD2010, XAD16N, XAD1600N, XAD 18, XAD1 180N, XAD7HP, DIADION® Sepabe, XAD761 ® HP20, HP20SS, HP21 , SP70, SP700, SP825L, SP850, CHP20, CHP50, SP207, HP2MGL, LEWATIT® AF 5, SEPLITE® CT10, LX20, LX 207, LXA8, LXA10, LXA17, LXA680, LXA1600, LXA1 180, LXA81, LXA816, LXA817, LXA8302, LXA88, LXS868, and AB-8. In another aspect, the hyper-crosslinked polymer is PUROSORB® PAD600 (polydivinylbenzene macroporous, sorbent resin, non-ionic form). In other aspects, the hyper-crosslinked polymer is a carbonaceous material, polystyrene, polyacrylic, polyacrylic ester, cation exchange resin, or polystyrene-divinylbenzene. Non-limiting examples include Amberlite (Rohm and Haas) XAD-2, XAD-4, XAD-7, XAD-16, XAD-18, XAD-1180, XAD-1600, XAD-2000, XAD-2010; Amberchrom (Toso Haas) CG-71m, CG-71c, CG-161m, CG161c; Diaion Sepabeads (Mitsubishi Chemicals) HP20, SP206, SP207, SP850, HP2MG, HP20SS, SP20MS; Dowex (Dow Chemical) XUS-40285, XUS-40323, XUS-43493 (also referred to as Optipore V493 (dry form) or Optipore L493 (hydrated form)), Optipore V503, Optipore SD-2; Hypersol Macronet (Purolite) MN-100, MN-102, MN-150, MN-152, MN-170, MN-200, MN-202, MN-250, MN-252, MN-270, MN-300, MN-400, MN-500, MN-502, Purosorb (Purolite) PAD 350, PAD 400, PAD 428, PAD 500, PAD 550, PAD 600, PAD 700, PAD 900, and PAD 950. In other aspects, the cross-linked polymer consists of a combination of either alpha, beta and / or gamma cyclodextrin crosslinked with either epichlorohydrin or trimethylolpropane glycidyl ether to form either water-insoluble or water-soluble cyclodextrin particles (see Colloids and Surfaces B: Biointerfaces, Volume 114, 1 February 2014, Pages 130-137). The cyclodextrin may also be functionalized with aromatic groups through a benzylation reaction and then cross-linked using a Friedel-Crafts reactions with a combination of dichloroethane / formaldehyde dimethyl acetal (see Chern. Sci., 2016, 7, 905). Animal Feed Also disclosed herein is an animal feed comprising compositions described herein. The animal feed may be solid (e.g., powder, granules, pellets), semi-solid (e.g. gel, ointment, cream, paste) or liquid (e.g. solutions, suspensions, emulsions). The animal feed may independently be solid, semi-solid (e.g., gel, ointment, cream, paste) or liquid (e.g. solutions, suspensions, emulsions). For example, the animal feed and the composition may both be liquid or both be semisolid or both be solid. Alternatively, the animal feed and composition may each be a different physical state. For example, the animal feed may be solid or semi-solid and the composition may be liquid. The composition may, for example, be used to "top-dress" (i.e., added on top) a ruminant feedlot ration or may be used to blend into a total mixed ration. In preferred aspects, compositions described are anti-methanogenic compositions. The composition may, for example, be added to the drinking water of the animal. In certain embodiments, the composition may be added to the drinking water of the animal immediately before ingestion, for example up to 1 hour before ingestion or up to 30 minutes before ingestion or up to 15 minutes before ingestion or up to 5 minutes before ingestion. The three main types of animal feed include roughages, concentrates and mixed feeds. In general, roughages contain a higher percentage of crude fiber and a lower percentage of digestible nutrients than concentrates. For example, roughages may be defined as containing equal to or greater than 20 wt% crude fiber and equal to or less than 60 wt% total digestible nutrients. Roughages may include, for example, dry roughages (e.g., hay, straw, artificially dehydrated forages containing at least 90 wt% dry matter), silages (formed from green forages such as grass, alfalfa, sorghum and corn and preserved in a silo at dry matter contents of 20 to 50 %), and pastures (e.g. green growing pastures providing forage that has a high water content and generally less than 30 % dry matter). The two basic types of roughages include grasses and legumes. Grasses are generally higher in fiber and dry matter than legumes. Legumes are generally higher in proteins, metabolizable energy, vitamins and minerals. Concentrates contain a relatively lower percentage of crude fiber and a higher percentage of digestible nutrients than roughages. For example, concentrates may be defined as containing less than 20 wt% crude fiber and greater than 60 wt% total digestible nutrients. Concentrates may include, for example, energy-rich grains and molasses. Corn, wheat, oats, barley and milo (sorghum grain) are energy-rich grains, containing about 70 to 80 wt% total digestible nutrients. Mixed feeds are generally a mixture of roughages and concentrates to provide "complete" balanced rations and may be either high or low in energy, protein or fiber. The disclosed compositions, for example, can be combined with animal feed in various amounts depending on the total amount of composition intended to be administered to the animal. The animal feed may, for example, comprise from about 0.0001 wt% to about 10 wt% of the disclosed compositions, based on the total dry weight of the animal feed. The animal feed may, for example, comprise from about 0.01 wt% to about 10 wt% of disclosed composition, based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.001 wt% to about 9.5 wt%, or from about 0.005 wt% to about 9 wt%, or from about 0.01 wt% to about 8.5 wt%, or from about 0.05 wt% to about 8 wt%, or from about 0.1 wt% to about 7.5 wt%, or from about 0.9 wt% to about 7 wt%, or from about 1 wt% to about 6 wt%, or from about 1.5 wt% to about 5.5 wt%, or from about 2 wt% to about 5 wt%, or from about 2.5 wt% to about 4.5 wt%, or from about 3 wt% to about 4 wt% disclosed composition based on the total dry weight of the animal feed. For example, the animal feed may comprise from about 0.4 wt% to about 9.5 wt%, or from about 0.5 wt% to about 9 wt%, or from about 0.6 wt% to about 8.5 wt%, or from about 0.7 wt% to about 8 wt%, or from about 0.8 wt% to about 7.5 wt%, or from about 0.9 wt% to about 7 wt%, or from about 1 wt% to about 6 wt%, or from about 1.5 wt% to about 5.5 wt%, or from about 2 wt% to about 5 wt%, or from about 2.5 wt% to about 4.5 wt%, or from about 3 wt% to about 4 wt% disclosed composition based on the total dry weight of the animal feed. In one embodiment, the disclosed composition is administered at a dose of preferably at least 16.67, 10, 5, 3, 2, 1, 0.5, 0.25 0.125 or 0.067wt% of the organic matter administered to the ruminant animal. For example, if a 450 kg ruminant animal (e.g., steer) consumes 2.5% to 3% of its body weight per day of feed, then the disclosed composition is administered at a dose proportional to the amount of organic matter administered to the ruminant. In the case of a 450 kg ruminant animal, and where 80% of the feed is organic matter, if the animal consumes about 2.5% of its body weight per day, then the disclosed composition is administered at a dose of about 0.27, 0.18, 0.09, 0.045, 0.0225, 0.01125 or 0.00603 kg per day to result in a dose at least 3, 2, 1, 0.5, 0.25 0.125 or 0.067wt% of the organic matter administered to the ruminant animal. In another embodiment, the disclosed composition is administered at a dose that provides 1 to 100 mg bromoform per kg dry matter intake, 1 to 90 mg bromoform per kg dry matter intake, 1 to 80 mg bromoform per kg dry matter intake, 1 to 70 mg bromoform per kg dry matter intake, 1 to 60 mg bromoform per kg dry matter intake, 1 to 50 mg bromoform per kg dry matter intake, 10 to 100 mg bromoform per kg dry matter intake, 10 to 90 mg bromoform per kg dry matter intake, 10 to 80 mg bromoform per kg dry matter intake, 10 to 70 mg bromoform per kg dry matter intake, 10 to 60 mg bromoform per kg dry matter intake, 10 to 50 mg bromoform per kg dry matter intake, 25 to 100 mg bromoform per kg dry matter intake, 25 to 90 mg bromoform per kg dry matter intake, 25 to 80 mg bromoform per kg dry matter intake, 25 to 70 mg bromoform per kg dry matter intake, 25 to 60 mg bromoform per kg dry matter intake, or 25 to 50 mg bromoform per kg dry matter intake. Any embodiment of the invention is meant to be illustrative only and is not meant to be limiting to the invention. Therefore, it should be appreciated that various other changes and modifications can be made to any embodiment described without departing from the spirit and scope of the invention. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference in their entirety for all purposes. EXAMPLES Example 1. Measurement of Bromoform with Gas Chromatography-Mass Spectroscopy Gas chromatography-mass spectroscopy (GCMS) was used to analyse the bromoform content in water solutions and in the stabilized solid samples that are the subject of this patent. A headspace technique is used where the sample placed inside of a 20 mL sample vial with a silicone septum is exposed to an elevated temperature. When the sample reaches equilibrium after a period, the vial is pressurized with helium gas and then a portion of this volume is analyzed by the GCMS. To quantify the amount of material, the temperature and volume of liquid / solid are chosen such that the concentration in the headspace at elevated temperature is proportional to the amount in the original unheated sample. For bromoform in water, 10 mL of solution is used and the temperature used is 70 °C; a temperature of 180 °C is used to analyse the bromoform content with the high surface area porous polymers (e.g., poly( divinylbenzene) where 600 mg of sample is used. For activated charcoal samples, 100 mg of the solid sample is added to a vial with 4 mL of chlorobenzene; under these conditions, the chlorobenzene competes with the bromoform for sorption sites effectively stripping the bromoform from the activated charcoal. 20 microliter samples of the chlorobenzene solutions of bromoform are then added to headspace vials which are analyzed at 150 °C where the entire contents of the vial are vaporized. At these elevated temperatures, the bromoform in its respective matrices has demonstrated a linear response using a standard addition method and the generation of numerous calibration curves. A Shimadzu GC-2030 gas chromatograph is used in conjunction with a HS-20NX headspace sampler module and a GCMS-QP2020NX single quadrupole mass spectrometer. A Shimadzu SH-I-5Sil column (with a silylene phase comprising a 5% diphenyl and 95% dimethyl polysiloxane). This column was chosen for its ability to separate various halomethanes (e.g., chloroform, bromochloromethane). For the headspace analyzer, the oven temperature used is dependent on the matrix (see above). Sample line and transfer line temperatures of 150 °C and 150 °C, respectively, where used. A shaking level of 4 was used for a 20-minute equilibrating time. The sample was pressurized for 1 minute to 55.2 kPA with helium and allowed to equilibrate for 0.50 minutes. Load and load equilibration times of 0.50 and 0.10 minutes, respectively, were used. An injection time of 1.50 minutes was used followed by a 4-minute needle flush time. The GC oven temperature is set to 50 °C initially and ramped to 150 °C at a 35 °C / minute rate, then to 200 °C at a 50 °C / minute rate and finally to 250 °C at a 70 °C / minute rate. A 50-ratio split injection is used with a gas pressure of 147.0 kPa and total column flow of 54.0 mL / minute (comprising 1.00 mL / minute column flow and 3.0 mL / minute purge flow). For the mass spectrometer, ion source and interface temperatures of230 °C and 220 °C, respectively, were used. The spectrometer is tuned using perfluorotributylamine to maximize the m / z 502 peak. A detector voltage of -0.1 kV relative to this tuning is used for this analysis. Example 2. Stability of 100 pM Bromoform in Water Background Bromoform at a concentration of 10 mM has been tested for its stability in water previously where it was found to very quickly evaporate into the atmosphere (i.e., within hours of putting it into the water). Here, a much smaller concentration of bromoform of 100 uM in water was analyzed to see if bromoform at this lower concentration is effective in remaining in the water solution for longer. This study was conducted as a basis for comparison with the bromoform stabilized on solid carriers. Materials and Methods Sample Preparation A 100 uM solution of bromoform was prepared in 500 ml of water. Approximately 12.6 mg of synthetic bromoform (equivalent volume) was pipetted into the water and thoroughly mixed for about a minute to ensure homogeneity. Experimental Parameters / Conditions The prepared solution was kept in an open bowl with a magnetic stirrer continuously stirring at approximately 50 rpm. The bowl was kept inside a fume cupboard to minimize bromoform stratification along the surface of the water since bromoform is of higher density than air. The airflow from the cupboard should have been able to reduce bromoform stratification from the water surface. Sample Collection The first sample was taken just after preparing the 100 uM solution to mark the starting concentration. At every 1-hour interval around 10 ml of sample was taken into a headspace vial and sealed using crimp caps. Samples collected for the first day were kept more frequently than for the other two days. This sample collection frequency was followed as it was seen before with a 10 mM concentration that bromoform in water is not stable and was seen to be lost in air in less than 24 hours. Results The headspace vials of the collected samples were run in GCMS. Due to the low concertation of bromoform in the water, no dilution was required for these samples. Following the parameters for water analysis relating to the gas chromatogram method, an appropriate method file was loaded and the results obtained were documented (see FIG. 1). The GCMS analysis was stopped after sample 9 because after sample 6 there were trace levels or no bromoform content registered in the GCMS read. As shown with the leftmost curve in FIG. 1, bromoform levels from the 100 uM aqueous solution had dissipated to trace amounts within the first 5 hours of measurement. Example 3. Stability of 10 wt.% Bromoform on PAD600 Background The initial testing utilized hyper-crosslinked poly(divinylbenzene), which is sold as PUROSORB™ PAD600 (polydivinylbenzene macroporous, sorbent resin, non-ionic form) (“PAD600”) by Purolite. This material has applications in the water and food processing industries. Materials and Methods Sample Preparation and GCMS Calibration Curves To a 100 mL solution of 10 mM bromoform (containing approximately 252 mg of bromoform), 2.268 grams of dried PAD600 resin beads was added and stirred for 24 hours in a closed jar. Subsequent, GCMS analysis of the water solution showed that the residual bromoform concentration was approximately 75 uM indicating near quantitative removal (i.e., 99.25 %) of bromoform from a 10 mM solution. After vacuum filtration, these samples were dried in an oven at 60°C for several hours before being placed in a fan-forced oven at 40°C to measure bromoform retention. To quantify the retention, a weight-by-weight dilution of 40-fold using the PAD600 polymer was carried out. Clear glass was used for the dilution process. This created a weight-by-weight dilution ratio of 1:40 for each sample (0.5 grams sample to 19.5 grams polymer). Each container was gently rotated to ensure thorough mixing of the sample and the polymer to produce a homogeneous diluted sample. Results FIG. 1 shows that a formulation comprising 10 wt.% bromoform and 90 wt.% PAD600 resin beads has superior stability relative to a water solution (compare the rightmost curve to the leftmost curve). At 40°C in a fan-forced oven, the retention was approximately 57% after 3 weeks; for comparison, with a dilute water solution, the retention had reduced to 50% in less than 2 hours. Example 4. Stability of 10 wt.% Bromoform on Activated Charcoal (Aldrich, C9157) Background The initial testing utilized activated charcoal, which is sold as C9157 by Sigma-Aldrich (Australia). This material has applications in the water and gas processing industries. It is also used in the medical industry to treat poisoning and marketed as a food additive / supplement. It is anticipated bromoform will readily and strongly sorb onto the surface of this material; with a high surface area typically ranging from approximately 900 to 3000 m2 / gram, the sorption capacity will be relatively high. Materials and Methods Sample Preparation and GCMS Calibration Curves To a 100 mL aqueous solution of 10 mM bromoform (containing approximately 252 mg of bromoform), 2.268 grams of dried C9157 activated charcoal is added and stirred for 24 hours in a closed jar. Subsequent, GCMS analysis of the water solution showed that the residual bromoform concentration was less than 1 uM indicating near quantitative removal (i.e., >99.99 %) of bromoform from a 10 mM solution. After isolating the activated charcoal using vacuum filtration, these samples were dried in an oven at 60°C for several hours. They were then placed into a fan-forced oven at 100°C in an accelerated aging experiment to confirm bromoform’s stability on activated charcoal. Results FIG. 1 shows that formulations comprising 10 wt.% and 15 wt.% bromoform on C9157 activated charcoal have superior stability relative to both a water solution and PAD600 resin beads (compare the rate of decline in bromoform retention of the middle two curves to the leftmost and rightmost curves). At 100°C in a fan-forced oven, the retention was approximately 53% after 6 days and 59% after 5 days for the 10 and 15 wt.% bromoform on active charcoal (C9154), respectively. For comparison, with a dilute water solution the retention had reduced to 50% in less than 2 hours. Example 5. Thermal Gravimetric Analysis (TGA) of Bromoform on Activated Charcoal Background Thermogravimetric analysis (TGA) or thermogravimetry (TG) is a technique where the mass of a polymer is measured as a function of temperature or time while the sample is subjected to a controlled temperature program in a controlled atmosphere. Materials and Methods TGA of bromoform extracted onto activated charcoal was performed with a NETZSCH thermobalance. The temperature in the thermobalance was varied from 25 °C to 400 °C with a starting temperature of 25 °C, then ramped to 100°C at 5°C / min, held for 30 minutes, then ramped to 200°C / min at 5°C / min, held for 30 minutes and finally ramped to 400 °C at 10 °C / min. Results An initial loss in mass of 4.51% was observed as the temperature increased from 35°C to 100°C. This change in mass was due to the loss of water as indicated by the differential scanning curve corresponding to this period. Not until the temperature increased from 200°C to 400°C was there partial vaporization of the bromoform on the activated charcoal corresponding to a loss in mass of 1.28% (see FIG. 2). The use of activated carbon in analytical chemistry applications suggest that high temperatures are necessary for its efficient thermal desorption. This is advantageous as the volatility is expected to drastically reduce. TGA confirmed strong retention of bromoform on activated charcoal even at elevated temperatures. Example 6. Determination of Bromoform Adsorption Isotherms for PAD600 Resin and Various Activated Charcoals Background Isotherms (e.g. Langmuir, Freundlich) are used to describe the chemical equilibria between analyte pressure (in the gas phase) and concentration (in a liquid phase) and surface coverage (or leading) at a given temperature. The Langmuir isotherm: 0 = bP / (l+bP) where theta is the fractional surface coverage, b is a chemical equilibrium constant, and P is the analyte concentration) can be used to describe behavior of bromoform on high surface area materials. This model is typically for suitable for monolayer adsorption and is typically less valid at higher loadings. The Freundlich isotherm: 0 = bP1 / n where theta is the fractional surface coverage, b is a chemical equilibrium constant, P is the analyte concentration and 1 / n describes the non-linearity of the sorption process, can be used to describe behavior of bromoform on high surface area materials. This model is typically used in a multilayer sorption situations and where the surface as heterogeneous sorption sites. The assumptions for these model are listed below and the above expression can be explained from both a kinetic and thermodynamic consideration. Typically, validation of an isotherm model is driven by experimental results. Inherent within this model, the following assumptions are valid specifically for the simplest case: the adsorption of a single sorbate onto a series of equivalent sites onto the surface of the solid. 1. The surface containing the sorbing sites is a perfectly flat plane with no corrugations (assume the surface is homogeneous). However, chemically heterogeneous surfaces can be considered to be homogeneous if the sorbate is bound to only one type of functional groups on the surface. 2. The sorbing gas sorbs into an immobile state. 3. All sites are energetically equivalent and the energy of adsorption is equal for all sites. 4. Each site can hold at most one molecule of A (mono-layer coverage only). 5. Minimal (or no) interactions between sorbate molecules on adjacent sites. When the interactions are ideal, the energy of side-to-side interactions is equal for all sites regardless of the surface occupancy. The chemical equilibrium constant b is temperature dependent and its relationship with temperature can be described by the Clausius-Clapeyron equation. For high temperatures, the value of b will be small (as adsorption is an exothermic process) and the shape of isotherm will approach a line at low pressure. For low temperatures, the value of b will be larger and the shape of the curve will have an asymptotic nature. This has implications when using GCMS headspace analysis to determine the weight percentage of bromoform in the activated charcoal matrix. At low temperatures, the pressure in the headspace will be relatively insensitive to the coverage fraction (due to the asymptotic shape of the isotherm) resulting in large errors in the analysis. This has been observed experimentally. Isotherms were analyzed for various forms of high surface carbon materials including activated charcoal, carbon black, and PAD600 to determine the amount of bromoform sorbing onto the material and the remaining amount left in solution. This analysis provided an estimate of the amount of bromoform that would be released in the rumen after administration of bromoform sorbed on the material in the form of an animal feed. A higher loading will generally lead to a higher equilibrium concentration in aqueous solution; however, the volatility characteristics in air will also be increased. As such, there is a compromise that needs to be considered given that bromoform will also react in the rumen to impart anti-methanogenic qualities. Materials and Methods The following materials were selected for analysis with bromoform extraction to determine an equilibrium adsorption isotherm: • Activated charcoal powder - C9157 (Sigma-Aldrich) and 161551 (Sigma-Aldrich); • Activated charcoal granules - C3014 (Sigma-Aldrich) • Carbon black - Vulcan XC72 (Cabot); and • Polydivinylbenzene resin beads - PUROSORB™ PAD600 (Purolite). Extraction of Bromoform onto High Surface Area Materials 10 millimoles (i.e., 2.52 grams or 0.875 mL) of bromoform was added to IL of water containing varying amounts of high surface area material (as listed above). As way of example, assuming 100% extraction, the addition of 22.68 g of high surface area would result in a material that consists of 10 wt.% bromoform. This material after filtration is dried under ambient conditions and then in an oven at 100°C to remove any residual water; this assumes that there is minimal loss at these temperatures. This was confirmed with activated charcoal as the Inventors were unable to do quantification with gas chromatography-mass spectrometry (GCMS) even with a headspace temperature of 200°C. To establish the isotherm curves for these materials, varying quantities of substrate were added to the solution with the residual bromoform concentration in the water solution being determined by GCMS. A mass balance was then used to determine the bromoform loading on the high surface material. Results The isotherm curves clearly show the deposition of bromoform from a 10 mM aqueous solution of bromoform onto the various substrates (see FIG. 3). Even for the three different variants of activated charcoal, there is significant variability in the sorption characteristics and binding affinity between the substrate’s surface and bromoform. The residual concentrations in aqueous solution are broadly indicative of the equilibrium concentration of bromoform that would be available in the rumen. Generally, concentrations between 10 and 20 wt.% are readily achievable with these substrates. When considering dosing, it is important to consider these equilibrium concentrations and not the absolute amount of bromoform. A dosage that uses a material with a higher loading will have higher release into the rumen compared to a dosage that uses a material with a lower loading even if the absolute amounts of bromoform are the same. It is important to realize that 100 mg of a 10 wt% loaded substrate will have enhanced release characteristics compared to 1000 mg of a 1 wt% loaded substrate. This is evident from the isotherm data and expected variations in desorption kinetics. The other consideration is the kinetics of the desorption process. The isotherm analysis does not provide insight on this and as such it is necessary to conduct in vitro and in vivo experiments to determine the availability of bromoform and the appropriate dosing using each of these formulations. Example 7. Liquid Phase Deposition and Vapor Phase Deposition of Bromoform onto Activated Charcoal Prior work for other applications (e.g., water treatment plants) has shown that activated carbon is a potent sorbent for volatile organic carbons (bromoform is a particular VOC) in both aqueous and gaseous environments. This is a beneficial property as it allows for either a liquid- solid extraction or vapor-solid extraction. As a component in a solid formulation, this approach is compatible with the approach of using fungi to produce bromoform using a vanadium haloperoxidase (VHPO) enzyme. Liquid-Phase Deposition High surface area material (e.g., activated charcoal or PAD600) is present in the water solution where the bromoform synthesis is being undertaken. The advantage is that this can be implemented very easily and a rotary mixer can be used to ensure a uniform deposition. The challenge is that other compounds volatile or non-volatile may be sorbed onto the high surface area substrate (e.g., activated charcoal). These would presumably be released when used as an animal feed. Furthermore, this process is not conducive to a continuous process as the biomass present in the VHPO-catalyzed production of bromoform would combine with the activated charcoal. Vapor-Phase Deposition Air is bubbled into the water solution where the bromoform synthesis is being undertaken. As bromoform is quite volatile under these circumstances, it can be sorbed in the vapor phase onto a stirred bed of high surface area material. It is important that this material is continuously mixed during this process to ensure the deposition process is uniform. This process is favored as continuous extraction can be achieved easily and only volatile components will be extracted. Preferred Forms of Activated Charcoal In both methods, larger size granules (i.e. 1 to 2 mm) may be favored. Coconut shell derivate activated charcoal seems to have many desirable qualities including being bio-derived, hardness (which would reduce powderization) during processing, availability in large quantiles, and the ability to sorb significant amounts of haloforms. Example 8. Assessment of Stabilized Formulations with Methanobrevibacter smithii bioassay Background Unprotected formulations of methane inhibitors, such as bromoform will be rapidly lost from the rumen due to the volatile nature of these compounds and their rate of passage through the rumen. Formulations that “entrap” bromoform for longer in the rumen are likely to provide higher levels of efficacy. However, these stabilized formulations will still need to be able to release the active compound to elicit a response. A simple bioassay using Methanobrevibacter smithii can be used to assess the effectiveness of the stabilized formulations in relation to the bioavailability of the inhibitor and its effect on methane production. Materials and Methods Culturing and Media Pure cultures of Methanobrevibacter smithii were inoculated from glycerol stocks into modified anaerobic BRN media (Maczulak et al., 1989, Miller et al., 1982) (30% clarified rumen fluid and Cysteine-HC1 as a reductant) in 10 ml Balch Tubes (Balch and Wolfe, 1976). The same media was used for all methane inhibition assays. Inoculated cultures were pressurized with UHP hydrogen gas to 150 kPa over pressure and incubated at 39°C with gentle shaking at 70 rpm on an orbital shaker. Cultures were regularly transferred into new media (1 / 100 dilution) every 48h (ODeoo approx. 0.3) and used as inoculum for assays with stabilized formulations. Culture headspace gases were analyzed for methane production and quantification after 24 and 48 hrs incubation. Stabilized Formulation Preparation For each formulation, a matrix control containing no bromoform and the bromoform charged matrix were assessed. As these formulations cannot be dissolved in a diluent, a weighted amount was added to each individual tube or bottle. A minimum amount of compound was chosen that would provide a final concentration of 5 uM of bromoform in a 100 ml bottle for the bromoform formulations. For 10 wt.% bromoform on activated charcoal, this corresponds to 1.26 mg of substrate. The matrix only formulation assays were performed in 10 ml tubes with the same weighed amount as for the 100 ml bottles, equating to a 10X increase in the matrix for these tubes compared with the bottles. For the activated charcoal matrix control, an equivalent amount of activated charcoal was used. Methanogen Assay All assays were set up in an anaerobic chamber (approx. 95% CO2 + 5% H2). Pre-warmed bulk modified BRN media (~ 2 L) was inoculated with 1:50 vol / vol of a 36-40h growing (ODeoo approx. 0.3) M. smithii culture and mixed thoroughly to ensure uniform distribution of methanogens and dispensed into replicate tubes or bottles with pre-weighted formulations. A M. smithii control culture (no additive) and a bromoform control culture (0.5 pM) were also used. All formulations were performed in duplicate cultures for both matrix and bromoform assays. All tubes and bottles were capped in the anaerobic chamber and pressurized with UHP Hydrogen (BOC, Australia) using a gassing-manifold and pressure gauge to 150 KPa overpressure. Tubes and bottles were incubated, at 39°C in an orbital shaker incubator at 70 rpm. Headspace pressures were recorded using microswitch pressure transducers (Cat# 185PC15DT, Honeywell, IL) connected to a datalogger (Wellman, Belgium), after 24h; if unchanged, allowed to be incubated until 48h. Methane and H2 analysis was done by drawing approximately 2 ml headspace gas in a gastight syringe with a 25G x 25mm needle (Terumo) and injected into a Shimadzu GC-2014 with a TCD detector as described by Gagen et al (Gagen et al., 2014) with N2 as carrier gas; CH4 and H2 calibration was done using a 5-level standard mixture of CH4, H2 & CO2 in N2 (Air Liquide, Australia) with linear R2 = > 0.99 for each gas standard. Gas percentages (CH4) were converted using headspace volumes and gas pressures and reported as micromoles of CH4 for 10ml or 100ml using Ideal Gas Law (PV = nRT). Results As seen in Table 1 below, the methane production from M. smithii was as predicted for the control cultures with a mean production of 328 pmol / 10 ml culture in 24 hrs, while the inclusion of bromoform at a concentration of 0.5 pM completely inhibited all growth and production of methane from M. smithii. There was no inhibition of growth or methane production for the control activated charcoal matrix tube. Control matrix tubes were at 10X the concentration of that of the bromoform matrix tubes. At a 5 pM nominal concentration, 10 wt.% bromoform on activated charcoal exhibited complete loss of methane production. Table 1. Methane production M. smithii methane production (pmol / 10 ml) ) Formulation Control1 Bromoform2 Comments Control 328 0 controls Bromoform on activated charcoal 315 0 100% inhibition 1 mean pmol / 10 ml culture 2 mean pmol / 100 ml culture Example 9. Assessment of Stabilized Formulations with In Vitro Fermentation Technique The in vitro fermentation technique (IVFT) was conducted as described in a previous publication (Journal of the Science of Food and Agriculture, 2014; 94: 1191-1196). Briefly, Bellco tubes were set up with 0.1 g of high fiber fermentation substrate (oaten chaff, ground to 5 pass 1 mm screen) and placed inside an anaerobic chamber (80% N2 / 10 % CO2 / 10% H2). The required amount of either pure bromoform stock or raw material stocks was added to the Bellco tube containing fermentation substrate. Following this, buffered sheep rumen fluid (10 mL) was added, and the tubes were sealed and crimped. A substrate control (oaten chaff only), and an inoculum control (rumen fluid only) were included, and all treatments were run in triplicate. Tubes 10 were then taken out of the chamber and incubated at 39°C for 24 hours with orbital shaking at 50 rpm. At the end of incubation, tubes were taken out, and total microbial gas produced was measured using a pressure transducer, as a proxy of overall microbial activity, before a sample of headspace gas was collected for methane analysis by gas chromatography. As seen in Table 2, at a nominal 5 pM and 10 pM concentration, 10 wt.% activated 15 charcoal exhibited methane production inhibition on par with direct bromoform addition. This is further confirmation that 10 wt.% activated charcoal will release from the activated charcoal surface into the rumen where it can inhibit methane production. Table 2. Average gas and methane production when the treatments were combined with oaten chaff substrate in the in vitro batch. Each treatment was tested in triplicate. Within the same column, treatments not connected by same letter are significantly different (p < 0.05). Significant reductions in methane are indicated with italicized font. Treatment Total gas (kPa) ch4 concentration (mL / 100 mL gas) ch4 production (mL / g DMi) % reduction compared to control kPa ch4 cone. ch4 prod. Substrate Control 74.4 de 8.1 ab 28.1 be Bromoform pM 0.5 74.0 e 8.6 ab 29.7 abc 1 -5 -5 1 76.0 bcde 9.4 a 32.8 abc -2 -4 -5 5 75.4 bcde 0.4 d 1.2 e -1 96 96 10 85.5 a 0.2 d 0.9 e -15 97 97 10 wt.% 0.5 81.6 abed 9.9 a 35.7 a -10 -10 -14 Bromoform 1 80.0 abede 9.8 a 35.0 ab -8 -10 -13 on 5 75.7 bcde 0.7 d 2.6 e -2 90 90 Activated Charcoal 10 81.1 abede 0.3 d 1.0 e -9 97 97 Example 10. Manufacture of Feed Pellets Containing Bromoform-Loaded Activated Charcoal Powder Using a peristaltic pump, 1 kg of bromoform was added over approximately 24 hours to a continuously stirred vessel containing 120 L of water and 7.5 kg of activated charcoal powder 10   (C9154). After 24 hours, the residual concentration of bromoform in the aqueous solution was confirmed by GCMS to be less than 20 pM (corresponding to approximately 0.6 g) confirming almost quantitative deposition of bromoform onto the activated charcoal matrix. Assuming some volatilization during this deposition response, the recipe was formulated based on an 11.5 wt.% loading of bromoform onto the activated charcoal powder. This bromoform loaded material was dried in a large drying chamber at 60°C for 72 hours. There was some heterogeneity in the deposition process as it was processed in two batches. For the second batch used for manufacturing of pellets, a loading factor of 13.0 wt.% was measured by GCMS. For the analysis of these samples, the splitter on the GCMS was turned off and the 5 detector voltage was increased to +0.05 kV above the tuning voltage was used to increase the sensitivity. After the pelleting process, there was approximately 37 % loss in bromoform content as measured by GCMS. The full recipe for the animal feed pellets containing bromoform-loaded activated charcoal is outlined in Table 3. 10 Table 3. Recipe for making pellets containing bromoform-loaded activated charcoal. Ingredient Inclusion rate (%) kg per 300 kg batch Ground wheat 21.350 64.050 Ground barley 40.000 120.000 Ground sorghum 20.000 60.000 Canola meal 10.000 30.000 Urea (stockfeed / prills) 1.000 3.000 Limestone fine 1.000 3.000 Salt 0.500 1.500 Beef / Dairy premix 0.100 0.300 Molasses 1.000 3.000 Water 4.000 12.000 Vegetable oil 1.000 3.000 AC (11.5% wt bromoform) 0.050 0.150 Total 100.000 300.000 Example 11. In Vitro Assessment of Inert Carriers to Deliver Antimethanogenic Compounds in Forage Based Diets Introduction 15           In Australia ruminant livestock contribute significantly to agricultural greenhouse gas emissions. Halogenated compounds such as bromoform (CHBn) have been shown to have potential as a feed additive for mitigating enteric methane but are inherently volatile due to their chemical characteristics. Nevertheless, if complexed into a stable carrier these compounds could be incorporated into various feed preparations and released in the rumen to achieve a direct abatement outcome and so contribute to reductions of agricultural emissions. The requirement for daily oral administration of compounds may limit utility in extensive production systems but readily aligns with intensive systems such as dairy and feedlot. The aim of this study was to identify a solid inert carrier used to stabilize the antimethanogenic compound bromoform that would deliver in vitro abatement and could progress to in vivo application. Materials and Methods Two solid inert carriers [activated carbon (AC), polymer resin (PR)] containing bromoform (15% w / w) were dosed daily in vitro at a low (L) or high (H) concentration, creating four treatments; AC-L, AC-H, PR-L and PR-H equivalent to 12.6 and 25.2 mg CHBn / kg DM for low and high doses, respectively. In this example as well as example 12, the polymer resin was Purolite PAD600 which is a crosslinked poly(divinylbenzene) resin. A substrate only treatment of 30 g ground oaten chaff was used as a control. Treatments were incubated in triplicate in 50 pm pore size nylon bags using an artificial rumen simulation technique (RUSITEC) for 14 days to assess methane mitigation. The RUSITEC system was initiated and maintained as described by Garcia et al., (2019). A constant flow of buffer into each fermenter maintained pH of 6.0 - 7.0. Gas produced by each fermenter was measured daily, sub sampled for methane quantification, fermentation fluid collected for analysis of VFA (volatile fatty acids) at three time points over 14 days and in vitro NDF (neutral detergent fiber), ADF (acid detergent fiber) and DM (dry matter) degradability calculated. Data were analyzed per timepoint using one-way ANOVA with treatments as fixed factors and methane concentration, production, total gas, and VFA profile as variables. A Tukey-Kramer HSD pairwise comparison was used when means differed (P<0.05). Results and Discussion All carrier types supported significant reductions in methane concentration and yield (P<0.05). The AC carrier delivered the greatest and most persistent mitigation effect regardless of concentration with mean reductions of 89-91% in the first week and despite withdrawal of AC-H during the second week, 97-99%, indicating a carry-over effect. Treatment PR-H demonstrated a mean reduction in methane yield of 76%, during the first week, however, this diminished to 41%, in the second week. There was a delay of at least 72 h to achieve 16-18% methane reductions for PRL compared with other carrier combinations that demonstrated an in vitro effect within 48 h. Carrier type did not influence (p>0.05) NDF, ADF or DM degradation compared with the control across sampling timepoints. Total mean VFA concentrations were similar (p>0.05) between all treatments (104 mmol / L) and control (101 mmol / L) across sampling timepoints. An anti-methanogenic activity of all treatments was apparent without a decrease in total gas production, total VFA concentration or substrate degradability in vitro. These preliminary results indicate that carrier type will influence the mitigation outcome. A 15% (w / w) CHBn for the PR may have exceeded its actual carrying capacity thereby explain the diminishing effect over time. The polymer resin in this study was less effective and less consistent in terms of effects on methane production in vitro. Conversely the concentration of CHBn on AC could be decreased given almost complete inhibition was achieved at the low concentration. Conclusion and Implications Bromoform stabilized onto an AC carrier demonstrated greater and more consistent effects on methane mitigation than PR, indicating it is a more promising candidate for progression to in vivo trials. The utility of AC to complex CHBn in a stable form requires further validation beyond 14 days. Ultimately the commercial use of inert solid carriers such as AC is dependent on the demonstration of consistent and minimal level of methane mitigation over an extended time period for either a feedlot or dairy application. Example 12. Testing the Effects of Stabilised Bromoform Feed Additives on In Vitro Fermentation Executive Summary This project consisted of two in vitro experiments designed to identify a preferred stabilized bromoform carrier material for progression to in vivo experiments. The first experiment examined methane production and rumen fermentation characteristics when analytical grade bromoform liquid (CO) or stabilized carrier formulations loaded with bromoform (C1-C3) were incubated in 10 mL tubes for 24-h with sheep rumen fluid and an oaten chaff substrate at 0, 0.5, 1, 5 and 10 uM. The stabilized formulations included activated charcoal loaded with 15% weight bromoform (Cl), a porous cross-linked polymer resin (PuroSorb™ PAD600) loaded with 10% weight bromoform (C2) and PAD600 loaded with 1% weight bromoform (C3). The most effective formulation was Cl, which significantly (P < 0.05) reduced methane concentration and yield by more than 90% at 5 uM and 10 uM concentrations and had statistically similar antimethanogenic activity to CO. Formulation C3 also had anti-methanogenic activity, reducing methane concentration and yield by 50% at 10 uM. C2 did not alter methane production at a statistically significant level at any of the dosing levels. The ratio of acetate:propionate was consistently reduced (P < 0.05) in treatments demonstrating significant methane reduction. The second experiment tested activated charcoal (AC) and polymer resin (PR) formulations at a low (L; 2.5 uM) and high (H; 5 uM) dose (AC-L and AC-H, PR-L and PR-H, respectively) using an artificial rumen simulation technique (RUSITEC) over two weeks to assess persistency of effects upon methane production and rumen fermentation characteristics. Both the AC and PR were loaded at 15% bromoform. All treatments reduced methane concentration and yield (P < 0.05). Treatments AC-L and AC-H had the greatest and most persistent effects on methane inhibition in vitro; mean reductions in methane concentration and yield were 89-91% in the first week and 97-99% in the second week. Although daily dosing of AC-H ceased during the second week, methanogenesis remained inhibited, indicating that a carry-over effect may exist. Treatment PR-H demonstrated a mean reduction in methane concentration and yield of 78% and 76%, respectively, during the first week, however, this diminished to 38 and 41%, respectively, in the second week. There was a delay of at least 72-h in reaching statistically significant methane reductions for PR-L, whereas methane decreased significantly for all other treatments after 24-h. Furthermore, the overall mean reduction in methane for PR-L (16-18%) was lower (P < 0.05) compared than other treatments. This was despite altering the dose of PR-L x 1.5 during the second week. The antimethanogenic activity of all treatments occurred without a decrease in total gas production, total VFA concentration and substrate dry matter (DM) disappearance in vitro. The persistent and greater impact on methane abatement, combined with an absence of negative effects on in vitro fermentation characteristics, suggests that AC formulations should be prioritized for progression to in vivo experiments. Background Halogenated compounds such as bromoform have potential as feed additives for mitigating methane emissions by ruminant livestock. These compounds can be produced synthetically, but they are also produced by living organisms. A well-publicized example of naturally produced halogenated compounds (organohalogens) is the seaweed Asparagopsis sps., which reduces methane emissions when included in the diets of sheep, beef and dairy cattle (Li et al., 2016; Roque et al., 2019; Kinley et al., 2020). Organohalogen production is not confined to seaweed. Many living organisms, including fungi, use haloperoxidase (HPO) enzymes to catalyze the biosynthesis of organohalogens (Wever & Barnett., 2017). Then inventors have developed an industrially scalable source of bromoform derived from a naturally occurring fungal isolate. Manufacturing involves liquid fermentation followed by biocatalysis, whereby bromoform is present in the liquid phase at completion of the process. During an initial project, the antimethanogenic activity of raw fungal extracts yielded completion of manufacturing were tested in vitro. During a 24-hr in vitro fermentation assay using oaten chaff as a substrate, both the supernatant (liquid fraction) and homogenate (liquid fraction plus biomass) had similar antimethanogenic activity compared to analytical bromoform. However, these sources of bromoform were highly volatile, indicating the raw liquid extracts are not suitable for application by the livestock industry at scale. In addition to the inherent volatility of aqueous bromoform, liquids have a high cost of transport and are not universally applicable across different feed manufacturing processes and delivery mechanisms for livestock. To address these limitations, the inventors have developed thermally stable, dry formulations as carriers for bromoform. The formulations utilize inert, high surface area materials that have significant bromoform sorption capacity. The high surface area materials are recognized as being safe for use in food supply chains and are relatively inexpensive. During the development of the formulations, it was demonstrated that the different high surface area materials, methods of loading, as well as the proportional loading of bromoform onto these materials, resulted in different stability profiles over time. It was hypothesized this may extend to different release characteristics of bromoform in the rumen environment. Hence, there may be differences in the efficacy of different high surface area materials loaded with bromoform when used for the purposes of enteric methane emissions reduction. Methodology The specific objectives of this in vitro trial were to: - Evaluate antimethanogenic activity and impact on rumen fermentation characteristics of four novel powdered formulations at several doses compared with analytical bromoform. - Assess the persistency of effects upon methane production and rumen fermentation characteristics of selected formulations. A two-step approach involved screening using a 24-h in vitro batch fermentation (IVFT) assay (Experiment 1) and an artificial rumen simulation technique (RUSITEC; Experiment 2) over two weeks. IVFT assay An IVFT batch fermentation assay (Durmic et al. 2014) was initially used to determine antimethanogenic activity and effects on rumen fermentation of the formulations. The assays were 24-h batch cultures using rumen fluid collected from three rumen fistulated sheep. Animals were managed according to protocol 2021 / ET001086 approved by the University of Western Australia Animal Ethics Committee. Briefly, 100 mg substrate (oaten chaff ground to pass a 1 mm screen) and the treatments were added to 30 mL vials one day prior to the assay and left overnight in an anaerobic chamber. The following day, 10 mL of rumen fluid inoculum (rumen fluid and buffer (McDougall, 1948) in a 1:1.5 (v / v) ratio) was added to each vial. The vials were stoppered, crimped and incubated with shaking at 39°C for 24-h. Controls consisted of a substrate control of 100 mg oaten chaff with buffer, and a negative control of buffered rumen fluid only. Each treatment was tested in triplicate. Total gas production was measured using a pressure transducer. Subsamples of the headspace were collected for methane quantification by gas chromatography. Samples of the fermentation fluid were taken for analysis of VFA and NH3 after 24 h. RUSITEC The RUSITEC system was set up and maintained as described by Garcia et al. (2019). Briefly, 600mL of rumen fluid inoculum (rumen fluid and buffer (McDougall, 1948) in a 1:1.5 (v / v) ratio) was added to each fermenter (n=16, 1 L total volume each). Oaten chaff was included in the fermenters in two small nylon bags (7x17 cm, 50 pm pore size), each containing 15 g DM, and the contents of one bag was replaced each day. A constant flow of buffer into the fermenters was used to maintain a pH of 6.0 - 7.0. The first 7 days consisted of a pre-treatment period for the fermentation to stabilize, with treatments added on Day 8 through to Day 21. The gas produced by each fermenter was measured daily and samples were collected for methane quantification (days 8-21). Samples of fermentation fluid were collected for analysis of VFA and NH3 at three times throughout the experiment (days 7, 15 and 21). VFA andNH3 Fermentation fluid was analyzed for VFA by the Department of Primary Industries and Regional Development Diagnostics and Laboratory Services (South Perth, WA) by a gas chromatography (GC) separation of VFA C2-C5 (Supelco Bulletin no.749D) using an external standard Supelco Volatile Free Acid Mix (Cat No. CRM46975, Sigma-Aldrich Australia) and an internal standard 0.9 mM 3-Methyl Valeric acid in 2.24 w / v% Phosphoric acid (Cat No. 222453 & 30417, Sigma-Aldrich Australia). Concentration of NH3 was measured spectrophotometrically using the Berthelot method (Smith & Murphy, 1993). Statistical Analysis Statistical differences between treatments were analyzed by one-way ANOVA in JMP 15.2.0 (SAS Institute, Inc.) with the Tukey-Kramer HSD post-hoc test to compare means. Data reported is arithmetic means ± SEM. Statistical analysis of the RUSITEC is detailed below. Experiment 1: IVFT Screening of Stabilised Formulations Background The objective of this experiment was to evaluate the antimethanogenic activity and effects upon rumen fermentation characteristics of the stabilized formulations using the in vitro batch fermentation technique (IVFT). The experiment compared efficacy of the novel formulations against an analytical bromoform standard when included at the same dose. Materials and Methods The IVFT assay was carried out as described above. Twelve different treatments were used tested. These consisted of three different formulations, Cl - C3, which had been prepared to provide four different concentrations of bromoform (0.5, 1,5, 10 pMj.The powdered formulations were; activated charcoal powder (Sigma-Aldrich, C9157, batch BCCG3575) loaded with 15% weight bromoform (Cl), a porous cross-linked polymer resin (PuroSorb™ PAD600) loaded with 10% weight bromoform (C2) and PAD600 loaded with 1% weight bromoform (C3). Each treatment was diluted with talc powder (Sibelco, Singapore; D98 29.9 pm, D50 9.3 pm) to achieve constant addition weight of 20 mg per in vial. The treatments and substrate were added to each vial and placed into an anaerobic chamber at 39°C for 24 h prior to commencement of the assay. In addition to a rumen fluid inoculum treatment only, a rumen fluid inoculum and substrate treatment, a control (CO) of amylene-stabilized analytical bromoform (Sigma Aldrich Cat. No. 36972) was used as a comparison. Analytical bromoform was prepared as a 1000 pM stock solution in DI water and added at four volumes (5, 10, 50 and 100 pL) to achieve the same four concentrations per assay volume of 10 mL. The bromoform standard (CO treatment) was added to the allocated vials just prior to the rumen fluid inoculum. Results In Vitro Gas Production and Methane Treatment Cl at 5 pM CHBr3 and Cl at 10 pM CHBr3 significantly inhibited methane concentration and yield by 90 - 97% compared with the substrate control (p<0.05, Table 1). There 5 was no statistical difference in efficacy between these two treatments. The bromoform standard, CO, was equally active, reducing methane concentration and yield by 96 - 97% at 5 pM and 10 pM (p<0.05), respectively. Treatment C3 at 10 pM CHBr3 reduced methane concentration and yield by 50 % (p<0.05). At 0.5 pM, treatments Cl and C3 increased methane yield by 12 to 19% (p<0.05). 10          Except for C2, all treatments increased total gas production compared to the substrate control (P<0.05), however, this did not occur in a dose dependent manner and the magnitude of any increase was small Table 4). The largest increase in total gas production over 24 h by 10% for C3 asl pM CHBr3 (p<0.05). 15 Table 4. IVFT methane and gas production (mean ± SE) when incubated with oaten chaff substrate. Within the same column, values with different superscripts are significantly different (p<0.05). Percentage reduction when compared to oaten chaff substrate control, negative values indicate an increase. DMi = dry matter incubated. Treatment Total gas volume (mL) CH4 concentration (mL / 100 mL gas) CH4 yield (mL / g DMi) % reduction Total CH4 CH4 gas cone, yield Substrate control 34.7de ± 0.27 8.1ab ± 0.97 28.1bc± 3.61 Bromoform 0.5 34.6e ± 0.23 8.6ab±0.88 29.7^13.05 0       -5      -5 (CO) 1 35.0bcde±0.20 9.4a ± 0.28 32.8abc ± 0.92 -1      -4      -5 5 34.9bcde ± Q29 0.4d±0.04 1.2e ± 0.12 -1     96    96 10 36.9a± 0.20 0.2d ± 0.00 0.9e ± 0.02 -6     97    97 Cl           0.5 36.1abcd±0.07 9.9a±0.02 35.7a± 0.02 -4     -10    -14 1 35.8abcde± 0.11 9.8a±0.12 35.0ab ± 0.40 -3     -10    -13 5 34.9bcde ± 0.48 0.7d±0.20 2.6e ± 0.66 -1     90    90 10 36.0abcde± 0.12 0.3d ±0.02 1.0e ± 0.07 -4     97    97 Treatment Total gas volume (mL) CH4 concentration (mL / 100 mL gas) CH4 yield (mL / g DMi) % reduction Total ch4 ch4 gas cone. yield C2 0.5 36.1abcd± 0.14 9.7a±0.29 35.3ab ± 1.18 -4 -8 -12 1 36.0abcde±0.17 9.3ab ± 0.44 33.4abc±1.54 -4 -3 -7 5 35.5abede±o.39 9.6a±0.15 33.9abc±0.40 -2 -7 -9 10 36.2abc± 0.22 9.5a±0.15 34.2ab±0.81 -4 -6 -10 C3 0.5 36.8a±0.34 10.2a±0.17 37.6a±0.78 -6 -13 -19 1 34.9bcde±0_25 9.3ab ± 0.23 32.7abc±1.00 -1 -4 -4 5 36.3ab ± 0.49 7.3b±0.58 26.5C±1.87 -5 19 15 10 34.8cde±0.34 4.5c±0.52 15.6d ± 1.98 0 50 50 VFA andNH3 The total VFA concentration was increased significantly at 0.5 pM for the bromoform standard and Cl, but not at other concentrations nor treatments. (p>0.05, Table 5). The bromoform 5 standard CO at 0.5 pM, Cl at 0.5 pM and C2 at 1 pM demonstrated increased acetate (p<0.05), whereas Cl atlO pM decreased acetate (p<0.05). Propionate was increased in CO and Cl at both 5 pM and 10 pM (p<0.05), respectively, accompanied by a significant reduction in the A:P ratio and a decrease in the concentration of NH3 (p<0.05). The A:P ratio was reduced, by C3 at 5 pM and 10 pM, respectively (p<0.05). 10 Table 5. IVFT VFA and NH3 concentrations (mean ± SE) when the treatments were incubated with oaten chaff substrate. Within the same column, values with different superscripts are significantly different (p<0.05). A:P = Acetate to propionate ratio. Treatment Total VFA Acetate Propionate Butyrate A:P NH3 mmol / L mg / L Substrate 83.0cd± 2.90 52.8cde± 1.87 19.3de ± 0.68 8.55ab ± 0.30 2.74ab ± 0.01 69.9a± 6.16 control Bromoform 0.5 94.2ab ± 2.51 61.1ab± 1.85 21.3bcd± 0.50 9.31a± 0.13 2.86a ± 0.02 57.5abc± 3.57 (CO) 1 87.8abcd ± 2.24 55.7bcd± 1.53 20.5de ± 0.50 9.02ab ± 0.18 2.71bc ± 0.02 60.8ab ± 1.63 5 84.6bcd±1.44 48.6ef ± 0.79 24.0ab ± 0.49 9.59a± 0.12 2.03ef ± 0.01 40.4cd± 0.71 10 85.0bcd±0.70 48.6ef±0.46 24.5a±0.19 9.56a±0.07 1.99ef ± 0.01 46.0bcd±0.90 Cl 0.5 97.4a± 1.58 63.3a± 1.01 22.1abcd± 0.42 9.39a± 0.12 2.86ab ± 0.01 55.3abcd± 5.15 1 91.9abc± 2.15 59.2abc± 1.34 21.0de ± 0.54 9.15a± 0.21 2.82ab ± 0.02 52.5abcd± 3.47 5 84.0bcd± 1.23 48.6ef ± 0.80 23.7abc ± 0.32 9.39a± 0.13 2.05e ± 0.01 38.0d ±4.19 10 82.7cd± 1.69 46.3f ± 1.18 24.3a±0.33 9.67a± 0.15 1.91f ± 0.01 43.9bcd± 1.15 C2 0.5 92.2abc ± 0.50 59.0abc ± 0.33 21.2cde ± 0.09 9.35a± 0.07 2.78ab ± 0.01 52.5abcd± 2.66 1 92.5abc ± 0.39 59.3ab ± 0.24 21.3cd± 0.12 9.37a± 0.03 2.79ab ± 0.05 52.3abcd± 0.58 5 90.2abcd ± 3.56 57.3abcd±2.79 21.1bcde ± 0.65 9.30a± 0.10 2.71abc ± 0.01 58.7abc ± 9.78 10 92.3abc ± 0.37 59.1abc± 0.23 21.4bcd± 0.13 9.29a± 0.05 2.76ab ± 0.02 55.9abcd± 1.87 C3 0.5 90.4abcd ± 1.22 57.8abcd ± 0.79 20.8de ± 0.40 9.23a± 0.05 2.78ab ± 0.08 54.4abcd± 0.58 WO 2025 / 141536                                   PCT / IB2024 / 063261 4— 00 Treatment Total VFA Acetate Propionate mmol / L 1     80.8d±2.99 52.1def ± 1.40 18.6e±0.90 5     89.0abcd±2.08 55.4bcd± 1.01 21.5bcd± 0.73 10   80.3d±2.11 48.6ef ± 1.26 20.3de ± 0.59 Butyrate A:P           NH3 mg / L 7.96b ± 0.57 2.81ab±0.04        59.1ab±1.03 9.29a±0.24 2.58c±0.01         57.1abc±4.82 8.90ab ± 0.22 2.39d±0.01         54.6abcd± 3.82 WO 2025 / 141536                                   PCT / IB2024 / 063261 Discussion Activated charcoal loaded with bromoform at 15% (Cl) was the most effective stabilized carrier, reducing methane concentration and yield by more than 90% when included at 5 pM and 10 pM CHBrs. This antimethanogenic effect was, however, not significantly different compared to analytical bromoform (CO). This suggests similar efficacy between CO and Cl; however, a limitation of the experimental procedure is that sub-sampling of gas for methane analysis occurs after 24 hr of incubation. The pattern of methane reduction over time is therefore unknown and such information would at least partially enable comparison of the rate of bromoform release between the stabilized carriers and against analytical bromoform. Furthermore, to assist making a direct comparison of methane abatement efficacy between the different carriers, treatments should have included different carriers when loaded with the same proportional amounts of bromoform. In the current study, activated charcoal was loaded at 15% bromoform in Cl, whereas bromoform was loaded onto PAD600 at either 10% (C2) or 1% (C3), which makes interpreting and comparing the results from activated charcoal and polymer resin treatments more difficult. Nonetheless, it is difficult to reconcile the large effects on methane abatement demonstrated in CO and C1 at 5 pM and 10 pM, with the apparent lack of effect for the polymer resin treatments, C2 and C3, especially when included at higher bromoform concentrations. The data also indicated that whilst activated charcoal had greater stability compared to loading bromoform onto PAD600 alone, polymer resin loading vastly improved stability compared to bromoform in a water solution. We therefore expected at worst, C2 and C3 would demonstrate broadly similar effects on methane production to CO, which is known to be highly volatile. In addition, we did not expect to see much effect of C3 on methane concentration or yield regardless of the bromoform concentration. This expectation is because bromoform loaded at 1% onto PAD600 is more tightly bound compared to 10% loading (C2 and C3). In contrast, the experimental results for C3 demonstrated an antimethanogenic effect at 10 pM CHBrs, which inhibited methane concentration and yield by up to 50% compared with the substrate control, and even greater inhibition compared to C2. The unexpected results for the polymer resin treatments (C2 and C3) may be explained by heterogeneity of the samples used in the experiment. The scale of this experiment required only the addition of micrograms of the stabilized formulations to achieve the desired bromoform concentrations. Consequently, the stabilized formulations were extended with talc powder, to attain a dose that could be accurately measured. The activated charcoal and talc were of similar particle size and likely achieved homogeneity. However, the polymer resin beads are considerably larger (300 - 1200 pm) and were ground before extending with talc powder. It is plausible that homogeneity of the diluted samples was compromised during this process, leading to these unexpected and variable results. There were no decreases in total VFA concentration across all treatments, indicating no adverse effects on in vitro fermentation. The four treatments which inhibited methane by more than 90% (CO and Cl at both 5 and 10 pM), were associated with significantly increased propionate and decreased NH3 in the fermentation fluid. These treatments also decreased the A:P ratio, as did C3 at 5 pM and C3 at 10 pM. Methane is the main sink of hydrogen in the rumen (Janssen 2010) but when methanogenesis is inhibited, propionate is considered to become the major hydrogen sink Ungerfeld, 2015). This is consistent with effects demonstrated in the current study. The lower NH3 concentrations observed in CO and Cl when methane production was inhibited suggests that that the breakdown of protein and deamination of dietary amino acids decreased, which may improve protein utilization efficiency by the animal. The increase in propionate and decrease in NH3 has been previously reported by Machado et al. (2016) when testing analytical bromoform in vitro. Overall NH3 concentrations were relatively low in this experiment because of the low-quality substrate, oaten chaff. The reductions of NH3 when methane was inhibited resulted in NH3 concentrations below 50 mg / L, which is considered the minimal threshold to maximize growth rates of rumen bacteria (Satter and Slyter, 1974). Hence it is possible that when methane is inhibited by ruminants consuming a low-quality forage diet, there is a greater risk of microbial protein synthesis declining in the absence on non-protein nitrogen supplementation. The significant antimethanogenic activity, combined with an absence of negative effects on rumen fermentation characteristics, of formulation C1 at concentrations of 5 and 10 pM CHBr3 over 24 h in vitro confirms that activated charcoal has potential as a carrier for fungal derived bromoform. Despite inconsistent experimental results amongst the polymer resin treatments, formulation C3 at 10 pM demonstrated an antimethanogenic effect of approximately 50%, suggesting that PAD600 is also a suitable candidate. Example 13. Further Testing the Effects of Stabilised Bromoform Feed Additives at Different Concentrations on In Vitro Fermentation Background The high surface area carriers, activated charcoal and the polymer resin PAD600, were demonstrated to significantly facilitate inhibition of methanogenesis when loaded with bromoform in vitro compared with an oaten chaff control. Activated charcoal loaded at 10% bromoform was the most active and had similar activity to analytical bromoform standard (> 90% reduction in methane at doses of 5 and 10 uM). The polymer resin, PAD600, at 1% loading of bromoform at the concentration inhibited methane by 50% when dosed at 10 10 uM compared with the control. Whilst experiment in Example 12 established that these formulations had an antimethanogenic effect over 24 hr, it is unknown whether the effect persists over a longer period. This experiment evaluated both activated charcoal and the PAD600 as carriers when included at 2.5 uM and 5 pM, respectively, using the rumen simulation technique, (RUSITEC methodology described above) to determine persistency over time in an open system and define any carry-over effect after withdrawal of the treatment from the system. Materials and Methods Treatments The carriers were activated charcoal (AC) and the polymer resin PAD600 (PR), which were both loaded with 15% analytical grade bromoform. During the experiment, the carriers were dosed at either a low (2.5 pM; L) or high (5 pM; H) concentration, creating 4 treatments; AC-L, AC-H, PR-L and PR-H. A substrate only treatment was included as a control during the experiment. Each treatment was replicated in triplicate across the fermenters (n = 15). The calculated concentrations of bromoform assumed the volume of fluid in each fermenter was maintained at 600 mL. When expressed as mg bromoform / kg of total DM substrate incubated daily (total 30 g DM oaten chaff), this equated to 12.6 and 25.2 mg bromoform / kg, respectively, for the low and high dose. The treatments were supplied in sealed 100 mL glass vials and stored at room temperature before and during the experiment. Consistent with the IVFT 24 hr experiment, the PAD600 treatments were ground prior to mixing with talc powder in an attempt to improve homogeneity of the formulated doses. The bromoform loaded carrier treatments were extended with talc powder so that the addition of 20 mg achieved the target bromoform concentration in each fermenter. The particle size of talc powder and AC was less than the nylon bag pore size, raising concerns that these materials would immediately wash out of the bags when submerged in fluid. Consequently, treatments were weighed out and placed into small envelopes created from folded ashless filter paper (Whatman Grade 2 cellulose filter papers, Cytiva, USA). Each envelope containing the designated treatment was placed into a nylon bag with the oaten chaff substrate 24 h prior to fermentation in each of the 16 RUSITEC vessels. RUSITEC Experiment The RUSITEC was managed as described by Garcia et al. (2019). Rumen fluid (240 mL) and buffer (360 mL) were added to each fermenter with one bag of digesta and one bag of oaten chaff substrate (15 g, ground to pass a 4 mm screen). After the pH had stabilized (~4 h), buffer was added to each fermenter at a rate of 30 mL / h using a peristaltic pump. The pH of the fermentation fluid was monitored daily and maintained between 6 and 7. On day 2, the bag of rumen digesta was removed and replaced with another containing oaten chaff. Every day thereafter, one nylon bag containing oaten chaff was refreshed (so each bag remained in the fermenter for 48 h). There were three periods in the RUSITEC trial: - Day 1 to 7 - pre-treatment period with oaten chaff substrate only (CONTROL). - Day 8 to 15 - first week of treatment, oaten chaff and treatments as described above (Table 6). - Day 16 to 22 - during the second week, control, AC-L and PR-H treatments were maintained as the first week. However, the PR-L dose rate was increased to the equivalent of 30 mg / d (x 1.5 dose from week 1). In addition, treatment AC-H was ceased to investigate the potential carry-over effect after withdrawal from the fermentation vessel. The pH, total gas production and methane concentration were measured daily. Samples of fermentation fluid were collected for measurement of VFA and NH3 concentrations at three timepoints; Day 8 (immediately before treatments commenced), 15 (after 7 days of treatment) and 22 (after 14 days of treatment). Table 6. Treatments added per RUSITEC fermenter. Treatment n Target bromoform concentration* (pM) Dosage Day 8-15 (mg / d) Dosage Day 16-22 (mg / d) Control 3 0 0 0 PR-L 3 2.5 20 30 AC-L 2* 2.5 20 20 PR-H 3 5.0 20 20 AC-H 3 5.0 20 0 ^Target bromoform concentration when 20 mg of treatment formulation added to fermenter maintained at 600 mL working volume *One fermenter containing an AC-L treatment was consistently different from the other two replicates and this data was excluded from the analysis In Vitro Substrate Disappearance The oaten chaff substrate was collected for apparent DM disappearance determination on Day 8 (immediately before treatments commenced), 15 (after 7 days of treatment), and 22 (after 14 days of treatment). On the day of collection, the nylon bag which had been in the fermenter for 48 h was removed, immediately placed on ice and rinsed under cold water until the run-off was clear. After cleaning, the bags were then placed in a 60°C oven for at least 48 h or until a constant weight was attained. The dried bags were weighed to calculate apparent dry matter (DM) disappearance based on weight differential on a DM basis. The residual dried oaten chaff substrate was ground through a 1 mm screen and analyzed for neutral detergent fiber (NDF), acid detergent fiber (ADF), inorganic ash (Van Soest et al., 1991). Organic matter (OM) was calculated by difference following analysis of ash content. Statistical Analysis The data was analyzed per timepoint using one-way ANOVA with treatments as fixed factors and methane concentration, methane production, total gas, fermentation and substrate parameters as variables. A Tukey-Kramer HSD pairwise comparison was used when means differed (p<0.05). Data points were removed from analysis if functional issues with the RUSITEC system occurred; namely where buffer lines were not flowing properly or if a leak was detected. Throughout the experiment, fermenter 1, which contained an AC-L treatment, was consistently different from the other two replicate vessels. Thus, the data for fermenter 1 was excluded from the analysis, leaving two replicates only for treatment AC-L. Results Gas and Methane An increase in mean total gas production during the first week of treatment was evident in with AC- H (P < 0.05; Table 4). Daily gas production of AC-H was greater than the control on Day 11 (p<0.05, FIG. 4A). All treatments significantly reduced methane concentration and yield compared to the substrate only control, irrespective of week (p<0.05, Table 7). Treatment PR-L significantly (p<0.05) reduced methane yield by 16% in both the first and second weeks of treatment. However, statistical differences for reductions methane concentration and yield did not occur until Day 11, 5 three days after treatment began (FIGs. 4B and 4C). Treatment PR-H reduced methane yield by 76% in the first week and 41% in the second week of treatment (p<0.05). The treatments AC-L and AC-H reduced methane concentration and yield by 89- 91% and 97-99% in the first and second weeks of treatment, respectively. The significant methane inhibition caused by AC-L, PR-H and AC-H occurred within the first 24 h 10 after treatments were added and was maintained throughout the two weeks, except for treatment PR-H, whereby methane yield increased and was similar to the control on Days 20 and 22 (FIG. 4C). Table 7. Total gas production, methane concentration, and methane yield (mean ± SE). Values 15 were statistically compared within the same column and time, values that do not share the same letter are significantly different (p<0.05). Percentage reduction when compared to oaten chaff substrate control. Second week of treatment = AC-L and PR-H added daily (continued at 20 mg), PR-L added daily (increased to 30 mg), AC-H treatment ceased. DMi = dry matter incubated. Treatment Gas production CH, concentration CH4 yield (mL / g DMi) % reduction (mL / 24h) (rnL / 100 rnL gas) CH4 CH4 yield cone. Pretreatment Control 829 ±38.4 5.21 ±0.23 1.43 ±0.06 - - PR-L 885 ±20.1 4.57 ±0.22 1.35 ±0.05 - - AC-L 843 ± 28.24 4.87± 0.11 1.37 ±0.04 - - PR-H 828 ± 34.2 4.25 ± 0.47 1.17± 0.14 - - AC-H 881 ±26.6 5.31 ±0.38 1.38 ±0.08 - - First week of treatment (Day 9 - 16) Control 879b±21.2 5.40a± 0.15 1.57a± 0.04 - - PR-L 882b ± 26.7 4.49b ± 0.13 1.32b ± 0.04 17 16 AC-L 932b ± 23.5 0.47c ± 0.17 0.15c± 0.07 91 90 Treatment Gas production (mL / 24h) CH4 concentration (rnL / 100 mL gas) CH4 yield (mL / g DMi) % reduction CH4 cone. CH4 yield PR-H 950b ± 24.5 1.21c± 0.21 0.37c± 0.06 78 76 AC-H 1066a±18.6 0.52c ± 0.23 0.17c ± 0.08 90 89 Second week of treatment (Day 17-22) Control 826 ±20.2 5.13a± 0.18 1.40a± 0.04 - - PR-L 848 ±28.8 4.20b ± 0.21 1.17b ± 0.06 18 16 AC-L 758 ±30.5 0.15d± 0.03 0.04d± 0.01 97 97 PR-H 795 ± 17.7 3.17c ± 0.26 0.83c± 0.06 38 41 AC-H 825 ±16.9 0.07d± 0.03 0.02d ± 0.01 99 99 In Vitro Substrate Disappearance All four treatments resulted in similar (p>0.05) NDF disappearance, ADF disappearance and in vitro DMD when compared to the control at all three sampling timepoints (Table 8). The 5 in vitro OMD for treatment AC-L was 9% lower than the control on Day 22 (p<0.05). Table 8. Percentage in vitro disappearance (mean ± SE) of oaten chaff substrate in the RUSITEC system at three time periods; the end of pre-treatment period (Day 8), the end of the first treatment week (Day 15), and the end of the second treatment week (Day 22). Within the same column and 10 timepoint, values not connected by same letter are significantly different (p<0.05). NDFD - NDF disappearance, ADFD - ADF disappearance, IVDMD - in vitro dry matter disappearance, OMD - in vitro organic matter disappearance. Treatment NDFD ADFD IVDMD IVOMD Day 8 Control 23 ±1.41 19 ±1.20 37 ±0.63 38ab± 0.96 PR-L 22 ± 0.44 20 ±0.51 36 ±0.73 37b ± 0.56 AC-L 22 ±1.34 19 ±0.89 38 ±0.61 39ab± 0.67 PR-H 22 ±1.33 19 ±1.34 37 ±0.62 38ab± 0.55 AC-H 25 ± 0.99 22 ±1.08 39 ±0.62 40a ± 0.51 Day 15 Treatment NDFD ADFD IVDMD IVOMD Control 25 ± 0.64 22 ± 0.75 42 ±0.58 44 ±0.34 PR-L 23 ± 0.99 20 ±1.40 41 ±0.28 43 ± 0.32 AC-L 23 ± 0.60 19 ±0.86 41 ±0.75 44 ±0.29 PR-H 22 ±1.50 19 ±0.99 40 ±0.65 42 ±0.81 AC-H 24 ±0.33 22 ± 0.62 42 ± 0.60 43 ± 0.48 Day 22 Control 23ab ± 0.96 18ab ± 1.59 41ab ± 0.39 46ab±0.30 PR-L 21b±0.57 15ab ± 0.22 40b ± 0.33 43bc± 0.78 AC-L 22b ± 0.64 16ab ± 1.35 40b ± 0.29 42c ± 1.07 PR-H 21b ± 0.91 14b±1.36 39b ± 0.52 44bc±0.39 AC-H 25a± 1.13 20a± 1.49 43a± 0.92 48a± 0.91 VFA and pH Total VFA concentrations were similar (p>0.05) between all treatments and control at all three sampling timepoints (Table 9). Compared to the control, acetate concentrations were reduced 5   in treatment AC-H on Day 15 and by AC-L on Day 22 (p<0.05). Treatments AC-L, PR-H and AC- H had lower acetate to propionate ratios on Day 15 compared with the control (p<0.05). Valerate concentrations were significantly higher compared to the control on Day 15 and Day 22 with both treatments AC-L and AC-H. The pH was similar between control and treatments at each sampling time. The concentration ofNH3 was 74% greater than the control (p<0.05) on Day 15 for treatment 10 AC-L. Table 9. Fermentation parameters (mean ± SE) in the liquid phase at three timepoints; the end of pre-treatment period, 6 d post treatment and 12 d post treatment. During the pre-treatment period, all fermenters represent control only. Within the same column and timepoint, values not connected by same letter are significantly different (p<0.05). Abbreviations: VFA = volatile fatty acids, A:P = acetate to propionate ratio, BCFA = branch-chained fatty acids. Treatment pH Total Acetate Propionate Butyrate Valerate Caproate BCFA A:P NH3 VFA mmol / L                                              mg / L Day 8 Control 6.3 ± 0.03 101 ±4.12 46.31 ±2.21 22.86 ± 1.28 19.70 ±0.77 8.46 ±0.39 2.31 ±0.13 1.70 ±0.06 2.03 ±0.07 10.39 ±0.98 PR-L 6.0 ± 114 ±3.80 52.74 ±0.50 24.28 ±0.69 23.26 ±2.74 9.46 ±0.62 2.86 ±0.55 1.90± 0.14 2.18 ±0.08 7.69 ±0.42 0.03 AC-L 6.3 ± 106 ±3.05 48.72 ±1.04 22.68 ±0.94 21.54 ±1.48 8.91 ±0.46 2.71 ±0.09 1.85 ±0.22 2.16±0.10 7.56 ±0.67 0.07 PR-H 6.2 ± 111 ±1.73 51.43 ±0.28 24.68 ±2.35 21.26 ±1.84 8.80 ±1.05 2.54 ±0.45 2.03 ± 0.28 2.12 ±0.18 8.25 ± 0.82 0.11 AC-H 6.2 ± 103 ±3.65 48.01 ±2.33 21.63 ± 1.93 20.92 ±0.43 8.11 ±0.22 2.71 ±0.31 1.69 ±0.04 2.24 ± 0.09 7.92 ± 0.29 0.12 Day 15 Control 6.2 ± 107 ±6.49 51.02a±4.10 21.74 ±3.47 21.18 ±0.52 6.92c±0.24 2.77bc± 3.42bc± 0.29 2.42a±0.24 7.87b ± 0.60 0.12 0.47 PR-L 6.3 ± 109 ±6.65 49.16a± 3.06 26.41 ±2.78 20.41 ±2.42 7.47bc± 1.20 2.46bc± 3.39bc± 0.14 1.88ab± 7.18b ± 0.46 WO 2025 / 141536                                   PCT / IB2024 / 063261 Treatment pH Total Acetate Propionate Butyrate Valerate Caproate BCFA A:P NH3 VFA mmol / L mg / L 0.08 0.19 0.09 AC-L 6.4 ± 119± 13.3 37.34ab ± 1.98 26.12 ±0.95 28.16 ±7.60 18.50a ±4.66 3.83ab ± 4.93ab ± 0.32 1.43b ± 0.13 13.70a ±2.74 0.03 0.32 PR-H 6.2 ± 96± 11.88 37.28ab ±4.97 26.82 ±2.95 16.28 ±2.05 8.01bc± 1.00 1.30c ±0.26 6.36a± 0.99 1.38 b± 0.03 9.92ab ± 0.56 0.06 AC-H 6.2 ± 92 ±5.10 29.97b ± 1.29 19.38 ±1.82 20.95 ±1.08 14.67ab ± 1.02 4.42a ±0.42 2.15c± 0.29 1.56b ± 0.10 7.5 8b ± 0.24 0.07 Day 22 Control 6.4 ± 96 ±3.45 44.49ab±1.05 23.17 ±3.50 17.23 ±0.86 6.10b ± 0.19 1.52 ±0.48 3.33b ± 0.33 2.01 ±0.31 4.44 ±2.08 0.03 PR-L 6.3 ± 110 ±6.80 50.16a± 3.05 29.50 ±2.45 18.45 ±2.15 6.66b ± 0.99 1.33 ±0.12 4.20b ± 0.32 1.71 ±0.05 2.90 ±1.98 0.08 AC-L 6.4 ± 95 ±4.06 31.85c± 1.02 23.1 ±0.43 19.54 ±1.93 12.35a ± 0.97 2.43 ± 0.22 5.28ab ± 0.62 1.38 ±0.06 6.10 ±2.75 0.04 PR-H 6.4 ± 97 ±3.21 37.49bc ±2.72 25.06 ±0.30 17.54 ±1.26 9.28ab ± 0.16 0.99 ±0.08 6.62a± 0.29 1.50 ±0.12 6.17 ±2.07 0.02 AC-H 6.2 ± 101 ±3.55 36.93bc ± 0.47 27.06 ±0.80 16.96 ±1.52 12.13a± 0.95 2.35 ±0.51 5.09ab ± 0.47 1.37 ±0.05 5.17± 1.98 0.04 WO 2025 / 141536                                   PCT / IB2024 / 063261 Discussion The carriers AC and PR both had significant antimethanogenic effects in the RUSITEC system, however, AC demonstrated greater efficacy in reducing methane. Treatments PR-H, ACL and AC-H caused a significant reduction in methane concentration and yield within 24 h. Treatment PR-H demonstrated a mean reduction of 77% in the first week, which lessened to reductions of 38-41% in the second week. Treatments AC-L and AC-H were stronger inhibitors of methane, causing reductions of 89-91% in the first week and 97-99% in the second week, and were not statistically different in activity. Treatment PR-L was the least active, causing significant reductions in methane concentration and yield within 72 h of addition, with average reductions between 16-18% in the first and second weeks. The antimethanogenic activity of AC-L and AC-H persisted for the duration of the two-week experiment. After daily dosing of AC-H ceased on Day 16, methane concentration and yield remained low in the second week. This persistent activity occurred without a decrease in total gas production or VFA concentrations. There were some shifts in the VFA profile with these treatments, including a lower acetate to propionate ratio after one week of treatment, and increases in valerate on both Day 15 and 22. The AC formulation did not affect substrate disappearance, apart from 9% lower in vitro OMD for the low dose on Day 22. This reduction was not seen with the higher dose of AC, nor did the low dose significantly affect other substrate disappearance measurements. These results indicate that the AC formulation did not affect the extent to which the microbial population was able to ferment the oaten chaff substrate. Coupled with the VFA concentrations and profile, this indicates there were no significant negative effects on fermentation. Treatment PR-H significantly reduced methane concentration and yield compared to the control until Day 20, when methane yield was similar to the control. On average, the reduction in methane yield caused by PR-H was 76% in the first week of treatment, compared with 41% in the second week. The dose rate of PR-L was increased by 50% on Day 16. Consequently, methane concentration and yield decreased compared with the control on Day 18, but did not alter the methane mitigation over the second week. The PR formulations did not demonstrate any negative effects on fermentation, with total gas and total VFA concentrations remaining similar to the control. The lower and less persistent activity of the PR formulations compared to the AC may be due to volatilization of bromoform. Although the samples were not measured at the end of the experiment to confirm the bromoform concentration, previous bench top studies have indicated that due to bromoform loaded onto PR at 10-20% w / w are less stable during storage than AC carriers at similar loading. Whilst the samples were refrigerated for storage, which generally lowers the risk of volatilization, some bromoform may have volatilized during the 24 h of exposure to laboratory temperatures whereby the treatment had been weighed into the filter paper envelopes and placed into the feed bags prior to placement into the fermenters. In agreement with the AC formulation, the PR had no effect on substrate disappearance, suggesting that fermentation was not affected by this formulation. This experiment has confirmed that the AC and PR carriers loaded at 15% bromoform have antimethanogenic activity in vitro which persisted for up to two weeks in the RUSITEC, however the PR carrier was associated with a diminishing response over time. The AC formulations were more effective than the PR, with both high and low doses inhibiting methane by more than 90% compared to the control. There appears to be an opportunity to further reduce the concentration of bromoform on AC given almost complete inhibition at 2.5 uM. After daily dosing of the high dose of AC ceased on Day 16, the methane inhibition persisted. The PR formulation also demonstrated significant antimethanogenic activity, with a maximum of 78% reduction in methane concentration in the first week with the high dose. Both AC and PR formulations reduced the amount of methane produced in the RUSITEC without affecting overall fermentation. Example 14. Validation of an Inert Carrier for Fungal Derived Bromoform to Deliver Mitigation Outcomes in Beef Cattle Introduction Strategies to reduce enteric methane emissions by targeting the key steps of methanogenesis are considered to have the most utility to achieve abatement targets for the livestock sector (Martinez- Fernandez et al., 2018). These inhibitors are either structural analogues of methyl-donor compounds (CHCh, CH2BrCl, CHBn) targeting the methyltransferase complex or analogues of methyl-coenzyme M inhibiting methyl-coenzyme M reductase enzyme. Bromoform is a halogenated methane analogue which inhibits methylcobalamine synthesis (Wood et al., 1968) and is recognized as a direct abatement tool for sheep and cattle (Li et al., 2016; Kinley et al., 2020). Technologies that significantly reduce enteric methane emissions from ruminant livestock are required for extensive grazing systems and the red meat supply chain to achieve a carbon neutral position by 2030 (Meat & Livestock Aust, CN2030). Roam Agricultural have developed a feed additive from the biosynthesis of bromoform by a fungal isolate to provide a viable mitigation pathway for the livestock sector. This pilot trial was a precursor to a larger replicated dose response study and assessed the efficacy and pattern of methane abatement when fungal derived bromoform combined with one of three inert carriers at two levels (10, 20% w / w) was fed to beef cattle consuming a forage diet compared to a control treatment (no carrier). Experimental Design The experimental protocol was approved by CSIRO Agriculture Animal Ethics Committee (ARA 23 / 01). Eight Droughtmaster steers with a (mean ±SD) live weight of 408 ± 24 kg were group housed in a yard and provided ad lib access to Rhodes grass (C. gayana) hay for an initial period of 4 days. The steers were then randomly assigned to one of two groups (A or B). Group A were immediately moved housed in individual pens and provided a chopped form of the same hay ad lib (~ 7.5 kg / d as fed at 0900) for seven days. Group B remained on hay under group housing in the yards for an additional 2 days, before moving into individual pens and fed as described for Group A. It was necessary to stagger the transition of groups into individual pens over time because there are only 4 methane chambers in total. During the period of individual housing, feed refusals for each animal from the previous day were collected and weighed prior to offering new feed. Dry matter content of feed offered was calculated by drying bulked samples collected throughout the trial period at 65°C for 48 h. Additional feed analysis was provided by NSW DPI, Wagga Wagga (Table 10). Following seven days in individual pens, four steers in each group were assigned to a control (CON) or carrier (16, C9, RP3) treatment and fed the basal hay diet supplemented once daily with 125 g canola meal for an additional three days in individual pens. For a further two days, canola meal only or containing one of the three carriers containing 10 % bromoform (w / w) fed at a rate equivalent of 30 mg bromoform / kg DM intake was provided in a separate feeder prior to 0900 to ensure consumption. After this period, each animal was placed into an open circuit respiration chamber and total methane production measured over 24 h. On the following day, canola meal only or containing one of the three carriers loaded at 20% (w / w bromoform) to deliver the equivalent of 30 mg / kg DM intake was provided in a separate feeder prior to 0900 to ensure consumption and total methane production measured for a further 24 h. After confinement in respiration chambers each animal was returned to a bulk pen and maintained on the basal diet ad lib. Table 10: Feed analysis for chopped Rhodes grass hay Dry Matter % 89.1 Total Crude Protein % 8.35 Neutral Detergent Fibre % 73 Acid Detergent Fibre % 39.5 Organic Matter % 89.35 Ash % 10.65 Metabolizable Energy, MJ / kg DM 6.95 Inert Carriers and Preparation Three forms of activated carbon (AC) we sourced from third party suppliers and designated as 16, C9 and RP3. Vapor phase deposition using analytical grade bromoform (as 96% tribromomethane and 1-3% ethanol as stabilizer, batch MKCS9626, Sigma-Aldrich® Merck KGaA, Darmstadt, Germany) was used to create two loadings of bromoform (10 or 20 % w / w) for each AC carrier. In brief, for each AC x loading, a known volume of CHBn was transferred to six, 20 mL glass vials and placed into a larger 350 mL glass jar containing 6.0 g (DM) AC. The jar was sealed and maintained at 40 °C for 12 h to allow the bromoform to be sorbed by the AC. Loading of bromoform was confirmed by weight differential of the AC after cooling to room temperature for 1 h. To determine the final concentration of bromoform loaded onto each AC five replicate samples were taken randomly from each of the batch loaded AC material to account for variation and assess homogeneity. A headspace solvent extraction method using chlorobenzene desorbed the bromoform into solution. Bromoform concentration in 20 uL aliquots were then analyzed by GCMS [(Shimadzu GC-2030 gas chromatograph in conjunction with a HS-20NX headspace sampler module and a GCMS-QP2020NX single quadrupole mass spectrometer) fitted with a Shimadzu SH-I-5Sil column (length 30 m, 0.25 mm ID, 0.25 pm film thickness), oven temperature was 70°C with sample line and transfer line temperatures maintained at 150 °C] and reported as the mean (±SD) for five replicate assays. Respiration Chamber Measurements Four open-circuit respiration chambers were used to determine total methane production from individual animals as described by Tomkins et al., (2016). Briefly, intake and exhaust air samples from four independent chambers with a volume of 23.04 m3, airflow of 3000 L / min, maintained at a negative pressure (-10.1 ± 0.14 Pa) and 2°C below ambient passed through a chemical drier and re-metered through independent rotameters before compositional analysis for CH4 and H2 (Servomex 4100 and Servomex Chroma, respectively, Servomex Group Ltd., Crowborough, UK). Enteric methane and hydrogen production (g / d) at STP was calculated by averaging each 24 h measurement. Enteric methane production was converted to daily methane yield (g / kg DMI) using individual animal intakes recorded over each 24 h period when confined in a chamber. Results & Discussion Mean measured concentration of bromoform for the 10% (100 mg / g) and 20% (200 mg / g) loaded AC was 100.8, 146.0, 131.6mg / gand225.9,239.6, 244.0 mg / g for C9,16 and RP3 carriers, respectively (Table 5). Mean (±SD) feed intake over consecutive days for animals supplemented with 10 or 20% loaded AC was 5.1 ± 0.5 and 5.2 ± 0.2 kg / d, respectively. Mean (±SD) feed intake for animals not supplemented (control) with an AC carrier was 5.0 ± 0.59 kg / d. Across all treatment groups mean DMI was within xl SD of the control mean. Mean methane yield (g / kg DMI) measured over 24 h periods, equivalent to one feeding event, was consistently lower for C9 compared to the other AC carriers at 10 or 20 % loading or control by up to 96% (g / kg DMI basis). The supplementation of 16 or RP3 resulted in abatement of 41-42% and 44-45%, respectively, irrespective of loading %. Hydrogen production (g / d) was consistently highest for C9 across both loading % compared with 16 or RP3 (Table 11). The mean methane yields observed for the control animals were not dissimilar to the predicted (Charmley et al., 2015) or reported values (Kennedy & Charmley 2012) for N beef cattle typically consuming a tropical grass hay diet. The pattern of total enteric methane production (g / d) was measured over two, 24 h periods post one feeding event at 0900. Relative to each feeding event, animals fed canola meal without an AC (control) demonstrated an increase in methane production followed by elevated but typically fluctuating levels indicating eructation over the subsequent 24 h (FIG. 5). By comparison, animals (n=2) fed the C9 carrier containing either 10 or 20% bromoform (w / w) demonstrated a clear reduction in methane production within 2 h consuming the material and a persistent inhibition over the following 22 h (FIG. 6). The persistent inhibition (> 3 h) of methane inhibition was not as apparent when animals were fed the 16 or RP3 carriers. A rapid reduction in initial emissions was reported for 16 irrespective of loading %. However, the continuity of abatement was inconsistent between animals and loading% (FIG. 7). One animal from Group A demonstrated a return to normal pre-feeding emissions comparable to the control animals within 5 h irrespective of loading %, whereas the animal from group B demonstrated a degree of abatement for up to 16 h post feeding for both 10 and 20% loaded material. Table 11: Mean (±SD)# DM intake, methane production and yield, hydrogen production and 5 yield for steers supplemented with one of three AC carriers containing bromoform at or 20% w / w Carrier Loading DM %(w / w)A n* intake kg / d Methane Hydrogen Production g / d Yield g / kg DMI Production g / d Yield g / kg DMI Control 0 4 5.0 ±0.6 119 ± 13.6 24 ± 1.7 0 0 C9 10.1 2 4.8 10.2 2.1 7.2 1.5 16 14.6 2 5.4 77.4 14.2 2.2 0.4 RP3 13.2 2 5.4 72.1 13.5 1.5 0.3 C9 22.6 2 5 4.8 1 7.9 1.6 16 23.9 2 5.3 74.1 13.8 1.9 0.4 RP3 24.4 2 5.3 68.9 13.1 1.7 0.3 # control group only indicates SD, A actual loading % (w / w) as determined by solvent extraction GCMS, *n = animal count x day Animals fed the RP3 material (n=2) also demonstrated inconsistent patterns in abatement of methane production relative to the feeding event and generally returned to baseline (pre feeding) 10 levels within 6-12 h post feeding (FIG. 8). Notably the animal from group A exhibited a gradual increase in methane production over time through the subsequent 20 h after a rapid (< 3 h) decrease in methane production irrespective of loading %. In comparison the animal from group B demonstrated a less rapid response to bromoform in the RP3 carrier over the first 3 h post feeding and returned to pre feeding methane production within 3 h post feeding for the 10% RP3 or 15 approximately 10 h post feeding for the 20% RP3 treatments. The patterns observed in methane production are not dissimilar to those previously reported where a compound introduced at feeding provides for almost immediate but short-term abatement. In general, mean total 24 h methane production values are reported in the literature and trends over time post feeding can be variable between animals reflecting individual feeding patterns. Nevertheless, the reduction in enteric methane commensurate with increases in hydrogen is consistent with the use of other antimethanogenic compounds which have a similar mode of action (Martinez et al., 2016, 2018). There were no obvious differences in the pattern of methane production between consecutive days (day 6 V 7 or day 9 V 10) for each of the three carriers suggesting that the change in loading of the bromoform from 10 to 20 % (w / w) does not affect the abatement response. The persistent level of abatement observed for C9 is substantial and appears independent of loading and repeatable over time. The actual dose rate of bromoform was estimated to be consistent across loading % as indicated by the amount of carrier fed daily (10% material ~ 1.45 g compared with 20% material ~ 0.75 g). The response cannot be attributed to a loading effect only. While the use of material over consecutive days introduces a time effect and the response may be compounding over time, the initial (day 6 or 9), repeatable (albeit n=2) and persistence abatement response > 3 h has not previously been reported. A statistically valid dose response trial using C9 at 10% loading is required. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth. REFERENCES Abbott, D. W., Aasen, I. M., Beauchemin, K. A., Grondahl, F., Gruninger, R., Hayes, M., Huws, S., Kenny, D. A., , S. J., & Kirwan, S. F. 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Claims

1. A composition comprising:i)     a haloform; andii)     a high surface area material;wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, ion exchange resins based on styrene and / or divinylbenzene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-based polymer, silica-based polymer, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof;if the high surface area material is a polymer then the polymer is cross-linked; andthe haloform is physically sorbed onto the high surface area material.

2. The composition of claim 1, wherein the high surface area material is a carbonaceous material selected from the group consisting of activated carbon (also known as activated charcoal), graphene, graphene oxide, and derivatives thereof.

3. The composition of anyone of claim 1 or 2, wherein the carbonaceous material is an activated carbon selected from the group consisting of powdered activated carbon (PAC), granular activated carbon (GAC), extruded activated carbon (EAC), bead activated carbon (BAC), impregnated carbon, polymer coated carbon, woven carbon, and combinations thereof.

4. The composition of claim 3, wherein the activated carbon comprises granular activated carbon (GAC) which may also be useful for reducing dust during feed manufacturing or delivery.

5. The composition of claims 3 or 4, wherein the activated carbon is derived from coconut shells, peat, coal, rice husks, wood, sawdust, corn cobs, or fruit pits.

6. The composition of any one of claims 3 to 5, wherein the activated carbon is pharmaceutical-grade activated carbon, food-grade activated carbon, industrial activated carbon, or environmental activated carbon.

7. The composition of any one of claims 3 to 6, further comprising a cyclodextrin,wherein the cyclodextrin has been sorbed onto the activated carbon prior to addition of the haloform to the composition and the haloform adsorbs preferentially to the cyclodextrin.

8. The composition of claim 7, wherein the cyclodextrin is an alpha, beta, or gamma cyclodextrin.

9. The composition of claim 7, wherein the cyclodextrin is a water insoluble highly crosslinked cyclodextrin based polymer.

10. The composition of claim 9, wherein the polymer is an epichlorohydrin and / or trimethylolpropane glycidyl ether crosslinked-cyclodextrin polymer.

11. The composition of any one of claims 7 to 10, wherein the cyclodextrin is sorbed onto the activated carbon through a water deposition method where the activated carbon is exposed to a saturated water solution of the cyclodextrin and then dried leaving the activated carbon coated in the cyclodextrin.

12. The composition of any one of claims 7 to 11, wherein the cyclodextrin is sorbed onto the activated carbon at a concentration of about 100 mg / g to about 700 mg / g.

13. The composition of claim 1, wherein the high surface area material is selected from the group consisting of Amberlite (Rohm and Haas) XAD-2, XAD-4, XAD-7, XAD-16, XAD-18, XAD-1180, XAD-1600, XAD-2000, XAD-2010; Amberchrom (Toso Haas) CG-71m, CG-71c, CG-161m, CG161c; Diaion Sepabeads (Mitsubishi Chemicals) HP20, SP206, SP207, SP850, HP2MG, HP20SS, SP20MS; Dowex (Dow Chemical) XUS-40285, XUS-40323, XUS-43493 (also referred to as Optipore V493 (dry form) or Optipore L493 (hydrated form)), Optipore V503, Optipore SD-2; Hypersol Macronet (Purolite) MN-100, MN-102, MN-150, MN-152, MN-170, MN-200, MN-202, MN-250, MN-252, MN-270, MN-300, MN-400, MN-500, MN-502, Purosorb (Purolite) PAD 350, PAD 400, PAD 428, PAD 500, PAD 550, PAD 600, PAD 700, PAD 900, PAD 950, and combinations thereof.

14. The composition of claim 13, wherein the high surface area material is PAD 600.

15. The composition of any one of claims 1-14, wherein the haloform is selected from the group consisting of bromoform, chloroform, iodoform, and combinations thereof.

16. The composition of claim 15, wherein the haloform is bromoform.

17. The composition of any one of claims 1 and 13 - 16, wherein the polymer is anorganic polymer, an inorganic polymer, or a hybrid polymer.

18. The composition of any one of claims 1-17, wherein the high surface area material has a surface area selected from the group consisting of about 1 to about 5000 m2 / g, about 100 to about 5000 m2 / g, about 200 about 5000 m2 / g, about 300 to about 5000 m2 / g, about 500 to about 5000 m2 / g, about 500 to about 4000 m2 / g, about 500 to about 3000 m2 / g, about 750 to about 3000 m2 / g, about 900 to about 3000 m2 / g, about 1000 to about 3000 m2 / g, and about 500 to about 1500 m2 / g.

19. The composition of any one of claims 1-18, wherein the said composition is an anti-methanogenic composition.

20. An animal feed composition comprising the composition of any one of claims 1 to 19.

21. The animal feed composition of claim 20, further comprising a cereal, starch, vegetable waste, vitamin, mineral, trace element, emulsifier, aromatizing product, binder, colorant, odorant, thickening agent, or a combination thereof.22 A method for reducing methane emission, the method comprising administering the composition of any one of claims 1 or 19 or the animal feed of claim 20 or 21 to a ruminant in an effective amount to reduce methane emission from the ruminant.

23. The method of claim 22, wherein the ruminant is a cow, bull, steer, ox, heifer, goat,sheep, giraffe, yak, deer, or antelope.

24. The method of claim 22 or 23, wherein the composition or animal feed is administered in an effective amount of about 1 to about 100 mg, about 5 to about 50 mg, or about 10 to about 40 mg of bromoform per kg dry matter.

25. The method of any one of claims 22 - 24, wherein the composition is an anti-methanogenic composition.

26. A process for preparing a composition, optionally an anti-methanogenic composition, wherein the process comprises:i) combining an aqueous solution comprising a haloform with a high surface area material to extract the haloform onto the high surface area material; andii) separating the high surface area material with the extracted haloform from the aqueous solution to prepare the composition;wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-based polymer, silica-based polymer, ion exchange resin, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof; andif the high surface area material is a polymer then the polymer is cross-linked.

27. A process for preparing a composition, optionally an anti-methanogenic composition, wherein the process comprises:i) bubbling air into an aqueous solution comprising a haloform to form a vapor phase with the haloform;ii)     passing the vapor phase with the haloform over a stirred bed of a high surface areamaterial to extract the haloform onto the high surface area material; andiii) collecting the high surface area material with the extracted haloform to prepare the anti-methanogenic composition;wherein the high surface area material is selected from the group consisting of a carbonaceous material, or a polymer, wherein the polymer is selected from the group consisting of polystyrene, polyacrylic, polyacrylic ester, styrene-divinylbenzene polymer, silicone-basedpolymer, silica-based polymer, ion exchange resin, poly(N-vinyl-2-pyrrolidone) (PVP)-based material, cyclodextrin-based material, and a derivative thereof; andif the high surface area material is a polymer then the polymer is cross-linked.

28. The process of claim 26 or 27, wherein the stirred bed of high surface area material is continuously mixed to ensure deposition of the haloform onto the high surface area material is substantially uniform.

29. The process of any one of claims 26 - 28, wherein the high surface area material is a carbonaceous material selected from the group consisting of activated carbon (also known as activated charcoal), graphene, graphene oxide, and derivatives thereof.

30. The process of any one of Claims 26 - 28, wherein the high surface area material is selected from the group consisting of Amberlite (Rohm and Haas) XAD-2, XAD-4, XAD-7, XAD-16, XAD-18, XAD-1180, XAD-1600, XAD-2000, XAD-2010; Amberchrom (Toso Haas) CG-71m, CG-71c, CG-161m, CGI61c; Diaion Sepabeads (Mitsubishi Chemicals) HP20, SP206, SP207, SP850, HP2MG, HP20SS, SP20MS; Dowex (Dow Chemical) XUS-40285, XUS-40323, XUS-43493 (also referred to as Optipore V493 (dry form) or Optipore L493 (hydrated form)), Optipore V503, Optipore SD-2; Hypersol Macronet (Purolite) MN-100, MN-102, MN-150, MN-152, MN-170, MN-200, MN-202, MN-250, MN-252, MN-270, MN-300, MN-400, MN-500, MN-502, Purosorb (Purolite) PAD 350, PAD 400, PAD 428, PAD 500, PAD 550, PAD 600, PAD 700, PAD 900, PAD 950, and a combination thereof.

31. The process of any one of claims 26 - 30, wherein the haloform is selected from the group consisting of bromoform, chloroform, iodoform, and combinations thereof.

32. The process of anyone of claims 26 - 31, wherein the composition is an anti-methanogenic composition.

33. The use of the composition of anyone of claims 1 - 19 or the animal feed of claim 20 or 21 for reducing methane emissions.

34. The use of claim 33, wherein said methane reduction is accomplished by administering the composition or the animal feed to a ruminant in an effective amount to reduce methane emission from the ruminant.

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