Anti-methanogenic compositions

Abstract

The invention relates to anti-methanogenic compounds having improved stability. In particular, the invention provides for a porous material-infused with at least one anti- methanogenic agent, methods of making the same, and the use of the infused-porous material in mitigating methane production in ruminant animals.

Description

ANTI-METHANOGENIC COMPOSITIONSFIELD OF THE INVENTION

[0001] The invention relates generally to compositions for reducing methane emissions in ruminant animals. The compositions comprise anti-methanogenic compounds having improved stability. In particular, the invention provides a porous material infused with one or more anti- methanogenic agents.BACKGROUND

[0002] Methanogens are microbes that produce methane as a metabolic by-product in hypoxic conditions. Such organisms are found in natural ecosystems (e.g. wetlands, oceans and lakes) and the gastrointestinal tract of vertebrates, such as ruminants.

[0003] Methane is the primary contributor to the formation of ground-level ozone, a hazardous air pollutant and greenhouse gas, exposure to which causes significant human mortality. Methane is also a powerful greenhouse gas, being approximately 28-34 times more potent at global warming than carbon dioxide. About 24% of the global methane emission is contributed by ruminant animals. Accordingly, strategies for reducing ruminant methane emissions have become an important focus to combat global warming.

[0004] Dietary supplementation and the use of feed additives are among the most promising methods for addressing enteric methane production in ruminants. For example, several seaweeds have been identified with the potential to reduce methane emissions from ruminants via feed supplementation, including (but not limited to) Asparcigopsis taxiformis, Alarici esculenia. Ascophyllum nodosum and Chondrus crispus.

[0005] Halogenated methane analogues, including but not limited to haloforms (e.g. chloroform, bromoform and iodoform), also have usefulness as anti-methanogenic agents. Specifically, exposure to halogenated methane analogues in the rumen results in an inhibition of an essential enzymatic reaction in methanogens halting their ability to produce methane. Halogenated methane analogues are therefore seen as an attractive feed supplement for reducing ruminant methane emissions.

[0006] Halogenated methane analogues are volatile and can degrade when exposed to biotic and / or abiotic factors. Suspension of halogenated methane analogues in a carrier such as oil, and storage of the mixture in the absence of air can delay halogenated methane analogue volatilisation anddegradation and improve shelf-life. However, practical application of oil-suspended halogenated methane analogues in processes involving exposure to air and other factors (e.g. in supplementing ruminant feed) remains problematic in terms of the stability, and subsequently controlling the administration and bioavailability of, halogenated methane analogues.

[0007] A need remains for improved anti-methanogenic formulations in which volatilisation to the atmosphere and / or degradation of anti-methanogenic compounds in the presence of biotic or abiotic factors is reduced compared to existing anti -methanogenic compound formulations.

[0008] Discussion or mention of any piece of prior art in this specification is not to be taken as an admission that the prior art is part of the common general knowledge of the skilled addressee of the specification.SUMMARY OF INVENTION

[0009] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.

[0010] In one aspect, provided herein is a meso- or micro- porous material infused with at least one anti-methanogenic agent.

[0011] In an embodiment, the infusion of the at least one anti -methanogenic agent into the meso- or micro- porous material protects the anti-methanogenic agent(s) against volatilisation and / or degradation.

[0012] In one aspect, provided herein is a composition comprising an infused meso- or micro- porous material according to the present invention.

[0013] In one aspect, provided herein is the use of an infused meso- or micro- porous material according to the present invention in the manufacture of a composition for mitigating methane production in a ruminant animal.

[0014] In one aspect, provided herein is the use of an infused meso- or micro- porous material according to the present invention for mitigating methane production in a ruminant animal.

[0015] In one aspect, provided herein is a composition for reducing methane production in a ruminant animal, said composition comprising a combination of a first anti-methanogenic agent and second anti-methanogenic agent, wherein the first anti-methanogenic agent and second anti- methanogenic agent act synergistically to reduce methane production in a ruminant animal.

[0016] In one aspect, provided herein is a method of mitigating methane production in a ruminant animal, comprising administering to the ruminant animal an effective amount of the composition as described herein.

[0017] Other aspects and embodiments of the invention will be evident from the following detailed description of various aspects of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1 shows the retention at days 0, 3 and 7 for bromoform suspended in canola oil (absent biochar). Letters above bars denote significant differences among treatments (Two-Way ANOVA, Tukey’s HSD, P<0.05).

[0019] Figure 2 shows the retention of bromoform at days 0, 3, 7, 14 and 28 in biochar-infused formulations F1-F4 (i.e., with Agspand FEEDCHAR® biochar (“FBC”) from Agspand (hardwood)) which each deliver 25 mg of bromoform per 5 g of formulation. Letters above bars denote significant differences among treatments (Two-Way ANOVA, Tukey’s HSD, P<0.05).

[0020] Figure 3 shows the retention of bromoform at days 0, 3, 7, 14 and 28 in biochar-infused formulations F5-F8 i.e., with High BET pinechar (softwood) from BioGrow (“PBC”) which each deliver 25 mg of bromoform per 5 g of formulation. Letters above bars denote significant differences among treatments (Two-Way ANOVA, Tukey’s HSD, P<0.05).

[0021] Figure 4 shows the retention of bromoform at day 7 for softwood biochar (i.e. High BET pinechar from BioGrow) infused formulations F1-F8 compared and oil controls (10-50 mg / g). Letters above bars denote significant differences among treatments (One-Way ANOVA, Tukey’s HSD, P<0.05).

[0022] Figure 5 shows retention of bromoform at days 0, 3 and 7 for bromoform in canola oil (absent biochar) control formulations which deliver 10-100 mg of bromoform per 1 g of formulation. Letters above bars denote significant differences among treatments (Two-Way ANOVA, Tukey’s HSD, P<0.05).

[0023] Figure 6 shows the retention of bromoform at days 0, 3, 7 and 14 in fixed ratio (4: 1) hardwood biochar (Agspand Feedchar®)-infiised formulations (F9-F13) designed to deliver 10- 100 mg / g of bromoform per 5 g of formulation. Letters above bars denote significant differences among treatments (Two-Way ANOVA, Tukey’s HSD, P<0.05).

[0024] Figure 7 shows the retention of bromoform at days 0, 3, 7 and 14 in fixed ratio (4: 1) biochar (BioGrow High BET Pinechar)-infiised formulations F14-F18 designed to deliver 10-100 mg ofbromoform per 5 g of formulation. Letters above bars denote significant differences among treatments (ANOVA, Tukey’s HSD, P<0.05).

[0025] Figure 8 shows the retention of bromoform activity at day 7 for formulations F9-F13 in hardwood biochar (Agspand Feedchar®) and F14-F18 in softwood biochar (BioGrow High BET Pinechar) compared to bromoform-in-oil controls (10-100 mg / g). Letters above bars denote significant differences among treatments (One-Way ANOVA, Tukey’s HSD, P<0.05).

[0026] Figure 9 shows the retention of bromoform in compositions of brewers grain (BG; 100 g) comprising bromoform in canola oil (BFO) infused in hardwood biochar Agspand Feedchar® (BC) (F19 and F21) compared to BFO absent biochar (F20 and 22). Content of bromoform in 100 g of brewers grain (“BG”) formulations. Results presented as averages ± standard error (n=3) and letters above bars denote significant differences among treatments (ANOVA, Tukey’s HSD, p<0.05).

[0027] Figure 10 shows the retention of bromoform in formulations comprising bromoform in canola oil (BFO) infused in a ratio of 4: 1 in hardwood biochar (Agspand Feedchar®; “BC”) (F24; “4BC: 1BFO”) and zeolite (“Zeol”) (F25; “4Zeol: lBFO”) versus control bromoform in oil (F23; “BFO”) heated at 75°C at 0, 60 and 120 min. Letters above bars denote significant differences among treatments (Two-Way ANOVA, Tukey’s HSD, P<0.05).

[0028] Figure 11 shows a comparison of bromoform release kinetics over 6 hours which is a 9: 1 ratio of pinechar (softwood) biochar to bromoform in oil (“9BC: 1BFO”), to deliver 1 mg bromoform per gram of formulation, compared to control (BFO) in Kansas buffer (pH 6.8) at 39°C.

[0029] Figure 12 shows a summary of bromoform and iodoform release from the composition as described herein formulated with Biochar Activated™ (“SC”)I from Soft Agriculture Pty Ltd (softwood) or pinechar (“PC”) from Biogrow (softwood) in a 4: 1 ratio with bromoform in oil (“BFO”), iodoform in oil (“IFO"), or both bromoform and iodoform in oil (“Mix”) formulated wet (“W”) (i.e. PCW and SCW) or dry (“D”) (i.e. PCD and SCD) and with and without surfactant (“S”) (PCS and SCS) (F26-F37; F54-55).

[0030] Figure 13 shows retention of bromoform activity in softwood biochar formulations (Biochar Activated™ (“SC”) from Soft Agriculture Pty Ltd) over a 7-day exposure to atmosphere at 25°C (F29 and F30 which were formulated to deliver 1 mg BF / g formulation).

[0031] Figure 14 shows total gas production from treatment with bromoform, iodoform, or both bromoform and iodoform in biochar at different concentrations (CON (F38-F41); Carrier A1-A5 (F42-F45; F56); Carrier B1-B5 (F46-F49; F57)).

[0032] Figure 15 shows methane production from treatment with bromoform, iodoform, or both bromoform and iodoform in biochar at different concentrations as a percentage of the negative control (CON-) (CON (F38-F41); Carrier A1-A5 (F42-F45; F56); Carrier B1-B5 (F46-F49; F57)). Letters above bars denote significant differences among treatments (PERMANOVA, pairwise tests, P<0.05).

[0033] Figure 16 shows methane production as a percentage of total gas production (% TGP) using 9BC: 1BFO formulations prepared using High BET Pinechar (Carrier A delivering 1 mg / g and Carrier B delivering 2 mg / g).

[0034] Figure 17 shows bromoform release from various meso- or micro- porous carriers after 4 hours in water at 39°C (%T0).

[0035] Figure 18 is a Van Krevelen plot of the 10 biochar samples from different commercial providers.

[0036] Figure 19 shows ATR-FTIR spectra of biochars BC 001-BC 010 (BC 001 (Activated charcoal), BC 002 (Biochar (Commodities)) BC 003 (Activated carbon), BC 004 (High BET Pinechar), BC_005 (Biochar Activated), BC_006 (Feedchar®), BC_007 (Activated carbon), BC 008 (Biochar (Carbon Chip)), BC 009 (Artisan biochar), BC 010 (Charcoal)).

[0037] Figure 20 shows SEM images of biochars BC_001-BC_010 (BC_001 (Activated charcoal), BC 002 (Biochar (Commodities)) BC 003 (Activated carbon), BC 004 (High BET Pinechar), BC_005 (Biochar Activated), BC_006 (Feedchar®), BC_007 (Activated carbon), BC 008 (Biochar (Carbon Chip), BC 009 (Artisan biochar), BC 010 (Charcoal)).

[0038] Figure 21 shows bromoform retention in 4BC: 1BFO and 9BC: 1BFO formulations (BC_001-BC_0010) after 7 days exposure to atmosphere at 25°C as a function of: BET surface area (Figure 21A); pore volume (Figure 21B); and biochar pore size (Figure 21C). (BC_001 (Activated charcoal), BC_002 (Biochar (Commodities)) BC_003 (Activated carbon), BC_004 (High BET Pinechar), BC 005 (Biochar Activated), BC 006 (Feedchar®), BC 007 (Activated carbon), BC 008 (Biochar (Carbon Chip), BC 009 (Artisan biochar), BC 010 (Charcoal)).

[0039] Figure 22 shows bromoform release from 4BC: 1BFO and 9BC: 1BFO formulations after 4 hours extraction in RO water at 39°C as a function of: biochar BET surface area (Figure 22A); pore volume (Figure 22B); and biochar pore size (Figure 22C). (BC_001 (Activated charcoal),BC 002 (Biochar (Commodities)) BC 003 (Activated carbon), BC 004 (High BET Pinechar), BC_005 (Biochar Activated), BC_006 (Feedchar®), BC_007 (Activated carbon), BC_008 (Biochar (Carbon Chip), BC 009 (Artisan biochar), BC 010 (Charcoal)).

[0040] Figure 23 shows bromoform retention in 4BC: 1BFO and 9BC: 1BFO formulations after 14 days exposure to atmosphere at 25°C as a function of BET surface area (Figure 23A), pore volume (Figure 23B); and mean pore size (Figure 23C).

[0041] Figure 24 is a comparative figure presenting bromoform retention (raw data - diamonds; beta regression (Model 1) - solid black line) in 4BC: 1BFO formulations after 14 days exposure to atmosphere at 25°C and bromoform release (raw data - squares; beta regression (Model 2) - dashed black line) from 4BC: 1BFO formulations after a 4 h extraction in RO water at 39°C, both as a function of BET surface area (light grey areas represent the 95% confidence intervals and the dotted black box encompasses 4BC: 1BFO formulations that have suitable retention and release properties) (Figure 24A); and comparative figure presenting bromoform retention (raw data - diamonds; beta regression (Model 3) - solid black line) in 9BC: 1BFO formulations after 14 days exposure to atmosphere at 25°C and bromoform release (raw data - squares; beta regression (Model 4) - dashed black line) from 9BC: 1BFO formulations after a 4 h extraction in reverse osmosis water at 39°C, both as a function of BET surface area (light grey areas represent the 95% confidence intervals and the dotted black box encompasses 9BC: 1BFO formulations that have suitable retention and release properties) (Figure 24B).

[0042] Figure 25 shows mean methane (ml / g digestible dry matter (DM)) produced (± standard error) by various feed compositions during in vitro gas production trials.

[0043] Figure 26 is a graph showing beta regression (y = 0. 1609x - 0.0013x2- 2.7736) was fitted (black line) to the methane mitigation data of treatment compositions that contained iodoform only (ASP5, ASP10, ASP15, ASP16, ASP20). The dashed line represents the plateau where, beyond this point, an improved model should predict that methane mitigation will remain constant or continue to increase. The shaded grey portion of the figure represents the 95% confidence interval.

[0044] Figure 27 is a graph showing beta regression (y = 4748x - 0.0076x2- 5.1709) was fitted (black line) to the methane mitigation data of treatment compositions that contained bromoform only (ASP1, ASP2, ASP3, ASP4). The dashed line represents the plateau where, beyond this point, an improved model fails to predict that methane mitigation will remain constant or continue to increase. The shaded grey portion of the figure represents the 95% confidence interval.

[0045] Figure 28 is a graph showing cumulative gas production (mL) over 24 h in treatments investigating bromoform dose response.

[0046] Figure 29 is a graph showing cumulative gas production (mL) over 24 h in treatments investigating iodoform dose response.

[0047] Figure 30 is a graph showing cumulative gas production (mL) over 24 h in treatments investigating the combined effect between bromoform and iodoform.

[0048] Figure 31 is a graph showing the synergistic effect of iodoform (mg / kg DM) when used in combination with bromoform to reduce methane in vitro. The dashed outline represents the concentrations of iodoform with the largest synergistic effects (~5 mg / kg DM).

[0049] Figure 32 is a graph showing the synergistic effect of bromoform (mg / kg DM) when used in combination with iodoform to reduce methane in vitro.DETAILED DESCRIPTIONDefinitions

[0050] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0051] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element is essential to the practice of the invention, unless otherwise indicated herein or clearly contradicted by context.

[0052] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0053] Except where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term ‘about’. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding conventions. The term "about" may be understood to refer to a range of + / - 10%, such as + / - 5% or + / - 1% or, + / - 0.1%.

[0054] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. For example, if a range is from about 1 to about 50, it is deemed to include, for example, 1, 7, 34, 46. 1, 23.7, 50, or any other value or range within the range.

[0055] The term “infuse” in the present context means that the meso- or micro- porous material carries the anti -methanogenic agent(s). Terms which have a consistent meaning, such as fill, imbue, permeate, ingrain, inoculate or suffuse, may be used interchangeably with ‘infuse’. Further, the term “infuse” encompasses scenarios where the meso- or micro- porous material may be partially or completely infused with the anti-methanogenic agent(s).

[0056] The term “biochar” is taken to mean a high-carbon residue material produced via pyrolysis; it is the direct thermal decomposition of biomass in the absence of oxygen (preventing combustion).

[0057] The term “charcoal” is taken to mean a porous black solid, consisting of an amorphous form of carbon, obtained as a residue when wood or other organic matter is heated in minimal oxygen in a process called pyrolysis.

[0058] “Activated carbon” is similar to common charcoal but is a high purity form of carbon with a very high surface area, characterised by microscopic pores. Impurities on the surface of the charcoal are removed during the production of activated carbon, greatly increasing its adsorption capacity. “Activated carbon” is also sometimes referred to as active carbon, activated charcoal or active coal.

[0059] “Pyrolysis” is the heating of an organic material, such as biomass, in the absence of oxygen, resulting in the production of “pyrolysed” materials. Biomass pyrolysis is conducted at high temperatures, usually at or above 500°C, providing enough heat to deconstruct the strong biopolymers. Because no oxygen is present combustion does not occur, rather the biomass thermally decomposes into combustible gases and charred material such as biochar, charcoal and activated carbon. “Slow pyrolysis” uses a slower heating rate and results in the production of biochar.

[0060] The term “anti-methanogenic agent” means any bioactive compound that reduces or substantially inhibits methanogenesis in a ruminant animal. Such a compound may be a halogenated methane analogue, a halogenated alkane or a halogenated organic acid, for example. An “anti-methanogenic agent” may include the following compounds: 3-NOP (3- nitrooxypropanol), dichloromethane, dibromomethane, bromochloromethane,bromodichloromethane, bromodiiodomethane, dibromoiodomethane, bromoiodomethane, chloroiodomethane, 2-bromoethanesulfonic acid, chloral hydrate, chloroform, iodoform, chloroethane, dichloroethane, tetrachloroethane, hexachloroethane, bromoethane, dibromoethane, tetrabromoethane, 1,2-dibromotetrachloroethane, iodoethane, diiodoethane, iodopropane, bromoform, carbon tetrachloride, carbon tetrabromide, carbon tetraiodide, or dibromochloromethane, or combinations thereof. Preferably, the anti-methanogenic agent is a haloform, particularly bromoform, chloroform or iodoform. As such, the term “anti -methanogenic agent” excludes compounds that do not reduce or do not substantially inhibit methanogenesis in a ruminant animal. Further, an “anti-methanogenic agent” may be derived (e.g. by extraction) from a macroalgae, preferably a green, brown, or red macroalgae. Preferably, the macroalgae is of the genus Asparcigopsis. particularly Asparcigopsis taxiformis or Asparcigopsis armata.

[0061] The term “biotic factor” refers to all biological elements in an ecosystem, and includes organisms from animals and humans, to plants, fungi, and bacteria.

[0062] The term “abiotic factor” refers to all non-living elements in an ecosystem, and includes light, water, temperature, air, nutrients and minerals.

[0063] The term “carrier” encompasses any vehicle in which an anti-methanogenic agent may be held and presented for formulation, including, but not limited to, solvents, emulsions, low-melting point waxes or viscous liquids, oil, coatings, dispersion agents, wetting agents, isotonic and absorption delaying agents and disintegrants.

[0064] The term “oil” means any non-polar, hydrophobic substance which is typically a liquid at ambient temperature and pressure. Oils may be derived from animals, vegetables, or petrochemicals, and typically have a high carbon and hydrogen content. The oil is preferably an edible oil, and preferably digestible by a ruminant animal. Typically, an oil of vegetable origin is extracted from the seeds or fruits of plants and is typically comprised primarily of triglycerides. The term "vegetable oil" is a generic term to indicate that the oil is of primarily or exclusively vegetable origin, and may comprise a mixture of one or more oils of vegetable origin or from differing origins (i.e. a blended oil). An edible oil may be selected from the group consisting of almond oil, apricot oil, argan oil, avocado oil, Brazil nut oil, canola oil, cashew oil, coconut oil, colza oil, com oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, pecan oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, and vegetable oil, or any combination thereof.

[0065] A “surfactant” in the context of the compositions disclosed herein is an amphiphilic molecule that has both hydrophobic and hydrophilic components. The hydrophobic tail is a hydrocarbon, fluorocarbon, or siloxane. Surfactants are typically classified based on their polar head as the hydrophobic tails are often similar. Non-ionic surfactants have a head group with no charge. Anionic surfactants have a negatively charged head and cationic surfactants have a positively charged head. Zwitterionic surfactants contain both positive and negative groups.

[0066] An “additive” in the context of the compositions disclosed herein is any additive used to favourably affect animal health and / or the environment. As such, an additive can be broadly classified into nutrients, such as amino acids, minerals, fats and vitamins, and non-nutrients, such as antibiotics, hormones, enzymes, prebiotics, yeast culture, pellet binder, or antioxidants.

[0067] As used herein, the phrases “preserving the stability” or “preserving the retention”, or grammatical variations thereof, when used to describe the stability of an anti-methanogenic agent means the volatilisation or degradation of the agent is reduced or substantially prevented when compared to a corresponding control.

[0068] As used herein, “mitigating methane production in a ruminant animal” means reducing or substantially eliminating methane production in said animal.

[0069] As used herein, the expressions "is for administration", "is to be administered" and "is prepared to be administered" have equivalent meanings. In other words, the statement that an active compound "is for administration" means that said active compound is formulated so that said active compound is in a state capable of exerting its activity.

[0070] The term “room temperature” or “ambient temperature” means normal conditions (i.e. storage or testing in a dry, clean, we 11 -ventilated area) at room temperature between about 15°C to about 25°C.

[0071] The term “atmospheric pressure” means average air pressure at sea level at ambient temperature; 1 atmosphere is equivalent to about 101,325 pascals.

[0072] The terms "administration concurrently" or "administering concurrently" or "coadministering" and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and / or delivered by separate routes either contemporaneously or simultaneously, or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By "simultaneously" is meant that the active agents are administered at substantially the same time, and preferably together in the same composition.

[0073] The terms "comprise", "comprises", "comprised" or "comprising", "including" or "having" and the like in the present specification and claims are used in an inclusive sense, that is to specify the presence of the stated features but not preclude the presence of additional or further features.

[0074] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of’ excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0075] Further examples of the invention are described below. However, it should be noted that the invention should not be limited to these examples, and that the invention is susceptible to variations, modifications and / or additions other than those specifically described, and it is to be understood that the invention includes all such variations, modifications and / or additions which fall within the scope of the claims.Preferred Embodiments

[0076] The present invention is predicated on the surprising finding that infusing a meso- or micro- porous material with at least one anti -methanogenic agent can preserve the stability of the agent(s) upon exposure of the infused meso- or micro- porous material to external biotic and / or abiotic factors. Further, it has been found that the ratio of meso- or micro- porous material to anti- methanogenic agent(s) in an infused meso- or micro- porous material can be a determinant of stability of the anti -methanogenic agent(s).

[0077] The present invention is also predicated on the surprising finding that combinations of two or more anti-methanogenic agents can act synergistically to mitigate methane production in a ruminant animal. Synergistic compositions can have numerous advantages. Primarily, reducing the amount of anti-methanogenic agent can provide an economic saving in terms of composition manufacture. Administering less anti -methanogenic agent to a ruminant animal but achieving the same outcome in methane mitigation has both economic and animal health benefits. Given variability in natural sources of anti-methanogenic agents, controlled manufacture of compositions to provide a synergistic outcome in ruminant methane abatement is preferable for consistent and effective results. By standardising the extraction, concentration, and combination of anti- methanogenic agents - such as specific halogenated compounds from seaweed - compositions may be optimised to maximise methane reduction while ensuring stability, safety, and efficacy.Synthetic anti -methanogenic agents also provide an alternative approach for producing synergistic compositions according to the present invention.

[0078] Synergistic compositions may provide for more predictable and scalable application in livestock, thereby contributing significantly to global efforts to reduce agricultural greenhouse gas emissions.

[0079] Of note, naturally occurring sources of anti -methanogenic agents (such as seaweeds) do not provide synergistic combinations of two or more anti-methanogenic agents, nor do such sources provide synergistic combinations in reliable and scalable amounts to affect methane abatement in ruminant animals. As such, compositions of the invention do not include unmodified natural sources of anti -methanogenic agents (such as seaweeds). Rather, compositions of the invention rely on extraction of anti-methanogenic agents from natural sources and their combination, or combination of anti-methanogenic agents of synthetic origin. As such, compositions of the invention are unique and distinct from those which might be naturally occurring.

[0080] According to one aspect, there is provided meso- or micro- porous material infused with at least one anti-methanogenic agent.

[0081] According to another aspect of the invention, there is provided a composition for reducing methane production in a ruminant animal, said composition comprising a combination of a first anti-methanogenic agent and second anti-methanogenic agent.

[0082] In an embodiment, the first anti-methanogenic agent and second anti-methanogenic agent are present in the composition in an amount which, when in use, acts synergistically to reduce methane production in a ruminant animal when compared to an additive methane reduction produced by corresponding controls of the first anti-methanogenic agent or second anti- methanogenic agent used independently.

[0083] In an embodiment, provided herein is a first composition comprising a first anti- methanogenic agent, and a second composition comprising a second anti-methanogenic agent for reducing methane production in a ruminant animal wherein, in use, the first and second compositions are combined.

[0084] In one embodiment, the first and second compositions are combined: prior to administration to a ruminant animal, or within the ruminant animal following administration.

[0085] In an embodiment, the ratio of the first anti-methanogenic agent to the second anti- methanogenic agent is from about 1.0:0.1 to about 1.0: 100.0. In an embodiment, the composition comprises less than about 25 mg / kg of dry matter (DM) of the first anti-methanogenic agent, preferably less than about 10 mg / kg DM of the first anti -methanogenic agent, more preferably less than about 5 mg / kg DM of the first anti-methanogenic agent.

[0086] In one embodiment, the meso- or micro- porous material suitable for the present invention is a charcoal, biochar, activated carbon, preferably plant-derived biochar including, but not limited to pine biochar, eucalyptus biochar, hardwood biochar, wheat straw biochar, sawdust biochar, rice husk biochar, walnut biochar, com stem biochar, cotton stem biochar, algal biochar, bamboo biochar, or mixtures thereof. In alternative embodiments, the meso- or micro- porous material may comprise cellulose, chitosan, lignin, polymers, natural fibres or aerogel, or mixtures thereof. Preferably, the biochar or other form of meso- or micro- porous material is in particulate form before infusing with the anti -methanogenic agent(s).

[0087] In an embodiment, the meso- or micro-porous material is pyrolysed.

[0088] Charcoal, biochar and activated carbon are carbon-rich materials produced from the pyrolysis of various feedstock biomasses (e.g., wood, coconut, grasses and seaweed) under oxygen-limited conditions. The yield and physical and chemical properties of the charcoal, biochar and activated carbon products is influenced by various production parameters, including biomass feedstock material, operating temperature, gas flowrate, residence time, furnace ramp rate and pressure. Importantly, these parameters influence pore characteristics (physical and chemical) leading to differences in the absorption / desorption of molecules between charcoals, biochars and activated carbons prepared by different protocols.

[0089] For example, high temperature pyrolysis encourages micropore formation, but excessive temperatures can lead to ash formation and hinder pore networks and reduce surface area. By contrast insufficiently low pyrolysis temperatures leads to poor development of microspores, incomplete volatilisation and pore blockages also leading to low surface area. Therefore, pyrolysis temperatures usually range between 400°C and 800°C with optimal heating rates between 10 and 30°C / min as too high a heating rate can result in low surface area due to pore blockages caused by particulates formed by carbonisation of volatile products. Residence time is also important, where surface area will increase through time up to 60 min but then may decrease.

[0090] Generally, the carbon content of pyrolysed materials increases with increasing pyrolysis temperature, whereas, in contrast, the oxygen element decreases with increasing pyrolysis temperature. This can be attributed to the volatilization processes in e.g. cellulose, hemicellulose,and lignin. It is generally expected for pyrolysed materials to form graphitic bonds and gain carbon as the highest intensity element because of decomposition of the biomass and carbonization during the pyrolysis.

[0091] Examples of meso- and micro- porous materials suitable for use with this invention include (but are not limited to) : biochar, charcoal, activated carbon, mesoporous carbon, wheat bran, barely bran, rice bran, rice husks, oat bran, cellulose and com cob, and mixtures thereof.

[0092] In an embodiment, the meso- or micro- porous material is an organic or inorganic material. In an embodiment, the meso- or micro- porous material is plant based. Preferably, meso- or micro- porous material is selected from the group consisting of charcoal, biochar, activated carbon, wheat bran, barely bran, rice bran, rice husks, oat bran, cellulose and com cob, and a mixture thereof.

[0093] In an embodiment, infused meso- or micro- porous material is derived from hardwood, soft wood, or coconut.

[0094] In an embodiment, the meso- or micro- porous material is biochar, charcoal, or activated carbon.

[0095] In an embodiment, the biochar is plant-derived including, but not limited to pine biochar, eucalyptus biochar, hardwood biochar, wheat straw biochar, sawdust biochar, rice husk biochar, walnut biochar, com stem biochar, cotton stem biochar, bamboo biochar, algal biochar or mixtures thereof.

[0096] In an embodiment, the meso- or micro- porous material may absorb from between about 50 % to about 5000 % its own weight. Preferably, the meso- or micro- porous material is infused with an amount of anti-methanogenic agent(s) such that the infused meso- or micro- porous material remains friable (i.e. where the surface of the meso- or micro- porous material is substantially dry or may be covered by a thin film of anti -methanogenic agent(s)). Alternatively, the meso- or micro- porous material is infused with an amount of anti-methanogenic agent(s) such that the infused meso- or micro- porous material has the consistency of a slurry or viscous liquid.

[0097] In an embodiment, the meso- or micro- porous material is inert and does not react with the infused anti-methanogenic agent(s). As such, materials which include reactive surfaces which act to degrade the anti-methanogenic agent(s) may be excluded. In particular, zeolite having a reactive surface, may be excluded.

[0098] In an embodiment, the infused meso- or micro- porous material of the present invention, comprises at least one halogenated methane analogue, a halogenated alkane or a halogenated organic acid as an anti-methanogenic agent. Preferably, the anti-methanogenic agent comprisesone or more compounds selected from the group consisting of: dichloromethane, dibromomethane, bromochloromethane, bromodichloromethane, bromodiiodomethane, dibromoiodomethane, bromoiodomethane, chloroiodomethane, 2-bromoethanesulfonic acid, chloral hydrate, chloroform, iodoform, chloroethane, dichloroethane, tetrachloroethane, hexachloroethane, bromoethane, dibromoethane, tetrabromoethane, 1,2-dibromotetrachloroethane, iodoethane, diiodoethane, iodopropane, bromoform, carbon tetrachloride, carbon tetrabromide, carbon tetraiodide and dibromochloromethane. Preferably, the one or more anti -methanogenic agents is a haloform, particularly bromoform, chloroform or iodoform, more preferably bromoform and / or iodoform. In an embodiment, the one or more anti-methanogenic agents is derived from a macroalgae, preferably a macroalgae of the genus Asparagopsis , and particularly Asparagopsis taxiformis or Asparagopsis armata. Methods of anti-methanogenic agent extraction from macroalgae are known to persons skilled in the art.

[0099] In an embodiment, the anti-methanogenic agent(s) is / are dissolved in a carrier to produce an anti-methanogenic agent-carrier (AC) solution.

[0100] The carrier may aid infusion of the dissolved anti -methanogenic agent(s) into the meso- or micro- porous material and, as such, the carrier may be a liquid, or low-melting point wax or viscous liquid.

[0101] In an embodiment, the liquid may be an oil.

[0102] In an embodiment, the anti -methanogenic agent(s) is / are dissolved in a carrier oil to produce an anti-methanogenic agent-oil (AO) solution. In an embodiment, the oil may be an edible oil such as vegetable oil or an edible oil blend. In a preferred embodiment, the oil is a canola oil or canola oil blend.

[0103] Regarding low-melting point waxes or viscous liquids, the melting point temperature is such that the wax or viscous liquid is free flowing at body temperature, preferably wherein body temperature is about or above 35°C. By contrast, low-melting point waxes or viscous liquids are less free flowing or solidify at temperatures less than body temperature (i.e. less than about 35°C).

[0104] Further, in an embodiment, the degree to which waxes or viscous liquids become more free flowing may provide for a sustained release of anti-methanogenic agent(s) from the meso- or micro- porous carrier. In alternative embodiments, the infused meso- or micro- porous carrier may be formulated in a wax, viscous liquid, capsule, tablet, bolus or other vehicle, to provide a sustained release of anti-methanogenic agent(s). In an alternative embodiment, the infused meso- or micro- porous carrier is not encapsulated or coated.

[0105] In an embodiment, the meso- or micro- porous material (MM) is infused with the AC (or AO) solution at a ratio of MM: AC of at least about 1: 1 to about 20: 1, preferably from about 4: 1 to about 9: 1.

[0106] In an embodiment, the meso- or micro- porous material (MM) is infused with the AC (or AO) solution at a ratio of MM:AC selected from the group consisting of: from about 1 : 1 to about 20: 1; from about 1 : 1 to about 15: 1; from about 1 : 1 to about 10: 1; from about 1 : 1 to about 9: 1; from about 1:2 to about 5:2; from about 1:2 to about 3:2; from about 3:2 to about 5:2; from about 2: 1 to about 20: 1; from about 2: 1 to about 15: 1; from about 2: 1 to about 10: 1; from about 2: 1 to about 9: 1; from about 3 : 1 to about 20: 1; from about 3 : 1 to about 15: 1; from about 3 : 1 to about 10: 1; from about 3: 1 to about 9: 1; from about 4: 1 to about 20: 1; from about 4: 1 to about 15: 1; from about 4: 1 to about 10: 1; from about 4: 1 to about 9: 1; from about 5: 1 to about 20: 1; from about 5: 1 to about 15: 1; from about 5: 1 to about 10: 1; from about 5: 1 to about 9: 1; from about 6: 1 to about 20: 1; from about 6: 1 to about 15: 1; from about 6: 1 to about 10: 1; from about 6: 1 to about 9: 1; from about 7: 1 to about 20: 1; from about 7: 1 to about 15: 1; from about 7: 1 to about 10: 1; from about 7: 1 to about 9: 1; from about 8 : 1 to about 20: 1; from about 8 : 1 to about 15: 1; from about 8 : 1 to about 10: 1; from about 8: 1 to about 9: 1; from about 9: 1 to about 20: 1; from about 9: 1 to about 15: 1; from about 9: 1 to about 10: 1; from about 10: 1 to about 20: 1; from about 10: 1 to about 15: 1; and from about 15: 1 to about 20: 1.

[0107] In an embodiment, the meso- or micro- porous material (MM) is infused with the AC (or AO) solution at a ratio of MM: AC of at least about 1: 1; preferably at least about 2: 1; preferably at least about 3: 1; preferably at least about 4: 1; preferably at least about 5: 1; preferably at least about 6: 1; preferably at least about 7: 1; preferably at least about 8: 1; preferably at least about 9: 1; preferably at least about 10: 1; preferably at least about 11: 1; preferably at least about 12: 1; preferably at least about 13: 1; preferably at least about 14: 1; preferably at least about 15: 1; preferably at least about 16: 1; preferably at least about 17: 1; preferably at least about 18: 1; preferably at least about 19 : 1 ; or preferably at least about 20: 1.

[0108] In an embodiment, the infused meso- or micro- porous material comprises pores with an average surface area, or a median surface area, as calculated by the Brunauer-Emmett-Teller (BET) method (Brunauer, S., Emmett, P. H., & Teller, E. (1938) Adsorption of Gases in Multimolecular Layers, Journal of the American Chemical Society, 60(2), 309-319), selected from the group consisting of: from about 0.1 m2 / g to about 1500 m2 / g; from about 0.1 m2 / g to about 1400 m2 / g; from about 0.1 m2 / g to about 1300 m2 / g; from about 0.1 m2 / g to about 1200 m2 / g; from about 0.1 m2 / g to about 1100 m2 / g; from about 0.1 m2 / g to about 1000 m2 / g; from about 0.1 m2 / gto about 900 m2 / g; from about 0.1 m2 / g to about 800 m2 / g; from about 0. 1 m2 / g to about 700 m2 / g; from about 0.1 m2 / g to about 600 m2 / g; from about 0.1 m2 / g to about 500 m2 / g; from about 0.1 m2 / g to about 450 m2 / g; from about 0.1 m2 / g to about 400 m2 / g; from about 0.5 m2 / g to about 1500 m2 / g; from about 0.5 m2 / g to about 1400 m2 / g; from about 0.5 m2 / g to about 1300 m2 / g; from about 0.5 m2 / g to about 1200 m2 / g; from about 0.5 m2 / g to about 1100 m2 / g; from about 0.5 m2 / g to about 1000 m2 / g; from about 0.5 m2 / g to about 900 m2 / g; from about 0.5 m2 / g to about 800 m2 / g; from about 0.5 m2 / g to about 700 m2 / g; from about 0.5 m2 / g to about 600 m2 / g; from about 0.5 m2 / g to about 500 m2 / g; from about 0.5 m2 / g to about 450 m2 / g; from about 0.5 m2 / g to about 400 m2 / g; from about 1 m2 / g to about 1500 m2 / g; from about 1 m2 / g to about 1400 m2 / g; from about 1 m2 / g to about 1300 m2 / g; from about 1 m2 / g to about 1200 m2 / g; from about 1 m2 / g to about 1100 m2 / g; from about 1 m2 / g to about 1000 m2 / g; from about 1 m2 / g to about 900 m2 / g; from about 1 m2 / g to about 800 m2 / g; from about 1 m2 / g to about 700 m2 / g; from about 1 m2 / g to about 600 m2 / g; from about 1 m2 / g to about 500 m2 / g; from about 1 m2 / g to about 450 m2 / g; from about 1 m2 / g to about 400 m2 / g; from about 1.5 m2 / g to about 1500 m2 / g; from about 1.5 m2 / g to about 1400 m2 / g; from about 1.5 m2 / g to about 1300 m2 / g; from about 1.5 m2 / g to about 1200 m2 / g; from about 1.5 m2 / g to about 1100 m2 / g; from about 1.5 m2 / g to about 1000 m2 / g; from about 1.5 m2 / g to about 900 m2 / g; from about 1.5 m2 / g to about 800 m2 / g; from about 1.5 m2 / g to about 700 m2 / g; from about 1.5 m2 / g to about 600 m2 / g; from about 1.5 m2 / g to about 500 m2 / g; from about 1.5 m2 / g to about 450 m2 / g; from about 1.5 m2 / g to about 400 m2 / g; from about 1.8 m2 / g to about 1500 m2 / g; from about 1.8 m2 / g to about 1400 m2 / g; from about 1.8 m2 / g to about 1300 m2 / g; from about 1.8 m2 / g to about 1200 m2 / g; from about 1.8 m2 / g to about 1100 m2 / g; from about 1.8 m2 / g to about 1000 m2 / g; from about 1.8 m2 / g to about 900 m2 / g; from about 1.8 m2 / g to about 800 m2 / g; from about 1.8 m2 / g to about 700 m2 / g; from about 1.8 m2 / g to about 600 m2 / g; from about 1.8 m2 / g to about 500 m2 / g; from about 1.8 m2 / g to about 450 m2 / g; from about 1.8 m2 / g to about 400 m2 / g; from about 2 m2 / g to about 1500 m2 / g; from about 2 m2 / g to about 1400 m2 / g; from about 2 m2 / g to about 1300 m2 / g; from about 2 m2 / g to about 1200 m2 / g; from about 2 m2 / g to about 1100 m2 / g; from about 2 m2 / g to about 1000 m2 / g; from about 2 m2 / g to about 900 m2 / g; from about 2 m2 / g to about 800 m2 / g; from about 2 m2 / g to about 700 m2 / g; from about 2 m2 / g to about 600 m2 / g; from about 2 m2 / g to about 500 m2 / g; from about 2 m2 / g to about 450 m2 / g; from about 5 m2 / g to about 400 m2 / g; from about 5 m2 / g to about 450 m2 / g; and from about 5 m2 / g to about 400 m2 / g.

[0109] In an embodiment, the infused meso- or micro- porous material comprises pores with an average surface area, or a median surface area, as calculated by the Brunauer-Emmett-Teller (BET) method, of about 1285.37 m2 / g, about 941.26 m2 / g, about 874.86 m2 / g, about 362.1 m2 / g,about 314.38 m2 / g, about 272.99 m2 / g, about 118.97 m2 / g, about 76. 21 m2 / g, about 6.95 m2 / g, and about 1.86 m2 / g.

[0110] In an embodiment, the infused meso- or micro- porous material comprises pores with an average pore volume, or a median pore volume, of from about 0.01 cm3 / g to about 1.5 cm3 / g, preferably from about 0.01 cm3 / g to about 1.3 cm3 / g, preferably from about 0.3 cm3 / g to about 0.5 cm3 / g, or preferably from about 0.045 cm3 / g to about 0.466 cm3 / g.

[0111] In an embodiment, the infused meso- or micro- porous material comprises pores with an average pore volume, or a median pore volume, selected from the group consisting of: from about 0.001 cm3 / g to about 1.10 cm3 / g; from about 0.001 cm3 / g to about 1.00 cm3 / g; from about 0.001 cm3 / g to about 0.90 cm3 / g; from about 0.001 cm3 / g to about 0.80 cm3 / g; from about 0.001 cm3 / g to about 0.70 cm3 / g; from about 0.001 cm3 / g to about 0.60 cm3 / g; from about 0.001 cm3 / g to about 0.50 cm3 / g; from about 0.002 cm3 / g to about 1.10 cm3 / g; from about 0.002 cm3 / g to about 1.00 cm3 / g; from about 0.002 cm3 / g to about 0.90 cm3 / g; from about 0.002 cm3 / g to about 0.80 cm3 / g; from about 0.002 cm3 / g to about 0.70 cm3 / g; from about 0.002 cm3 / g to about 0.60 cm3 / g; from about 0.002 cm3 / g to about 0.50 cm3 / g; from about 0.003 cm3 / g to about 1.10 cm3 / g; from about 0.003 cm3 / g to about 1.00 cm3 / g; from about 0.003 cm3 / g to about 0.90 cm3 / g; from about 0.003 cm3 / g to about 0.80 cm3 / g; from about 0.003 cm3 / g to about 0.70 cm3 / g; from about 0.003 cm3 / g to about 0.60 cm3 / g; from about 0.003 cm3 / g to about 0.50 cm3 / g; from about 0.004 cm3 / g to about 1.10 cm3 / g; from about 0.004 cm3 / g to about 1.00 cm3 / g; from about 0.004 cm3 / g to about 0.90 cm3 / g; from about 0.004 cm3 / g to about 0.80 cm3 / g; from about 0.004 cm3 / g to about 0.70 cm3 / g; from about 0.004 cm3 / g to about 0.60 cm3 / g; from about 0.004 cm3 / g to about 0.50 cm3 / g; from about 0.005 cm3 / g to about 1.10 cm3 / g; from about 0.005 cm3 / g to about 1.00 cm3 / g; from about 0.005 cm3 / g to about 0.90 cm3 / g; from about 0.005 cm3 / g to about 0.80 cm3 / g; from about 0.005 cm3 / g to about 0.70 cm3 / g; from about 0.005 cm3 / g to about 0.60 cm3 / g; from about 0.005 cm3 / g to about 0.50 cm3 / g; from about 0.006 cm3 / g to about 1.10 cm3 / g; from about 0.006 cm3 / g to about 1.00 cm3 / g; from about 0.006 cm3 / g to about 0.90 cm3 / g; from about 0.006 cm3 / g to about 0.80 cm3 / g; from about 0.006 cm3 / g to about 0.70 cm3 / g; from about 0.006 cm3 / g to about 0.60 cm3 / g; from about 0.006 cm3 / g to about 0.50 cm3 / g; from about 0.007 cm3 / g to about 1.10 cm3 / g; from about 0.007 cm3 / g to about 1.00 cm3 / g; from about 0.007 cm3 / g to about 0.90 cm3 / g; from about 0.007 cm3 / g to about 0.80 cm3 / g; from about 0.007 cm3 / g to about 0.70 cm3 / g; from about 0.007 cm3 / g to about 0.60 cm3 / g; from about 0.007 cm3 / g to about 0.50 cm3 / g; from about 0.008 cm3 / g to about 1.10 cm3 / g; from about 0.008 cm3 / g to about 1.00 cm3 / g; from about 0.008 cm3 / g to about 0.90 cm3 / g; from about 0.008 cm3 / g to about 0.80 cm3 / g; from about 0.008 cm3 / g to about 0.70 cm3 / g; from about 0.008 cm3 / g to about 0.60 cm3 / g; from about 0.008 cm3 / g to about 0.50 cm3 / g;from about 0.009 cm3 / g to about 1.10 cm3 / g; from about 0.009 cm3 / g to about 1.00 cm3 / g; from about 0.009 cm3 / g to about 0.90 cm3 / g; from about 0.009 cm3 / g to about 0.80 cm3 / g; from about 0.009 cm3 / g to about 0.70 cm3 / g; from about 0.009 cm3 / g to about 0.60 cm3 / g; from about 0.009 cm3 / g to about 0.50 cm3 / g; from about 0.010 cm3 / g to about 1.10 cm3 / g; from about 0.010 cm3 / g to about 1.00 cm3 / g; from about 0.010 cm3 / g to about 0.90 cm3 / g; from about 0.010 cm3 / g to about 0.80 cm3 / g; from about 0.010 cm3 / g to about 0.70 cm3 / g; from about 0.010 cm3 / g to about 0.60 cm3 / g; from about 0.010 cm3 / g to about 0.50 cm3 / g; from about 0.020 cm3 / g to about 1.00 cm3 / g; from about 0.030 cm3 / g to about 1.00 cm3 / g; from about 0.035 cm3 / g to about 1.00 cm3 / g; from about 0.040 cm3 / g to about 1.00 cm3 / g; from about 0.050 cm3 / g to about 1.00 cm3 / g; from about 0.060 cm3 / g to about 1.00 cm3 / g; from about 0.020 cm3 / g to about 0.50 cm3 / g; from about 0.030 cm3 / g to about 0.50 cm3 / g; from about 0.035 cm3 / g to about 0.50 cm3 / g; from about 0.040 cm3 / g to about 0.50 cm3 / g; from about 0.050 cm3 / g to about 0.50 cm3 / g; and from about 0.060 cm3 / g to about 0.50 cm3 / g.

[0112] In an embodiment, the infused meso- or micro- porous material comprises pores with an average pore volume, or a median pore volume, selected from about 1.088 cm3 / g, preferably about 0.466 cm3 / g, preferably about 0.388 cm3 / g, preferably about 0.193 cm3 / g, preferably about 0.179 cm3 / g, preferably about 0.157 cm3 / g, preferably about 0.055 cm3 / g, preferably about 0.045 cm3 / g, preferably about 0.005 cm3 / g, or preferably about 0.003 cm3 / g.

[0113] In an embodiment, the infused meso- or micro- porous material has an average pore size, or a median pore size ranging from about 1 nm to about 1 pm, preferably from about 1 nm to about 10 nm, preferably from about 1.498 nm to about 10.532 nm, preferably from 1 nm to about 5nm, or preferably from about 1.498 nm to about 3.387 nm.

[0114] In an embodiment, the infused meso- or micro- porous material has an average pore size, or a median pore size selected from the group consisting of: from about 1.0 nm to about 10 nm; from about 1.0 nm to about 9.0 nm; from about 1.0 nm to about 8.0 nm; from about 1.0 nm to about 7.0 nm; from about 1.0 nm to about 6.0 nm; from about 1.0 nm to about 5.0 nm; from about 1.0 nm to about 4.5.0 nm; from about 1.0 nm to about 4.0 nm; from about 1.0 nm to about 3.5 nm; from about 1. 1 nm to about 10 nm; from about 1.1 nm to about 9.0 nm; from about 1.1 nm to about 8.0 nm; from about 1.1 nm to about 7.0 nm; from about 1.1 nm to about 6.0 nm; from about 1.1 nm to about 5.0 nm; from about 1.1 nm to about 4.5.0 nm; from about 1.1 nm to about 4.0 nm; from about 1. 1 nm to about 3.5 nm; from about 1.2 nm to about 10 nm; from about 1.2 nm to about 9.0 nm; from about 1.2 nm to about 8.0 nm; from about 1.2 nm to about 7.0 nm; from about 1.2 nm to about 6.0 nm; from about 1.2 nm to about 5.0 nm; from about 1.2 nm to about 4.5.0 nm;from about 1.2 nm to about 4.0 nm; from about 1.2 nm to about 3.5 nm; from about 1.3 nm to about 10 nm; from about 1.3 nm to about 9.0 nm; from about 1.3 nm to about 8.0 nm; from about1.3 nm to about 7.0 nm; from about 1.3 nm to about 6.0 nm; from about 1.3 nm to about 5.0 nm; from about 1.3 nm to about 4.5.0 nm; from about 1.3 nm to about 4.0 nm; from about 1.3 nm to about 3.5 nm; from about 1.4 nm to about 10 nm; from about 1.4 nm to about 9.0 nm; from about1.4 nm to about 8.0 nm; from about 1.4 nm to about 7.0 nm; from about 1.4 nm to about 6.0 nm; from about 1.4 nm to about 5.0 nm; from about 1.4 nm to about 4.5.0 nm; from about 1.4 nm to about 4.0 nm; from about 1.4 nm to about 3.5 nm; from about 1.5 nm to about 10 nm; from about1.5 nm to about 9.0 nm; from about 1.5 nm to about 8.0 nm; from about 1.5 nm to about 7.0 nm; from about 1.5 nm to about 6.0 nm; from about 1.5 nm to about 5.0 nm; from about 1.5 nm to about 4.5.0 nm; from about 1.5 nm to about 4.0 nm; and from about 1.5 nm to about 3.5 nm.

[0115] In an embodiment, the infused meso- or micro- porous material has an average pore size, or a median pore size of about 10.532 nm, preferably about 3.387 nm, preferably about 2.351 nm, preferably about 2.31 nm, preferably about 2.284 nm, preferably about 2.133 nm, preferably about 1.979 nm, preferably about 1.848 nm, preferably about 1.773 nm, or preferably about 1.498 nm.

[0116] Preferably, the meso- or micro- porous material may be particulate. That is, the meso- or micro- porous material may comprise or consist of a plurality of particles. In an embodiment, the meso- or micro- porous material may be broken down (e.g. by grinding, milling or crushing) to form a plurality of particles. In an embodiment, the meso- or micro- porous material comprises or consists of a plurality of milled particles.

[0117] In an embodiment, the average particle size, or median particle size, of the plurality of particles of the meso- or micro- porous material is from about 100 pm to about 5000 pm, preferably from about 100 pm to about 4000 pm, preferably from about 100 pm to about 3000 pm, preferably from about 100 pm to about 2000 pm, preferably from about 100 pm to about 1000 pm, preferably from about 100 pm to about 500 pm or preferably from about 100 pm to about 250 pm.

[0118] In an embodiment, the average particle size, or the median particle size, of the plurality of particles is from about 100 pm to about 2 cm (20,000 pm), preferably from about 100 pm to about1.5 cm (15,000 pm), preferably from about 100 pm to about 1 cm (10,000 pm), or preferably from about 100 pm to about 0.5 cm (5000 pm).

[0119] In an embodiment, the average particle size, or the median particle size, of the plurality of particles is selected from the group consisting of: from about 50 pm to about 200 pm; from about 50 pm to about 300 pm; from about 50 pm to about 400 pm; from about 50 pm to about 500 pm; from about 50 pm to about 600 pm; from about 50 pm to about 700 pm; from about 50 pm toabout 800 un; from about 50 pirn to about 900 pun; from about 50 pim to about 1000 pun; from about 75 pim to about 200 pim; from about 75 pim to about 300 pim; from about 75 pim to about 400 pim; from about 75 pim to about 500 pim; from about 75 pim to about 600 pim; from about 75 pim to about 700 pim; from about 75 pim to about 800 pim; from about 75 pim to about 900 pim; from about 75 pim to about 1000 pim; from about 90 pim to about 200 pun; from about 90 pun to about 300 pun; from about 90 pun to about 400 pun; from about 90 pun to about 500 pun; from about 90 pun to about 600 pun; from about 90 pun to about 700 pun; from about 90 pun to about 800 pun; from about 90 pun to about 900 pun; from about 90 pun to about 1000 pun; from about 100 pun to about 200 pun; from about 100 pun to about 300 pun; from about 100 pun to about 400 pun; from about 100 pun to about 500 pun; from about 100 pun to about 600 pun; from about 100 pun to about 700 pun; from about 100 pun to about 800 pun; from about 100 pun to about 900 pun; from about 100 pun to about 1000 pun; from about 150 pun to about 200 pun; from about 150 pun to about 300 pun; from about 150 pun to about 400 pun; from about 150 pun to about 500 pun; from about 150 pun to about 600 pun; from about 150 pun to about 700 pun; from about 150 pun to about 800 pun; from about 150 pun to about 900 pun; from about 150 pun to about 1000 pun; from about 200 pun to about 300 pun; from about 200 pun to about 400 pun; from about 200 pun to about 500 pun; from about 200 pun to about 600 pun; from about 200 pun to about 700 pun; from about 200 pun to about 800 pun; from about 200 pun to about 900 pun; from about 200 pun to about 1000 pun; from about 200 pun to about 200 pun; from about 250 pun to about 300 pun; from about 250 pun to about 400 pun; from about 250 pun to about 500 pun; from about 250 pun to about 600 pun; from about 250 pun to about 700 pun; from about 250 pun to about 800 pun; from about 250 pun to about 900 pun; from about 250 pun to about 1000 pun; from about 300 pun to about 400 pun; from about 300 pun to about 500 pun; from about 300 pun to about 600 pun; from about 300 pun to about 700 pun; from about 300 pun to about 800 pun; from about 300 pun to about 900 pun; from about 300 pun to about 1000 pun; from about 350 pun to about 400 pun; from about 350 pun to about 500 pun; from about 350 pun to about 600 pun; from about 350 pun to about 700 pun; from about 350 pun to about 800 pun; from about 350 pun to about 900 pun; from about 350 pun to about 1000 pun; from about 400 pun to about 500 pun; from about 400 pun to about 600 pun; from about 400 pun to about 700 pun; from about 400 pun to about 800 pun; from about 400 pun to about 900 pun; from about 400 pun to about 1000 pun; from about 450 pun to about 500 pun; from about 450 pun to about 600 pun; from about 450 pun to about 700 pun; from about 450 pun to about 800 pun; from about 450 pun to about 900 pun; from about 450 pun to about 1000 pun; from about 500 pun to about 600 pun; from about 500 pun to about 700 pun; from about 500 pun to about 800 pun; from about 500 pun to about 900 pun; from about 500 pun to about1000 pun; from about 500 pim to about 500 pun; from about 500 pun to about 600 pun; from about 500 pun to about 700 pun; from about 500 pun to about 800 pun; from about 500 pun to about 900 un; from about 500 pun to about 1000 pun, from about 250 pun to about 2 cm (20,000 pun); from about 250 pun to about 1.8 cm; from about 250 pun to about 1.6 cm; from about 250 pun to about 1.5 cm; from about 250 pun to about 1.4 cm; from about 250 pun to about 1.3 cm; from about 250 pun to about 1.2 cm (20,000 pun); from about 250 pun to about 1.1 cm; from about 250 pun to about 1.0 cm (10,000 pun); from about 500 pun to about 2 cm (20,000 pun); from about 500 pun to about 1.8 cm; from about 500 pun to about 1.6 cm; from about 500 pun to about 1.5 cm; from about 500 pun to about 1.4 cm; from about 500 pun to about 1.3 cm; from about 500 pun to about 1.2 cm (20,000 pun); from about 500 pun to about 1.1 cm; from about 500 pun to about 1.0 cm (10,000 pun); from about 750 pun to about 2 cm (20,000 pun); from about 750 pun to about 1.8 cm; from about 750 pun to about 1.6 cm; from about 750 pun to about 1.5 cm; from about 750 pun to about 1.4 cm; from about 750 pun to about 1.3 cm; from about 750 pun to about 1.2 cm (20,000 pun); from about 750 pun to about 1.1 cm; from about 750 pun to about 1.0 cm (10,000 pun); from about 1000 pun to about 2 cm (20,000 pun); from about 1000 pun to about1.8 cm; from about 1000 pun to about 1.6 cm; from about 1000 pun to about 1.5 cm; from about 1000 pun to about 1.4 cm; from about 1000 pun to about 1.3 cm; from about 1000 pun to about1.2 cm (20,000 pun); from about 1000 pun to about 1.1 cm; from about 1000 pun to about 1.0 cm (10,000 pun); from about 1500 pun to about 2 cm (20,000 pun); from about 1500 pun to about1.8 cm; from about 1500 pun to about 1.6 cm; from about 1500 pun to about 1.5 cm; from about 1500 pun to about 1.4 cm; from about 1500 pun to about 1.3 cm; from about 1500 pun to about1.2 cm (20,000 pun); from about 1500 pun to about 1.1 cm; from about 1500 pun to about 1.0 cm (10,000 pun); from about 2000 pun to about 2 cm (20,000 pun); from about 2000 pun to about1.8 cm; from about 2000 pun to about 1.6 cm; from about 2000 pun to about 1.5 cm; from about 2000 pun to about 1.4 cm; from about 2000 pun to about 1.3 cm; from about 2000 pun to about1.2 cm (20,000 pun); from about 2000 pun to about 1.1 cm; from about 2000 pun to about 1.0 cm (10,000 pun); from about 2500 pun to about 2 cm (20,000 pun); from about 2500 pun to about1.8 cm; from about 2500 pun to about 1.6 cm; from about 2500 pun to about 1.5 cm; from about 2500 pun to about 1.4 cm; from about 2500 pun to about 1.3 cm; from about 2500 pun to about1.2 cm (20,000 pun); from about 2500 pun to about 1.1 cm; from about 2500 pun to about 1.0 cm (10,000 pun); from about 3000 pun to about 2 cm (20,000 pun); from about 3000 pun to about1.8 cm; from about 3000 pun to about 1.6 cm; from about 3000 pun to about 1.5 cm; from about 3000 pun to about 1.4 cm; from about 3000 pun to about 1.3 cm; from about 3000 pun to about1.2 cm (20,000 pun); from about 3000 pun to about 1.1 cm; from about 3000 pun to about 1.0 cm(10,000 pm); from about 3500 pm to about 2 cm (20,000 pm); from about 3500 pm to about1.8 cm; from about 3500 pm to about 1.6 cm; from about 3500 pm to about 1.5 cm; from about 3500 pm to about 1.4 cm; from about 3500 pm to about 1.3 cm; from about 3500 pm to about1.2 cm (20,000 pm); from about 3500 pm to about 1.1 cm; from about 3500 pm to about 1.0 cm (10,000 pm); from about 4000 pm to about 2 cm (20,000 pm); from about 4000 pm to about1.8 cm; from about 4000 pm to about 1.6 cm; from about 4000 pm to about 1.5 cm; from about 4000 pm to about 1.4 cm; from about 4000 pm to about 1.3 cm; from about 4000 pm to about1.2 cm (20,000 pm); from about 4000 pm to about 1.1 cm; from about 4000 pm to about 1.0 cm (10,000 pm); from about 4500 pm to about 2 cm (20,000 pm); from about 4500 pm to about1.8 cm; from about 4500 pm to about 1.6 cm; from about 4500 pm to about 1.5 cm; from about 4500 pm to about 1.4 cm; from about 4500 pm to about 1.3 cm; from about 4500 pm to about1.2 cm (20,000 pm); from about 4500 pm to about 1.1 cm; from about 4500 pm to about 1.0 cm (10,000 pm); from about 5000 pm to about 2 cm (20,000 pm); from about 5000 pm to about1.8 cm; from about 5000 pm to about 1.6 cm; from about 5000 pm to about 1.5 cm; from about 5000 pm to about 1.4 cm; from about 5000 pm to about 1.3 cm; from about 5000 pm to about1.2 cm (20,000 pm); from about 5000 pm to about 1.1 cm; about 5000 pm to about 1.0 cm (10,000 pm) and from about 1000 pm to about 5000 pm.

[0120] The physical and chemical properties of microporous and mesoporous materials may influence the absorption / desorption of molecules from their surfaces. Microporous materials are materials with pore diameters of less than 2 nm (20 angstroms) while mesoporous materials are materials with pore diameters of between 2 nm - 50 nm. Common examples of microporous materials include zeolites, metal-organic frameworks, biochars and activated carbons, while mesoporous materials include mesoporous carbon, activated carbon and various metal oxides.

[0121] In an embodiment, the meso- or micro- porous material has a carbon content of from about 50 % to about 99 %, preferably from about 50 % to about 95 %, preferably from about 50 % to about 90 %, or preferably from about 55.3 % to about 89. 18 %.

[0122] In an embodiment, the meso- or micro- porous material has a carbon content selected from the group consisting of: from about 50 % to about 100 %; from about 50 % to about 99 %; from about 50 % to about 95 %; from about 50 % to about 90 %; from about 50 % to about 85 %; from about 50 % to about 80 %; from about 55 % to about 100 %; from about 55 % to about 99 %; from about 55 % to about 95 %; from about 55 % to about 90 %; from about 55 % to about 85 %; from about 55 % to about 80 %; from about 60 % to about 100 %; from about 60 % to about 99 %; from about 60 % to about 95 %; from about 60 % to about 90 %; from about 60 % to about 85 %; fromabout 60 % to about 80 %; from about 65 % to about 100 %; from about 65 % to about 99 %; from about 65 % to about 95 %; from about 65 % to about 90 %; from about 65 % to about 85 %; from about 65 % to about 80 %; from about 70 % to about 100 %; from about 70 % to about 99 %; from about 70 % to about 95 %; from about 70 % to about 90 %; from about 70 % to about 85 %; from about 70 % to about 80 %; from about 75 % to about 100 %; from about 75 % to about 99 %; from about 75 % to about 95 %; from about 75 % to about 90 %; from about 75 % to about 85 %; from about 75 % to about 80 %; from about 80 % to about 100 %; from about 80 % to about 99 %; from about 80 % to about 98 %; from about 80 % to about 97 %; from about 80 % to about 96 %; from about 80 % to about 95 %; from about 80 % to about 90 %; from about 80 % to about 89 %; from about 80 % to about 88 %; from about 80 % to about 87 %; from about 80 % to about 86 %; from about 80 % to about 85 %; from about 85 % to about 100 %; from about 85 % to about 99 %; from about 85 % to about 98 %; from about 85 % to about 97 %; from about 85 % to about 96 %; from about 85 % to about 95 %; from about 85 % to about 94 %; from about 85 % to about 93 %; from about 85 % to about 92 %; from about 85 % to about 91 %; from about 85 % to about 90 %; from about 90 % to about 100 %; from about 90 % to about 99 %; from about 90 % to about 98 %; from about 90 % to about 97 %; from about 90 % to about 96 %; from about 90 % to about 95 %; from about 90 % to about 94 %; from about 90 % to about 93 %; from about 90 % to about 92 %; from about 90 % to about 91 %; from about 95 % to about 100 %; from about 95 % to about 99 %; from about 95 % to about 98 %; from about 95 % to about 97 %; and from about 95 % to about 96 %.

[0123] In an embodiment, the meso- or micro- porous material has a carbon content of about 89.78 %, preferably about 89. 18 %, preferably about 79.37 %, preferably about 79. 1 %, preferably about 79.07 %, preferably about 75.11 %, preferably about 74.94 %, preferably about 66.34 %, preferably about 62.78 %, or preferably about 55.3 %.

[0124] In an embodiment, the meso- or micro- porous material has a hydrogen content of from about 0.5 % to about 5.0 %, preferably from about 0.5 % to about 4.0 %, preferably from about 0.5 % to about 3.5 %, or preferably from about 0.53 % to about 3.48 %.

[0125] In an embodiment, the meso- or micro- porous material has a hydrogen content selected from the group consisting of: from about 0.25 % to about 5.0 %; from about 0.25 % to about 4.0 %; from about 0.25 %to about 3.5 %; from about 0.25 %to about 3.0 %; from about 0.25 % to about 2.5 %; from about 0.25 % to about 2.0 %; from about 0.4 % to about 5.0 %; from about 0.4 % to about 4.0 %; from about 0.4 %to about 3.5 %; from about 0.4 %to about 3.0 %; from about 0.4 % to about 2.5 %; from about 0.4 % to about 2.0 %; from about 0.5 % to about 5.0 %; from about 0.5 % to about 4.0 %; from about 0.5 % to about 3.5 %; from about 0.5 % to about 3.0 %; fromabout 0.5 % to about 2.5 %; from about 0.5 % to about 2.0 %; from about 0.6 % to about 5.0 %; from about 0.6 % to about 4.0 %; from about 0.6 % to about 3.5 %; from about 0.6 % to about 3.0 %; from about 0.6 % to about 2.5 %; from about 0.6 % to about 2.0 %; from about 0.7 % to about 5.0 %; from about 0.7 %to about 4.0 %; from about 0.7 %to about 3.5 %; from about 0.7 % to about 3.0 %; from about 0.7 % to about 2.5 %; from about 0.7 % to about 2.0 %; from about 0.8 % to about 5.0 %; from about 0.8 % to about 4.0 %; from about 0.8 % to about 3.5 %; from about 0.8 % to about 3.0 %; from about 0.8 % to about 2.5 %; from about 0.8 % to about 2.0 %; from about 0.9 % to about 5.0 %; from about 0.9 % to about 4.0 %; from about 0.9 % to about3.5 %; from about 0.9 % to about 3.0 %; from about 0.9 % to about 2.5 %; from about 0.9 % to about 2.0 %; from about 1 .0 % to about 5.0 %; from about 1.0 % to about 4.0 %; from about 1.0 % to about 3.5 %; from about 1.0 % to about 3.0 %; from about 1.0 % to about 2.5 %; and from about 1.0 % to about 2.0 %.

[0126] In an embodiment, the meso- or micro- porous material has a nitrogen content of from about 0.05 % to about 1.5 %, preferably from about 0.05 % to about 1.1 %, preferably from about 0.05 % to about 1.1 %, or preferably from about 0. 10 % to about 1.08 %.

[0127] In an embodiment, the meso- or micro- porous material has a nitrogen content selected from the group consisting of: from about 0.05 %to about l.5 %; from about 0.05 %to about l.4 %; from about 0.05 %to about 1.3 %; from about 0.05 %to about 1.2 %; from about 0.05 % to about 1.1 %; from about 0.05 % to about 1.0 %; from about 0.05 % to about 0.9 %; from about 0.05 % to about 0.8 %; from about 0.05 % to about 0.5 %; from about 0.06 % to about 1.5 %; from about 0.06 %to about 1.4 %; from about 0.06 %to about 1.3 %; from about 0.06 %to about 1.2 %; from about 0.06 %to about 1.1 %; from about 0.06 %to about 1.0 %; from about 0.06 %to about 0.9 %; from about 0.06 % to about 0.8 %; from about 0.06 % to about 0.5 %; from about 0.07 % to about1.5 %; from about 0.07 % to about 1.4 %; from about 0.07 % to about 1.3 %; from about 0.07 % to about 1.2 %; from about 0.07 % to about 1.1 %; from about 0.07 % to about 1.0 %; from about 0.07 %to about 0.9 %; from about 0.07 %to about 0.8 %; from about 0.07 %to about 0.5 %; from about 0.08 %to about 1.5 %; from about 0.08 %to about 1.4 %; from about 0.08 %to about 1.3 %; from about 0.08 % to about 1.2 %; from about 0.08 % to about 1.1 %; from about 0.08 % to about 1.0 %; from about 0.08 % to about 0.9 %; from about 0.08 % to about 0.8 %; from about 0.08 % to about 0.5 %; from about 0.09 % to about 1.5 %; from about 0.09 % to about 1.4 %; from about 0.09 % to about 1.3 %; from about 0.09 % to about 1.2 %; from about 0.09 % to about 1.1 %; from about 0.09 %to about 1.0 %; from about 0.09 %to about 0.9 %; from about 0.09 %to about 0.8 %; from about 0.09 % to about 0.5 %; from about 0.1 % to about 1.5 %; from about 0.1 % to about 1.4 %; from about 0.1 % to about 1.3 %; from about 0.1 % to about 1.2 %; from about 0.1 % toabout 1.1 %; from about 0.1 %to about 1.0 %; from about 0.1 %to about 0.9 %; from about 0.1 % to about 0.8 %; and from about 0.1 % to about 0.5 %.

[0128] In an embodiment, the meso- or micro- porous material has a nitrogen content of about 1.08 %, preferably about 0.87 %, preferably about 0.46 %, preferably about 0.28 %, preferably about 0.19 %, preferably about 0.15 %, preferably about 0.13 %, preferably about 0.12 %, preferably about 0. 12 %, or preferably about 0.1 %.

[0129] In an embodiment, the meso- or micro- porous material has an oxygen content of equal to or less than 25.0 %, preferably equal to or less than 20.0 %, preferably equal to or less than 18.37 %, preferably equal to or less than 15 %, preferably equal to or less than 10 %, preferably equal to or less than 5 %, or preferably equal to or less than 2 %.

[0130] In an embodiment, the meso- or micro- porous material has an oxygen content selected from equal to or less than 18.37 %, preferably equal to or less than 18.07 %, preferably equal to or less than 15.6 %, preferably equal to or less than 13.72 %, preferably equal to or less than 13.03 %, preferably equal to or less than 8.18 %, preferably equal to or less than 7.26 %, preferably equal to or less than 4.18 %, preferably equal to or less than 2.49 %, preferably less than 1 %, preferably less than 0.5 %, or preferably less than 0. 1 %.

[0131] In an embodiment, the meso- or micro- porous material has an oxygen content selected from the group consisting of: from about 0.1 % to about 25.0 %; from about 0.1 % to about 23.0 %; from about 0.1 % to about 21.0 %; from about 0.1 % to about 20.0 %; from about 0.1 % to about 19.0 %; from about 0.1 % to about 18.0 %; from about 0.1 % to about 17.0 %; from about 0.1 % to about 16.0 %; from about 0.1 %to about 15.0 %; from about 0.3 % to about 25.0 %; from about 0.3 % to about 23.0 %; from about 0.3 % to about 21.0 %; from about 0.3 % to about 20.0 %; from about 0.3 %to about 19.0 %; from about 0.3 %to about 18.0 %; from about 0.3 %to about 17.0 %; from about 0.3 % to about 16.0 %; from about 0.3 % to about 15.0 %; from about 0.5 % to about 25.0 %; from about 0.5 % to about 23.0 %; from about 0.5 % to about 21.0 %; from about 0.5 % to about 20.0 %; from about 0.5 %to about 19.0 %; from about 0.5 % to about 18.0 %; from about 0.5 %to about 17.0 %; from about 0.5 %to about 16.0 %; from about 0.5 %to about 15.0 %; from about 0.7 % to about 25.0 %; from about 0.7 % to about 23.0 %; from about 0.7 % to about 21.0 %; from about 0.7 % to about 20.0 %; from about 0.7 % to about 19.0 %; from about 0.7 % to about 18.0 %; from about 0.7 % to about 17.0 %; from about 0.7 % to about 16.0 %; from about 0.7 % to about 15.0 %; from about 0.9 %to about 25.0 %; from about 0.9 % to about 23.0 %; from about 0.9 %to about 21.0 %; from about 0.9 %to about 20.0 %; from about 0.9 %to about 19.0 %; from about 0.9 %to about 18.0 %; from about 0.9 %to about 17.0 %; from about 0.9 %to about 16.0 %;from about 0.9 % to about 15.0 %; from about 1.0 % to about 25.0 %; from about 1.0 % to about 23.0 %; from about 1.0 % to about 21.0 %; from about 1.0 % to about 20.0 %; from about 1.0 % to about 19.0 %; from about 1.0 %to about 18.0 %; from about 1.0 % to about 17.0 %; from about 1.0 %to about 16.0 %; from about 1.0 %to about 15.0 %; from about 1.5 %to about 25.0 %; from about 1 .5 % to about 23.0 %; from about 1.5 % to about 21.0 %; from about 1.5 % to about 20.0 %; from about 1.5 % to about 19.0 %; from about 1.5 % to about 18.0 %; from about 1.5 % to about 17.0 %; from about 1.5 % to about 16.0 %; from about 1.5 % to about 15.0 %; from about 2.0 % to about 25.0 %; from about 2.0 % to about 23.0 %; from about 2.0 % to about 21.0 %; from about 2.0 %to about 20.0 %; from about 2.0 %to about 19.0 %; from about 2.0 %to about 18.0 %; from about 2.0 %to about 17.0 %; from about 2.0 % to about 16.0 %; from about 2.0 %to about 15.0 %; from about 2.5 % to about 25.0 %; from about 2.5 % to about 23.0 %; from about 2.5 % to about 21.0 %; from about 2.5 % to about 20.0 %; from about 2.5 % to about 19.0 %; from about 2.5 % to about 18.0 %; from about 2.5 % to about 17.0 %; from about 2.5 % to about 16.0 %; and from about 2.5 % to about 15.0 %.

[0132] In an embodiment, the meso- or micro- porous material has an oxygen content from about 1 % to about 20 %, preferably from about 2 % to about 20 %, preferably from about 1 % to about 18.37 %, or preferably from about 2.49 % to about 18.37 %.

[0133] In an embodiment, the meso- or micro- porous material has an ash content of from about 0.5 % to about 65 %, preferably from about 1 % to about 60 %, preferably from about 2 % to about 50 %, preferably from about 2 % to about 49.07 %, preferably from about 2 % to about 10 %.

[0134] In an embodiment, the meso- or micro- porous material has an ash content selected from the group consisting of: from about 0.5 % to about 65 %; from about 0.5 % to about 60 %; from about 0.5 % to about 55 %; from about 0.5 % to about 50 %; from about 0.5 % to about 40 %; from about 0.5 % to about 35 %; from about 0.5 % to about 30 %; from about 0.5 % to about 25 %; from about 0.5 %to about 20 %; from about 0.5 %to about 10 %; from about 1.0 % to about 65 %; from about 1.0 % to about 60 %; from about 1.0 % to about 55 %; from about 1.0 % to about 50 %; from about 1.0 % to about 40 %; from about 1.0 % to about 35 %; from about 1.0 % to about 30 %; from about 1.0 % to about 25 %; from about 1.0 % to about 20 %; from about 1.0 % to about 10 %; from about 1.5 % to about 65 %; from about 1.5 % to about 60 %; from about 1.5 % to about 55 %; from about 1.5 % to about 50 %; from about 1.5 % to about 40 %; from about 1.5 % to about 35 %; from about 1.5 % to about 30 %; from about 1.5 % to about 25 %; from about 1.5 % to about 20 %; from about 1.5 %to about 10 %; from about 1.6 %to about 50 %; from about 1.6 %to about 40 %; from about 1.6 % to about 35 %; from about 1.6 % to about 30 %; from about 1.6 % to about 25 %;from about 1.6 % to about 20 %; from about 1.6 % to about 10 %; from about 1.7 % to about 50 %; from about 1.7 % to about 40 %; from about 1.7 % to about 35 %; from about 1.7 % to about 30 %; from about 1.7 % to about 25 %; from about 1.7 % to about 20 %; from about 1.7 % to about 10 %; from about 1.8 % to about 50 %; from about 1.8 % to about 40 %; from about 1.8 % to about 35 %; from about 1.8 % to about 30 %; from about 1.8 % to about 25 %; from about 1.8 % to about 20 %; from about 1.8 %to about 10 %; from about 1.9 %to about 50 %; from about 1.9 %to about 40 %; from about 1.9 % to about 35 %; from about 1.9 % to about 30 %; from about 1.9 % to about 25 %; from about 1.9 % to about 20 %; from about 1.9 % to about 10 %; from about 2.0 %to about 65 %; from about 2.0 % to about 60 %; from about 2.0 % to about 55 %; from about 2.0 % to about 50 %; from about 2.0 % to about 40 %; from about 2.0 % to about 35 %; from about 2.0 % to about 30 %; from about 2.0 % to about 25 %; from about 2.0 % to about 20 %; and from about 2.0 % to about 10 %.

[0135] In an embodiment, the meso- or micro- porous material has an ash content of less than or equal to 49.07 %; less than or equal to 29.37 %; less than or equal to 16.81 %; less than or equal to 11.72 %; less than or equal to 9.24 %; less than or equal to 6.55 %; less than or equal to 2.88 %; less than or equal to 2.24 %; less than or equal to 2.2 %; less than or equal to 2.02 %, less than or equal to 1.0 %, or less than or equal to 0.5 %.

[0136] In an embodiment, the infused meso- or micro- porous material comprises at least about 1 mg of the anti-methanogenic agent(s) (dissolved in a carrier) per g of the infused meso- or micro- porous material. Preferably, the infused meso- or micro- porous material comprises about 1 mg to about 500 mg, more preferably about 5 mg to about 100 mg of the anti -methanogenic agent(s) per g of the infused meso- or micro- porous material.

[0137] In an embodiment, the infused meso- or micro- porous material comprises an amount of the anti-methanogenic agent(s) (dissolved in a carrier) per g of the infused meso- or micro- porous material selected from the group consisting of: from about 1 mg to about 500 mg; from about 1 mg to about 400 mg; from about 1 mg to about 300 mg; from about 1 mg to about 200 mg; from about 1 mg to about 100 mg; from about 1 mg to about 50 mg; from about 1 mg to about 25 mg; from about 1 mg to about 20 mg; from about 1 mg to about 10 mg; from about 1 mg to about 5 mg; from about 2 mg to about 500 mg; from about 2 mg to about 400 mg; from about 2 mg to about 300 mg; from about 2 mg to about 200 mg; from about 2 mg to about 100 mg; from about 2 mg to about 50 mg; from about 2 mg to about 25 mg; from about 2 mg to about 20 mg; from about 2 mg to about 10 mg; from about 2 mg to about 5 mg; from about 3 mg to about 500 mg; from about 3 mg to about 400 mg; from about 3 mg to about 300 mg; from about 3 mg to about 200 mg; from about3 mg to about 100 mg; from about 3 mg to about 50 mg; from about 3 mg to about 25 mg; from about 3 mg to about 20 mg; from about 3 mg to about 10 mg; from about 3 mg to about 5 mg; from about 4 mg to about 500 mg; from about 4 mg to about 400 mg; from about 4 mg to about 300 mg; from about 4 mg to about 200 mg; from about 4 mg to about 100 mg; from about 4 mg to about 50 mg; from about 4 mg to about 25 mg; from about 4 mg to about 20 mg; from about 4 mg to about 10 mg; from about 4 mg to about 5 mg; from about 5 mg to about 500 mg; from about 5 mg to about 400 mg; from about 5 mg to about 300 mg; from about 5 mg to about 200 mg; from about 5 mg to about 100 mg; from about 5 mg to about 50 mg; from about 5 mg to about 25 mg; from about 5 mg to about 20 mg; from about 5 mg to about 10 mg; from about 5 mg to about 5 mg; from about 1 mg to about 6 mg; from about 2 mg to about 6 mg; and from about 3 mg to about 6 mg.

[0138] In an embodiment, the infused meso- or micro- porous material comprises about 5 mg of anti-methanogenic agent(s) per g of the infused meso- or micro- porous material.

[0139] In an embodiment, the meso- or micro- porous material is further infused with a surfactant. In an alternative embodiment, the infused meso- or micro- porous material excludes a surfactant.

[0140] A surfactant, when added to a substance, reduces the substances surface tension. It was found that some meso- or micro- porous materials bind the infused anti-methanogenic agent(s) very strongly, limiting (or completely inhibiting) the release of the anti-methanogenic material from the meso- or micro- porous material back into the environment. Examples include meso- or micro- porous materials with High BET pores and large pore volumes, such as activated carbons. Activated carbons are pyrolysed at temperatures higher than biochars and charcoals and have a higher purity. In such cases, it may be desirable to include a surfactant in the composition to facilitate release of the anti -methanogenic agent(s).

[0141] In an embodiment, the addition of a surfactant to meso- or micro- porous material infused with anti-methanogenic agent(s) increases the release rate of the infused anti-methanogenic agent(s). The amount of surfactant added to the meso- or micro- porous material and be controlled in order to optimise the release rate of the anti-methanogenic agent(s) from the infused meso- or micro- porous material. The amount required will vary depending on the type of meso- or micro- porous material and its retention versus release relationship of the anti -methanogenic agent(s).

[0142] In an embodiment, the surfactant is an anionic surfactant selected from the group consisting of: ammonium lauryl sulfate, sodium lauryl sulfate (sodium dodecyl sulfate, SLS, or SDS), sodium laureth sulfate (sodium lauryl ether sulfate or SLES), sodium myreth sulfate and carboxylate salts (soaps), such as sodium stearate.

[0143] In an embodiment, the surfactant is a cationic surfactant selected from the group consisting of: cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, and dioctadecyldimethylammonium bromide (DODAB).

[0144] In an embodiment, the surfactant is a zwitterionic surfactant selected from the group consisting of: phospholipids, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelins, lauryldimethylamine oxide and myristamine oxide.

[0145] In an embodiment, the surfactant is a non-ionic surfactant selected from the group consisting of: Triton X-100, poloxamers, glycerol monostearate, glycerol monolaurate, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, Tween 20 (polysorbate 20), Tween 40 (polysorbate 40), Tween 60 (polysorbate 60) and Tween 80 (polysorbate 80).

[0146] In an embodiment, the surfactant is selected from the group consisting of: triton X-100; poloxamers; glycerol monostearate; glycerol monolaurate; sorbitan monolaurate; sorbitan monostearate; sorbitan tristearate; Tween 20 (polysorbate 20); Tween 40 (polysorbate 40); Tween 60 (polysorbate 60); Tween 80 (polysorbate 80); ammonium lauryl sulfate; sodium lauryl sulfate; sodium laureth sulfate; sodium myreth sulfate; sodium stearate; phospholipids; phosphatidylserine; phosphatidylethanolamine; phosphatidylcholine; sphingomyelins; lauryldimethylamine oxide; and myristamine oxide; or combinations thereof.

[0147] In an embodiment, the surfactant is Tween 20 (polysorbate 20) or Tween 80 (polysorbate 80), preferably Tween 20 (polysorbate 20).

[0148] In an embodiment, the meso- or micro- porous material comprises up to about 2 % w / w surfactant (i.e. 20 mg of surfactant per g of dry weight meso- or micro- porous material when measured prior to oven drying the meso- or micro- porous material and prior to infusing the meso- or micro- porous material with the anti-methanogenic agent(s)), preferably up to about 1.5 % w / w surfactant (i.e. 15 mg of surfactant per g of dry weight meso- or micro- porous material), preferably up to about 1.0 % w / w surfactant (i.e. 10 mg of surfactant per g of dry weight meso- or micro- porous material).

[0149] In an embodiment, the meso- or micro- porous material comprises up to about 2 % w / w surfactant, preferably up to about 1.8 % w / w, preferably up to about 1.7 % w / w, preferably up to about 1.6 % w / w, preferably up to about 1.5 % w / w, preferably up to about 1.4 % w / w, preferably up to about 1.3 % w / w, preferably up to about 1.2 % w / w, preferably up to about 1.1 % w / w, preferably up to about 1.0 % w / w, preferably up to about 0.9 % w / w, preferably up to about 0.8 %w / w, preferably up to about 0.7 % w / w, preferably up to about 0.6 % w / w, or preferably up to about 0.5 % w / w, per g of dry weight meso- or micro- porous material when measured prior to oven drying the meso- or micro- porous material and prior to infusing the meso- or micro- porous material with the anti -methanogenic agent(s).

[0150] In an embodiment, the meso- or micro- porous material comprises from about 0.1 % w / w to about 2 % w / w surfactant, or preferably from about 0.5 % w / w to about 1 % w / w surfactant, per g of dry weight meso- or micro- porous material when measured prior to oven drying the meso- or micro- porous material and prior to infusing the meso- or micro- porous material with the anti- methanogenic agent(s).

[0151] In an embodiment, the meso- or micro- porous material comprises a surfactant (% w / w) in an amount per g of dry weight meso- or micro- porous material when measured prior to oven drying the meso- or micro- porous material and prior to infusing the meso- or micro- porous material with the anti-methanogenic agent(s) selected from the group consisting of: from about 0.1 % w / w to about 0.2 % w / w; from about 0.1 % w / w to about 0.3 % w / w; from about 0.1 % w / w to about 0.4 % w / w; from about 0.1 % w / w to about 0.5 % w / w; from about 0.1 % w / w to about 0.6 % w / w; from about 0.1 % w / w to about 0.7 % w / w; from about 0.1 % w / w to about 0.8 % w / w; from about 0.1 % w / w to about 0.9 % w / w; from about 0.1 % w / w to about 1.0 % w / w; from about 0.1 % w / w to about 1.1 % w / w; from about 0.1 % w / w to about 1.2 % w / w; from about 0.1 % w / w to about 1.3 % w / w; from about 0.1 % w / w to about 1.4 % w / w; from about 0.1 % w / w to about 1.5 % w / w; from about 0.1 % w / w to about 1.6 % w / w; from about 0.1 % w / w to about 1.7 % w / w; from about 0.2 % w / w to about 1.8 % w / w; from about 0.2 % w / w to about 1.9 % w / w; from about 0.1 % w / w to about 2.0 % w / w; from about 0.2 % w / w to about 1.0 % w / w; from about 0.2 % w / w to about 1.1 % w / w; from about 0.2 % w / w to about 1.2 % w / w; from about 0.2 % w / w to about 1.3 % w / w; from about 0.2 % w / w to about 1.4 % w / w; from about 0.3 % w / w to about 1.4 % w / w; from about 0.3 % w / w to about 1.5 % w / w; from about 0.3 % w / w to about 1.6 % w / w; from about 0.3 % w / w to about 1.7 % w / w; from about 0.3 % w / w to about 1.8 % w / w; from about 0.3 % w / w to about 1.9 % w / w; from about 0.3 % w / w to about 2.0 % w / w; from about 0.4 % w / w to about 1.0 % w / w; from about 0.4 % w / w to about 1.1 % w / w; from about 0.4 % w / w to about 1.2 % w / w; from about 0.4 % w / w to about 1.3 % w / w; from about 0.4 % w / w to about 1.4 % w / w; from about 0.4 % w / w to about 1.4 % w / w; from about 0.4 % w / w to about 1.5 % w / w; from about 0.4 % w / w to about 1.6 % w / w; from about 0.4 % w / w to about 1.7 % w / w; from about 0.4 % w / w to about 1.8 % w / w; from about 0.4 % w / w to about 1.9 % w / w; from about 0.4 % w / w to about 2.0 % w / w; from about 0.5 % w / w to about 1.0 % w / w; from about 0.5 % w / w to about 1.1 % w / w; from about 0.5 % w / w to about 1.2 % w / w; from about 0.5 % w / w to about 1.3 % w / w; from about 0.5 % w / wto about 1.4 % w / w; from about 0.5 % w / w to about 1.4 % w / w; from about 0.5 % w / w to about1.5% w / w; from about 0.5 % w / w to about 1.6% w / w; from about 0.5 % w / w to about 1.7% w / w; from about 0.5 % w / w to about 1.8 %w / w; from about 0.5 %w / w to about 1.9 %w / w; from about 0.5 % w / w to about 2.0 % w / w; from about 0.6 % w / w to about 1.0 % w / w; from about0.6 %w / w to about 1.1 %w / w; from about 0.6 %w / w to about 1.2 %w / w; from about0.6 %w / w to about 1.3 %w / w; from about 0.6 %w / w to about 1.4 %w / w; from about0.6 %w / w to about 1.4 %w / w; from about 0.6 %w / w to about 1.5 %w / w; from about0.6 %w / w to about 1.6 %w / w; from about 0.6 %w / w to about 1.7 %w / w; from about0.6 %w / w to about 1.8 %w / w; from about 0.6 %w / w to about 1.9 %w / w; from about0.6 %w / w to about 2.0 %w / w; from about 0.7 %w / w to about 1.0 %w / w; from about0.7 %w / w to about 1.1 %w / w; from about 0.7 %w / w to about 1.2 %w / w; from about0.7 %w / w to about 1.3 %w / w; from about 0.7 %w / w to about 1.4 %w / w; from about0.7 %w / w to about 1.4 %w / w; from about 0.7 %w / w to about 1.5 %w / w; from about0.7 %w / w to about 1.6 %w / w; from about 0.7 %w / w to about 1.7 %w / w; from about0.7 %w / w to about 1.8 %w / w; from about 0.7 %w / w to about 1.9 %w / w; from about0.7 %w / w to about 2.0 %w / w; from about 0.8 %w / w to about 1.0 %w / w; from about0.8 %w / w to about 1.1 %w / w; from about 0.8 %w / w to about 1.2 %w / w; from about0.8 %w / w to about 1.3 %w / w; from about 0.8 %w / w to about 1.4 %w / w; from about0.8 %w / w to about 1.4 %w / w; from about 0.8 %w / w to about 1.5 %w / w; from about0.8 %w / w to about 1.6 %w / w; from about 0.8 %w / w to about 1.7 %w / w; from about0.8 %w / w to about 1.8 %w / w; from about 0.8 %w / w to about 1.9 %w / w; from about0.8 %w / w to about 2.0 %w / w; from about 0.9 %w / w to about 1.0 %w / w; from about0.9 %w / w to about 1.1 %w / w; from about 0.9 %w / w to about 1.2 %w / w; from about0.9 %w / w to about 1.3 %w / w; from about 0.9 %w / w to about 1.4 %w / w; from about0.9 %w / w to about 1.4 %w / w; from about 0.9 %w / w to about 1.5 %w / w; from about0.9 %w / w to about 1.6 %w / w; from about 0.9 %w / w to about 1.7 %w / w; from about0.9 %w / w to about 1.8 %w / w; from about 0.9 %w / w to about 1.9 %w / w; from about0.9 %w / w to about 2.0 %w / w; from about 1.0 %w / w to about 1.0 %w / w; from about1.0 %w / w to about 1.1 %w / w; from about 1.0 %w / w to about 1.2 %w / w; from about1.0 %w / w to about 1.3 %w / w; from about 1.0 %w / w to about 1.4 %w / w; from about1.0 %w / w to about 1.4 %w / w; from about 1.0 %w / w to about 1.5 %w / w; from about1.0 %w / w to about 1.6 %w / w; from about 1.0 %w / w to about 1.7 %w / w; from about1.0 %w / w to about 1.8 %w / w; from about 1.0 %w / w to about 1.9 %w / w; from about1.0 % w / w to about 2.0 % w / w; and from about 0.4 % w / w to about 1.0 % w / w.

[0152] In an embodiment, other additives which may impact the release kinetics of the anti- methanogenic agent(s) include, molasses, carnauba wax and salts.

[0153] In an embodiment, infusion of the anti-methanogenic agent(s) into the meso- or micro- porous material substantially prevents volatilisation or degradation of the anti-methanogenic agent(s) when the infused meso- or micro- porous material is exposed to biotic and / or abiotic factors when compared to an equivalent weight of anti-methanogenic agent(s) absent the biochar exposed to identical conditions. Preferably, volatilisation and / or degradation of the anti- methanogenic agent(s) is measured in air at ambient temperature and pressure over at least 7 days after infusion when compared to an equivalent weight of anti-methanogenic agent(s) absent the meso- or micro- porous material exposed to identical conditions. In this context, ‘substantially prevents’ preferably means at least about 50 %, preferably at least about 60 %, of the anti- methanogenic agent(s) is / are retained in the infused biochar by day 7 compared to an equivalent weight of anti-methanogenic agent(s) absent biochar.

[0154] In an embodiment, the retention of at least about 50 % of the anti-methanogenic agent(s) is achieved when: the anti-methanogenic agent(s) is / are dissolved in an oil carrier to produce an anti-methanogenic agent-oil (AO) solution, wherein the concentration of the agent(s) is at least about 5 mg anti- methanogenic agent(s) per g of oil, preferably from 5 mg to 200 mg anti-methanogenic agent(s) per g of oil, the meso- or micro- porous material (MM) is infused with the AO solution at a ratio of MM:A0 of at least about 1 g of MM to 1 g of AO (1: 1) to about 20: 1, preferably from about 1: 1 to about 10: 1, more preferably from about 4: 1 to about 9: 1.

[0155] In an embodiment, the infused meso- or micro- porous material is charcoal, biochar or activated carbon infused with at least one anti-methanogenic agent dissolved in oil, wherein the charcoal, biochar or activated carbon is in the form of a plurality of particles, wherein the plurality of particles have an average particle size, or a median particle size, of about 100 pm to about 1000 pm, preferably about 150 pm to about 400 pm, wherein the meso- or micro- porous material has an average pore volume, or median pore volume, of from about 0.001 cm3 / g to about 1.2 cm3 / g, or preferably from about 0.003 cm3 / g to about 1.1 cm3 / g, wherein the infused meso- or micro- porous material comprises about 1 mg to about 500 mg anti- methanogenic agent(s) per g of the infused meso- or micro- porous material, andwherein infusion of anti-methanogenic agent(s) into the meso- or micro- porous material substantially prevents volatilisation and / or degradation of the anti-methanogenic agent(s) when exposed to biotic and / or abiotic factors.

[0156] In another aspect, there is provided a composition comprising the anti -methanogenic agent- infused biochar of the present invention. In an embodiment, a composition encompasses a specific dosage form such as a liquid suspension, tablet, capsule, pellet, bolus, or lick block. Alternatively, a composition may be a result of the incorporation of the infused biochar of the invention into an animal feed, such as is one or more of cereal straws, legume straws, canola straws, cereal hays, legume hays, grass hays, com stalks / stover, silage, brewers grain, palm kernel, or mixtures thereof.

[0157] In an embodiment, the composition is incorporated into animal feed, wherein the composition is formulated to deliver at least about 5 mg of the anti-methanogenic agent(s) per kg of animal feed intake. Preferably, the composition is formulated to deliver about 25 mg of the anti-methanogenic agent(s) per kg of animal feed intake.

[0158] In an embodiment, the animal feed is selected from the group consisting of: cereal straws; legume straws; canola straws; cereal hays; legume hays; grass hays; com stalks / stover; silage; spent brewers grain; palm kernel; and mixtures thereof.

[0159] In a preferred embodiment, the composition is not encapsulated by a coating. Preferably, the infused meso- or micro- porous material retains the infused anti-methanogenic agent(s) in the absence of a protective coating for at least 7 days, preferably at least 14 days.

[0160] In an embodiment, the meso- or micro- porous material is inert and does not react with the infused anti-methanogenic agent(s). As such, materials which include reactive surfaces which act to degrade the anti-methanogenic agent(s) may be excluded. In particular, zeolite having a reactive surface may be excluded.

[0161] Zeolite mainly consists of silicon, aluminium and oxygen, and contains several metal or H+ ions with ion exchange properties.

[0162] It is postulated that mineral impurities in zeolite result in chemical decomposition of bromoform. Thus, porous materials which are inert (e.g. charcoal, biochar, activated carbon etc, or other porous materials lacking chemically destructive surfaces or impurities) are expected to protect bromoform from chemically active constituents in feed formulations.

[0163] In an embodiment, the meso- or micro- porous material infused with anti -methanogenic agent(s) and, optionally, a surfactant, is substantially free from or excludes added protein, such as, but not limited to, non-enzymatically browned bypass protein. “Added protein” refers to anyexternal source of protein or peptide which is not derived from, or originally present within, the meso- or micro- porous material.

[0164] In an embodiment, the meso- or micro- porous material infused with anti -methanogenic agent(s) and, optionally, a surfactant, is substantially free from, or excludes, biochar in catalytic systems. Biochar provides a large surface area material and can be used as a catalyst support (e.g., metal ions / metal complexe s / metal particles) providing well dispersed active sites for catalytic degradation reactions. Such biochar / catalyst systems for the specific purpose of degrading halogenated organic molecules would work against the invention and are therefore excluded.

[0165] In another aspect, there is provided a method of mitigating methane production in a ruminant animal, wherein the ruminant animal is administered an effective amount of the composition as described herein. In an embodiment, the composition is infused biochar as described herein. In an embodiment, the infused biochar or composition is administered directly into the rumen (e.g. by intraruminal injection or by administration of an intraruminal device). Alternatively, the infused biochar or composition is ingested by a ruminant animal.

[0166] In another aspect, there is provided a method of manufacturing an infused meso- or micro- porous material, the method comprising the steps of: providing one or more anti-methanogenic agent(s), and infusing the anti-methanogenic agent(s) into a meso- or micro- porous material to produce the infused meso- or micro- porous material.

[0167] In an embodiment, the method of manufacturing an infused meso- or micro- porous material may further comprise: providing a meso- or micro- porous material, and converting the meso- or micro- porous material into a plurality of meso- or micro- porous particles prior to infusing the meso- or micro- porous particles with anti-methanogenic agent(s), preferably wherein the converting comprises grinding, cutting, pressing, crushing or milling.

[0168] In another aspect, there is provided a method of producing infused meso- or micro- porous material, comprising: heating meso- or micro- porous material for a period of time, milling the meso- or micro- porous material to produce a plurality of particles,combining an amount of a mixture of at least one anti-methanogenic agent in a carrier to the plurality of meso- or micro- porous particles to produce infused meso- or micro- porous material. Preferably, the meso- or micro- porous material is heated to 50°-60°C for 24-48 hours.

[0169] Preferably, the plurality of particles have an average particle size, or a median particle size, of from about 100 pm to about 1000 pm.

[0170] In an embodiment, the composition comprises a combination of a first anti -methanogenic agent and second anti-methanogenic agent. Preferably the composition comprises from about 0.5 mg / kg DM to about 21 mg / kg DM of the first anti -methanogenic agent, preferably about 1 mg / kg DM to about 8 mg / kg DM of the first anti-methanogenic agent. Preferably the composition comprises less than about 25 mg / kg DM of the second anti-methanogenic agent, preferably less than about 10 mg / kg DM of the second anti -methanogenic agent, further preferably less than about 5 mg / kg of DM of the second anti -methanogenic agent.

[0171] In an embodiment, the composition comprises from about 0.5 mg / kg DM to about 5 mg / kg DM of the second anti-methanogenic agent, preferably about 1 mg / kg DM to about 3 mg / kg DM of the second anti-methanogenic agent.

[0172] In an embodiment, when in use, the first anti -methanogenic agent and second anti- methanogenic agent are combined in an amount to act synergistically to reduce methane production in a ruminant animal when compared to an additive methane reduction produced by corresponding controls comprising the first anti-methanogenic agent or second anti-methanogenic agent used independently.

[0173] In an embodiment, when in use, the combination of the compositions is such that the ratio of the first anti-methanogenic agent to the second anti-methanogenic agent is from about 1.0:0. 1 to about 1.0: 100.0, preferably about 1.0:0. 1 to about 1.0:50.0, further preferably about 1.0:0. 1 to about 1.0:20.0, and further preferably about 1.0:0.1 to about 1.0: 10.0. More preferably, the ratio of the first anti-methanogenic agent to the second anti-methanogenic agent is about 1.0:0.1, about 1.0: 1.0, about 1.0:2.0, about 1.0:3.0, about 1.0:4.0, about 1.0:5.0, about 1.0:6.0, about 1.0:7.0, about 1.0:8.0, about 1.0:9.0, about 1.0: 10.0, about 1.0: 11.0, about 1.0: 12.0, about 1.0: 13.0, about 1.0: 14.0, about 1.0: 15.0, about 1.0: 16.0, about 1.0: 17.0, about 1.0: 18.0, about 1.0: 19.0, about1.0:20.0, about 1.0:21.0, about 1.0:22.0, about 1.0:23.0, about 1.0:24.0, about 1.0:25.0, about1.0:26.0, about 1.0:27.0, about 1.0:28.0, about 1.0:29.0, about 1.0:30.0, about 1.0:31.0, about1.0:32.0, about 1.0:33.0, about 1.0:34.0, about 1.0:35.0, about 1.0:36.0, about 1.0:37.0, about1.0:38.0, about 1.0:39.0, about 1.0:40.0, about 1.0:41.0, about 1.0:42.0, about 1.0:43.0, about1.0:44.0, about 1.0:45.0, about 1.0:46.0, about 1.0:47.0, about 1.0:48.0, about 1.0:49.0, or about 1.0:50.0.

[0174] In an embodiment, a first composition comprises a first anti -methanogenic agent. Preferably the composition comprises less than about 25 mg / kg DM of the first anti-methanogenic agent, preferably less than about 10 mg / kg DM of the first anti -methanogenic agent, further preferably less than about 5 mg / kg DM of the first anti-methanogenic agent.

[0175] In an embodiment, the first composition comprises from about 0.5 mg / kg DM to about 21 mg / kg DM of the first anti-methanogenic agent, preferably about 1 mg / kg DM to about 8 mg / kg DM of the first anti-methanogenic agent. More preferably, the first composition comprises about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 mg / kg DM of the first anti-methanogenic agent.

[0176] In an embodiment, a second composition comprises a second anti-methanogenic agent. Preferably the composition comprises less than about 25 mg / kg DM of the second anti- methanogenic agent, preferably less than about 10 mg / kg DM of the second anti-methanogenic agent, more preferably less than about 5 mg / kg DM of the second anti-methanogenic agent.

[0177] In an embodiment, the second composition comprises from about 0.5 mg / kg DM to about 5 mg / kg DM of the second anti-methanogenic agent, preferably about 1 mg / kg DM to about 3 mg / kg DM of the second anti-methanogenic agent. More preferably, the second composition comprises about 1, 1.5, 2, 2.5 or 3 mg / kg DM of the second anti-methanogenic agent.

[0178] In yet another aspect, there is provided a method of reducing methane production in a ruminant animal, the method comprising administering an effective amount of: a composition comprising the combination of a first anti-methanogenic agent and second anti- methanogenic agent, or a first composition comprising a first anti-methanogenic agent, and a second composition comprising a second anti-methanogenic agent for reducing methane production in a ruminant animal wherein, in use, the first and second compositions are combined, and wherein methane reduction is measured in comparison to a corresponding control untreated animal.

[0179] In an embodiment, methane production is reduced in the ruminant animal by at least about 30%, preferably at least about 60%, in comparison to the control animal. Further preferably, methane production is reduced in the ruminant animal by at least about 35%, at least about 45%, at least about 50%, or at least about 55%.

[0180] In an embodiment, methane production is reduced in the ruminant animal by up to about 100%, preferably up to about 96%, in comparison to the control animal. Further preferably, methane production is reduced in the ruminant animal by up to about 97%, 98%, or 99%.

[0181] In an embodiment, administration to a ruminant animal is by consumption of any substance, composition or dosage form capable of delivering a composition of the invention. This may be a liquid, suspension, emulsion, tablet, capsule, pellet, bolus, lick block, or animal feed, comprising a composition of the invention.

[0182] In an embodiment, the first anti -methanogenic agent or second anti -methanogenic agent is a compound selected from the group consisting of: dichloromethane, dibromomethane, diiodomethane, bromochloromethane, bromodichloromethane, bromodiiodomethane, dibromoiodomethane, bromoiodomethane, chloroiodomethane, 2-bromoethanesulfonic acid, chloral hydrate, chloroform, iodoform, chloroethane, dichloroethane, tetrachloroethane, hexachloroethane, bromoethane, dibromoethane, tetrabromoethane, l,2dibromotetrachloroethane, iodoethane, diiodoethane, iodopropane, bromoform, carbon tetrachloride, carbon tetrabromide, carbon tetraiodide and dibromochloromethane, and combinations thereof, but wherein the first anti-methanogenic agent and second anti -methanogenic agent are not identical.

[0183] In an embodiment, the first anti -methanogenic agent or second anti-methanogenic agent are haloforms.

[0184] In an embodiment, the first anti-methanogenic agent is iodoform.

[0185] In an embodiment, the second anti-methanogenic agent is bromoform.

[0186] In an embodiment, the first anti-methanogenic agent is iodoform in an amount from about 1 mg / kg DM to about 25 mg / kg DM (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg / kg DM), and the second anti-methanogenic agent is bromoform in an amount from about 0.5 mg / kg DM to about 25 mg / kg DM (such as about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg / kg DM), wherein the first anti-methanogenic agent and second anti-methanogenic agent are present in the composition in an amount to act synergistically to reduce methane production in a ruminant animal when compared to an additive methane reduction produced by corresponding controls of the first anti-methanogenic agent or second anti-methanogenic agent used independently.

[0187] In yet another aspect, there is provided a method of reducing methane production in a ruminant animal, the method comprising administering an effective amount of:a composition comprising iodoform in an amount from about 1 mg / kg of dry matter (DM) to about 25 mg / kg DM, and a composition comprising bromoform in an amount from about 0.5 mg / kg DM to about 25 mg / kg DM, wherein the combination of iodoform and bromoform acts synergistically to reduce methane production in a ruminant animal when compared to an additive methane reduction produced by corresponding controls of iodoform or bromoform used independently.

[0188] In an embodiment, the composition comprising iodoform and the composition comprising bromoform are administered simultaneously or sequentially.

[0189] In an embodiment, iodoform and bromoform are admixed in the same composition.

[0190] In an embodiment, anti -methanogenic agents useful in preparing compositions of the invention are in substantially purified form. Specifically, the anti-methanogenic agents may be isolated or extracted from their natural sources (e.g. Asparagopsis seaweed) or made synthetically.

[0191] In yet another aspect, there is provided a method of manufacturing a composition for reducing methane production in a ruminant animal, said method comprising combining a first anti- methanogenic agent and second anti-methanogenic agent. In this regard, the composition comprises the features substantially as described above. In an embodiment, the first anti- methanogenic agent and second anti-methanogenic agent combined in the composition in an amount which, when in use, acts synergistically to reduce methane production in a ruminant animal when compared to an additive methane reduction produced by corresponding controls of the first anti-methanogenic agent or second anti-methanogenic agent used independently. In an embodiment, the composition comprises one or more further anti-methanogenic agents and / or additives.

[0192] According to one aspect, there is provided a meso- or micro-porous material infused with at least one anti-methanogenic agent. In an embodiment a first meso- or micro-porous material infused with a first anti-methanogenic agent may be combined with a second meso- or micro- porous material infused with a second anti-methanogenic agent to provide a combination of anti- methanogenic agents. Preferably, the combination, when in use in a ruminant animal, acts synergistically to reduce methane production compared to the additive reduction achieved by the first and second meso- or micro-porous material when in use in a ruminant animal independently.

[0193] In an alternative embodiment, a combination of two or more anti -methanogenic agents is infused into a meso- or micro-porous material.In an embodiment, the infused meso- or micro-porous material comprises at least about 1 mg of the anti-methanogenic agent(s) (dissolved in a carrier) per g of the infused meso- or micro-porous material. Preferably, the infused meso- or micro-porous material comprises from about 1 mg to about 1500 mg of the anti -methanogenic agent(s) per g of the infused microporous material. More preferably, the infused meso- or micro-porous material comprises from about 1.86 mg to about 1285.37 mg, or from about 50 mg to about 500 mg, or from about 100 mg to about 400 mg, or from about 5 mg to about 100 mg of the anti -methanogenic agent(s).

[0194] In an embodiment, infusion of the anti -methanogenic agent(s) into the meso- or micro- porous material substantially prevents volatilisation or degradation of the anti -methanogenic agent when the infused meso- or micro-porous material is exposed to biotic and / or abiotic factors when compared to an equivalent weight of anti-methanogenic agent absent the biochar exposed to identical conditions. Preferably, volatilisation and / or degradation of the anti -methanogenic agent is measured in air at ambient temperature and pressure over at least 7 days after infusion when compared to an equivalent weight of anti-methanogenic agent absent the meso- or micro-porous material exposed to identical conditions. In this context, ‘substantially prevents’ preferably means at least about 50 %, preferably at least about 60 %, of the anti -methanogenic agent is retained in the infused biochar by day 7 compared to an equivalent weight of anti-methanogenic agent absent biochar.

[0195] In an embodiment, the retention of at least about 50 % of the anti-methanogenic agent(s) is achieved when: the anti-methanogenic agent is dissolved in an oil carrier to produce an anti-methanogenic agentoil (AO) solution, wherein the concentration of the agent is at least about 5 mg anti-methanogenic agent per g of oil, preferably from 5 mg to 200 mg anti-methanogenic agent per g of oil, the meso- or micro-porous material (MM) is infused with the AO solution at a ratio of MM: AO of at least about 1 g of MM to 1 g of AO (1 : 1) to about 20: 1, preferably from about 1 : 1 to about 10: 1, preferably from about 4: 1 to about 9: 1.

[0196] In alternative embodiments, compositions according to the invention may be administered directly into the rumen (e.g. by intraruminal injection or by administration of an intraruminal device).

[0197] Further examples of the invention are described below. However, it should be noted that the invention should not be limited to these examples, and that the invention is susceptible to variations, modifications and / or additions other than those specifically described, and it is to beunderstood that the invention includes all such variations, modifications and / or additions which fall within the scope of the claims.EXAMPLESTable 1: Exemplary FormulationsTable 2: Porous Material Types and SuppliersExample 1: Testing the Ratio of Biochar to Anti- Methanogenic AgentMaterials'.

[0198] Bromoform (SKU: 241032, Sigma Aldrich), canola oil (Australian, Countdown), Supelco 15 mL vials (SKU: 27161, Sigma Aldrich), 50 mL falcon tubes, 1.5 mL Eppendorf tubes, 2 mL amber Shimadzu GCMS vials (Cat#. 220-97331-31, Shimadzu), methanol (SKU: 34860, SigmaAldrich), Feedchar® with a Brunauer-Emmett-Teller (BET) surface area determination of 265m2 / g (Agspand Pty Ltd, Tasmania), pine biochar with BET of 386m2 / g (Biogrow Ltd, NZ).

[0199] An experiment was conducted to compare the stability of bromoform-in-oil suspension infused into a biochar (BC) compared to the bromoform-in-oil suspension absent biochar.

[0200] Bromoform was dissolved in canola oil at 10 mg, 12.5 mg, 25 mg and 50 mg of bromoform per g of oil, following which the bromoform-in-oil (BFO) suspensions were infused into Feedchar® biochar (FBC) or pinechar biochar (PBC) in accordance with formulations listed in Table 3.Table 3. BC:BFO Formulations* = Ratio is g of biochar per g of BFOFBC = Feedchar® (Agspand Pty Ltd, Tasmania) (hardwood)PBC = High BET Pine biochar (Biogrow Ltd, NZ) (softwood)

[0201] In the above, a ratio of BC:BFO of 1: 1 defines a ratio of 1 g of BC for every 1 g of BFO, as an example. The last column in the table shows that for each formulation, the BFO concentration and corresponding BC:BFO ratio was such that there was a total of 5 mg / g (25 mg per 5 g) bromoform for each gram of infused biochar.

[0202] Methodology: BFO formulations were prepared by first preparing 40 g of a stock BFO solution with a bromoform concentration of 50 mg / g by adding 2 g of bromoform to 38 g canolaoil. This formulation was then used to prepare 20 g BFO solutions with bromoform concentrations according to the following:10 mg / g = 4 mL stock BFO plus 16 g canola oil,12.5 mg / g = 5 g stock BFO plus 15g canola oil, and25 mg / g = 10 g stock BFO plus 10g canola oil.

[0203] Infused BFO in biochar formulations were prepared on a 20 g scale to accommodate 3 replicates of 1 g each time point. Each formulation was thoroughly mixed with a spatula (~ 60rpm) for a fixed time of 2 min to provide a substantially homogeneous formulation where the BC remained friable.

[0204] Samples (1 g) were accurately weight and transferred to Supelco 15 mL vials prior to either capping and storing (day 0 = DO) or placement in a Panasonic climate-controlled cabinet set at a constant temperature of 25°C with no light (D3, D7, D14 and D28). At each time point, samples (n=3) were capped and stored at -20°C until analysis by gas chromatography mass spectrometer (GCMS).

[0205] Samples for GCMS analysis were extracted into 40 mL of methanol for 20 h with agitation using an orbital shaker table (100 rpm) prior to a 1 in 10 dilution with methanol containing 5 pg / mL naphthalene as internal standard. To reach appropriate concentrations of bromoform in the analytical solutions, the entire 1 g of the infused biochar formulation samples was extracted, while only 200 mg of BFO control formulations was extracted. All analytical samples were subjected to centrifugation (5 min at 13,000 rpm) prior to analysis. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Stabilwax capillary column (30 m x 0.25 mm i.d., 0.25 pm film thickness) using helium as carrier at column flow rate of 1.25 mL / min. The GCMS was used in splitless mode where 2 pL of sample was injected. The GCMS oven program was as follows, 1 min at 40°C, ramping at 16°C / min to 250°C, followed by a 2 min hold at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 180°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software.Results'.

[0206] Figure 1 shows that bromoform retention over the 7-day exposure period at ambient conditions for the control treatments (bromoform in oil at concentrations of 10 - 50 mg / g absent biochar) significantly reduced by D7 (i.e. ~ 60% reduction compared to DO) for all concentrations. Statistical significance was determined by Two-Way ANOVA and Tukey’s HSD where P<0.05.Leters above the bars denote significant differences - thus, the % bromoform retention at DO for the 10 mg / g concentration was significantly different to the same concentrations at D3 and D7.

[0207] Figure 2 shows bromoform retention in biochar (Feedchar®)-infused formulations F1-F4 over the 28-day exposure to ambient conditions. The interaction between formulation and days exposed to ambient conditions was significant (ANOVA, p<0.001), with lower bromoform retention with days exposed to ambient conditions in formulations with lower biochar to BFO ratios (e.g., 1: 1, 3:2) than formulations with higher biochar to BFO ratios (e.g., 4: 1 and 9: 1). Notably, over 7 days of exposure there was no significant change in bromoform content in formulations F3 and F4 when compared to DO. Bromoform retention in formulations Fl and F2 dropped by about 30-35 % by D7, which was significantly lower than at DO, and F3 and F4 at D7, albeit not as severe as the reduction in retention with BFO absent biochar at D7 (Fig. 1). At day 28 there was a significant loss of bromoform detected in all formulations compared to DO.

[0208] Figure 3 shows bromoform retention in pine biochar-infused formulations F5-F8, over the 28-day exposure to ambient conditions. An interaction between formulation and days exposed to ambient conditions was not statistically significant (ANOVA, p=0.134). Nevertheless, at day 28 there was a trend emerging where bromoform retention increased with higher biochar to BFO ratios (e.g., 9PBC: 1BFO) similarly to the Feedchar®-infiised formulations shown in Figure 2 (F3 and F4). At day 28 formulation F8 (i.e., 9PBC: 1BFO) had significantly (ANOVA Tukey’s HSD, p=0.006) higher bromoform retention (78.3 %) than F5 (47.1 %). Note that at day 28 bromoform retention was significantly lower than DO in all formulations except formulation F8 (i.e., 9BC: 1BFO).

[0209] Figure 4 shows that, overall, retention of bromoform at day 7 was significantly higher for biochar formulations (F1-F8) than the control bromoform in canola oil (BFO absent biochar) treatments. While Formulation F3 showed the highest retention which was statistically significant over Fl, F2, and F7 and the BFO (absent biochar controls), the performance of F3 was statistically equivalent to that of Formulations F4-F6 and F8. For at least formulations F3 and F4, bromoform retention by biochar infusion was improved at day 7 (-0-10 % loss) compared to bromoform in oil controls (>60 % Fig.4).Conclusions day 7:

[0210] Infused BFO in biochar significantly improved the retention of bromoform by 100-200% of BFO formulations over a 7-day exposure to ambient conditions. Notably, all ratios of BC:BFO significantly improved retention.

[0211] The formulations with higher ratios (4BC: 1BFO and 9BC: 1BFO) retained more bromoform activity over 7 days and 14 days.

[0212] There was no significant loss of bromoform (ANOVA) from the formulations 4BC: 1BFO and 9BC: 1BFO after 7 days compared to losses of >60 % for BFO absent biochar.

[0213] There is an observable difference after 7 days between biochar types with higher retention in FBC (Feedchar®) compared to PBC (pinechar) at higher ratios of BC:BFO, and higher retention in PBC compared to FBC at lower ratios of BC:BFO. A difference in the effect of biochar type is hypothesised to have a basis on the surface area of the biochar (m2 / g = BET) as a parameter affecting retention. To this point, PBC (pinechar) has a higher BET (386 m2 / g) compared to FBC (Feedchar®) (265 m2 / g) and therefore holds more oil per gram of biochar. This is expected to be absorbed into biochar with greater BET surface area.Conclusions day 28:

[0214] There was significant differentiation between formulations after 28 days with the highest ratio of biochar to BFO having the highest retention of bromoform (>75 %) for both Agspand Feedchar® and BioGrow Pine char. Retention decreased markedly at lower ratios of BC:BFO and the formulation of 9BC: 1BFO is the preferred formulation at 28 days.

[0215] A loss of bromoform of <25 % supports the use of the 9BC: 1BFO formulation for feed systems requiring a shelf life beyond 7 days.Example 2: Further Testing the Ratio of Biochar to Anti-Methanogenic Agent

[0216] The methodology of Example 1 was used in a further experiment to test the BFO infused biochar formulations up to 100 mg according to the formulations shown in Table 4. In this experiment the BC:BFO ratio was maintained at 4: 1, and the total bromoform per g of formulation varied depending on the BFO starting concentration.Table 4. BC:BFO Formulations* = Ratio is g of biochar per g of BFOFBC = Feedchar® (Agspand Pty Ltd, Tasmania)PBC = Pine biochar (Biogrow Ltd, NZ)

[0217] Figure 5 shows control bromoform -in-oil formulations from lO mg / g to lOO mg / g, and bromoform retention over a 7-day exposure to ambient conditions. Retention was consistently and significantly low (32-38%) at D7 compared to DO for all concentrations (Two-Way ANOVA, Tukey’s HSD, P<0.05). This is similar to the data presented in Figure 1 which also shows a -60% loss in BFO absent biochar.

[0218] Figure 6 shows the results of bromoform retention with biochar-infused formulations F9- F13. Over the 14-day exposure, there was no interaction detected between formulation and days exposed to ambient conditions (ANOVA, p=0.871). No significant loss of bromoform from formulations F9-13 was detected over a 7-day exposure to ambient conditions. Significant losses detected at day 14 from F10, F12 and Fl 3, while losses from Fl l approached significance (Tukey’s HSD, p=0.071).

[0219] Figure 7 shows the results of bromoform retention with biochar-infused formulations Fl 4- F18. There was no interaction between formulation and days exposed to ambient conditions (ANOVA, P=0.321). No statistically significant loss of bromoform was detected for formulations F 14- 18 over the 7-day exposure to ambient conditions, albeit that there is a reduced bromoformretention of formulations F15 and Fl 6, with losses from F15 at day 7 approaching significance (ANOVA, Tukey’s HSD, p=0.051).

[0220] Figure 8 shows that the retention of bromoform at day 7 was significantly higher for biochar-infused formulations (F9-F 18, >82 % retention) than the control bromoform in canola oil (BFO absent biochar) treatments (<39 % retention). There were significant differences detected, with formulations F9, F1O, F11 and F 18 retaining significantly higher bromoform activity (100 %) than F15 (82 %). There was also a significant difference between formulations F9, Fl 1 and F18 compared to Fl 6.Conclusions'.

[0221] At a fixed ratio of 4: 1 BC:BFO (4 g BC + 1 g BFO) using Agspand Feedchar® there was no loss of bromoform after a 7 day period regardless of the concentration of bromoform within the BFO using a concentration range of 10 mg / g - 100 mg / g of bromoform in canola oil. A similar result was achieved using BioGrow Pine char after 7 days.

[0222] By comparison, the loss of bromoform from oil absent biochar was > 60 % after 7 days.

[0223] The stabilisation effect found for bromoform is expected for other haloforms (e.g. chloroform and iodoform) and halogenated methane analogues (e.g. dichloromethane, bromochloromethane, dibromochloromethane, dibromoiodomethane, diiodobromomethane). These related molecules all have similar chemical characteristics (periodically similar elements, shape, size, bond types, and bond polarities) leading to similar physical properties and therefore will likely undergo chemical interaction with the mixture of carriers explored for bromoform. It is also likely that other halogenate alkanes (e.g. chloroethane, bromoethane, iodoethane, chloropropane, bromopropane and iodopropane) will be stabilised by these mixtures.Example 3: Stability of Bromoform in Brewers Grain (BG)Background:

[0224] Brewers grain (BG) is a by-product of beer brewing comprising residual malted barley with a dry matter content of 23 % and a protein content of 22 % dry weight. Brewers grain and formulations thereof (e.g. palm kernel (PKBG)) are used as feed supplements for various animal production systems. It was of interest to investigate the possibility of using BG as a delivery mechanism for bromoform in oil (BFO). Targeted formulations of BG, incorporating BFO or BFO infused into biochar (BC), were trialled to investigate the stability of bromoform in these formulations when exposed to ambient conditions typical of ensilage / animal feeding scenarios.Materials'.

[0225] Bromoform (SKU: 241032, Sigma Aldrich), canola oil (Australian, Countdown), 50 mL falcon tubes, 1.5 mL Eppendorf tubes, 2 mL amber Shimadzu GCMS vials, methanol (SKU: 34860, Sigma Aldrich), Feedchar® (Agspan, Tasmania), crystallisation dishes (250 mL), and brewers grain (Lion Brewery, NZ).Methods:

[0226] Formulations F19-F22 were prepared as outlined in Table 5.Table 5. Ingredients for BG formulations

[0227] BFO and BFO infused Feedchar® were prepared according to Example 1. BG compositions incorporating BFO or BFO infused Feedchar® were exposed to controlled ambient conditions using Panasonic climate-controlled cabinet set to a temperature of 25 °C. The compositions were sampled (~2 g accurately weighed) on days 0, 3 and 7, and stored in 50 mL Falcon tubes at -20°C prior to extraction and GCMS analysis.

[0228] Samples for GCMS analysis were extracted into 40 mL of methanol over a 20h period prior to 1: 1 dilution with methanol containing 10 pg / mL naphthalene as internal standard. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Stabilwax capillary column (30m x 0.25mm i.d., 0.25pm film thickness) using helium as carrier at column flow rate of 1.25mL / min. The GCMS was used in splitless mode where 1 pL of sample was injected. The GCMS oven program was as follows, 1 min at 40°C, ramping at 16°C / min to 250°C, followed by a 2 min hold at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 180°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software.Results:

[0229] Figure 9 shows that compositions comprising BFO absent biochar (F20 and F22) almost immediately lost bromoform at DO and were completely devoid of bromoform by day 7. Bycontrast, compositions comprising BFO infused into Feedchar® (F19 and F21) experienced substantially no loss of bromoform over the 7-day testing period (retaining >80 % bromoform).Conclusion:

[0230] Infusion of bromoform into biochar, such as Feedchar® (hardwood biochar) protects against bromoform loss when the infused biochar is incorporated into brewers grain. Bromoform absent biochar is likely degraded and / or volatilises.Example 4: Reactivity of Porous Materials Towards Halogenated Methane Analogues (e.g. Haloforms)Background:

[0231] Processes used in the production of animal feeds (such as pellets or lick blocks) often include steps with elevated temperatures, sometimes up to 100°C, in complex mixtures containing minerals known to degrade halogenated organics. Therefore, the litmus test for anti-methanogenic agent-infused porous materials and the scope of their application is their performance at elevated temperature. It was postulated that some porous substrates may act as efficient catalysts for oxidative degradation of organic compounds. To investigate this possibility, the stability of bromoform in bromoform-infused biochar was compared to bromoform-infused porous zeolite at elevated temperatures (i.e., 75°C), bromoform in oil (25 mg / g) as a control, over 120min.Materials:

[0232] Bromoform (SKU: 241032, Sigma Aldrich), canola oil (Australian, Coundown), Supelco 15 mb vials (SKU: 27161, Sigma Aldrich), Supelco 22mL vials (SKU: 27161, Sigma Aldrich), (SKU: 27162, Sigma Aldrich), 2 mL amber Shimadzu GCMS vials (Cat#. 220-97331-31, Shimadzu), n-hexane (SKU: 1043712500, Sigma Aldrich), Feedchar® (from Agspand), zeolite (88-95 % Clinoptilolite, supplied by NZeolite).Methods:

[0233] Porous substrate / BFO formulations were designed to deliver 25 mg of bromoform as a 5 % inclusion into a 100 g pelletised ration targeting >98 % methane abatement in sheep eating 1 kg dry matter per day. Porous formulations of BFO were prepared in a ratio of 4 parts porous substrate (either Feedchar® biochar (BC) or zeolite (Zeol)) to 1-part BFO (25 mg / g) (F23-F25). These formulations were prepared at a scale of 100 g / batch and are hereon referred to as either, BFO (F23), 4BC: 1BFO (F24), and 4Zeoll:BFO (F25). The content of bromoform in these formulations was monitored with GCMS at 0, 60 and 120 min when exposed to the atmosphere at a temperature of 75°C.

[0234] 0.5g of BFO (F23), 4BC 1 :BFO (F24) or 4Zeol: 1BFO (F25) was added to a pre-heated open top 15mL test tube and heated in a hot water bath set at 75°C using a temperature controlled heater stirrer (Heidolph Hei-Connect Hotplate Stirrer, fitted with a temperature sensor). Treatments were sampled at each time point (i.e. 0, 60, 120 min) and each treatment was replicated (n=5).

[0235] On completion of treatments, samples were immediately extracted with n-hexane (20 mb) with stirring for 20 h at room temperature. Note, accurately weighed subsamples of BFO (-100 mg) were extracted, while accurately weighed samples (~0.500g) of 4BCTBFO (F24) and 4Zeol: lBFO (F25) were extracted. Extracts were sequentially diluted, first by 1 in 10 with n- hexane, followed by 1 in 10 with a 5 pg naphthalene / mL n-hexane solution. These analytical samples were analysed with GCMS.

[0236] All analytical samples were subjected to centrifugation (5 min at 13,000 rpm) prior to analysis. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Rxi-5Sil MS silica capillary column (30 m x 0.25 mm i.d., 0.25 pm film thickness) using helium at column flow rate of 1 mL / min as the carrier gas. The GCMS was used in splitless mode where 1 pL of sample was injected. The GCMS oven program was as follows, 1 min at 40°C, ramping at 15°C / min to 150°C to 8.3 min, then ramping at 35°C / min to 320°C at 13.2 min, followed by a 3 min hold into the injection port at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 250°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software.

[0237] Time zero (TO) measurements were equivalent between formulations (i.e. BFO (F23), 4BC: 1BFO (F24) and 4Zeol: 1BFO (F25)) and were adjusted to represent 100% BF recovery across different formulations.Results'.

[0238] Figure 10 shows that there was a significant difference detected in bromoform retention within and between formulations F23-F25 over the 120 minute exposure to 75°C. Notably, the interaction between formulation and time exposed to 75°C was significant (ANOVA, p<0.001), with lower bromoform retention in the BFO infused zeolite formulation (F25, 17.2%) over time compared to the BFO control (F23, 96.6%) and the BFO-infused biochar (F24, 90.8 %).Conclusion:

[0239] The infusion of BFO (25 mg / g) into biochar does not change (reduce or increase) bromoform losses from non-absorbed BFO at temperatures up to 75 °C for 120 minutes. By contrast, infusion of BFO (25 mg / g) into zeolite chemically degraded the bromoform. It is postulated that mineral impurities in zeolite result in chemical decomposition of bromoform. Thus, porous materials which are inert (e.g. charcoal, biochar, activated carbon etc, or other porous materials lacking chemically destructive surfaces or impurities) are expected to protect bromoform from chemically active constituents in feed formulations.Example 5: Effect of Biochar Type, Particle Size, and Additives on Haloform Release Kinetics in Aqueous Conditions from HFO (Haloform in Oil) Formulations

[0240] Bromoform and iodoform release kinetics with the HFO formulation was investigated over six different experiments. The effect of biochar type, biochar particle size, and additives (i.e., water, surfactant on haloform (i.e., bromoform or iodoform) release kinetics in aqueous conditions from the HFO formulation was investigated.

[0241] The effect of biochar type and / or pretreatment regime (dry (D) vs pre-wetting (W) vs surfactant (S)) on the release rate kinetics was determined (Figure 12).

[0242] The rationales for manipulating these parameters are as follows,• Commercial biochars have different absorption / desorption properties based on the wood feedstock material, production methods and particle size (i.e., milled or unmilled).• Additives compete with surface binding sites and influence wetting properties, effecting the absorption / desorption properties of biochar in aqueous conditions such as rumen fluid.• Different haloforms have unique physicochemical properties including, polarity and solubility which will influence their adsorption and desorption properties in formulations with biochar.Materials:

[0243] Bromoform (SKU: 241032, Sigma Aldrich), iodoform (SKU: 109452, Sigma Aldrich), naphthalene (SKU: 40053, Sigma Aldrich), canola oil (Australian, Countdown), falcon tubes, 1.5mL Eppendorf tubes, 2mL amber Shimadzu GCMS vials (Cat#. 220-97331-31, Shimadzu), methanol (SKU: 34860, Sigma Aldrich), pine biochar with BET of 386m2 / g (Biogrow Ltd, NZ) and Biochar Activated™ (Soft Agriculture Pty Ltd, Australia).Methods:[0243a] To test the effect of biochar type (i.e., Biochar Activated™ (Soft Agriculture Pty Ltd, Australia) versus Pinechar (Biogrow Ltd)) and pre -treatments (i.e., dry, wet, surfactant) on haloform release kinetics in aqueous conditions from the HLO formulation 4BC: 1HLO (HLO = haloform in oil) formulations were prepared according to Table 6 as follows:1) Biochars (100 pm-1000 pm) were oven dried at 60°C for 48 h prior to use. This biochar dry biochar was used to prepare dry formulations (i.e., F27, L30, L33, L36) and surfactant amended formulations (i.e., L28, L31, L34, F37).2) Wet biochar was prepared by the slow addition of 1 part water (50 g) to 4 parts dry biochar (200 g). Dry biochar (200 g) was weighed into the mixing bowl (Part No.9) of a KitchenAid Blender followed by the slow addition (-120 sec) of water (50 g) while mixing on the lowest setting (i.e., No.l) with the flex edge beater (Part No. 14). The product was mixed for a further 5 min and allowed to equilibrate over 24h at room temperature prior to use. This biochar was used to prepare wet formulations (i.e., F26, F29, F32, F35).3) Surfactant treated biochar was prepared by the slow addition of 2 parts surfactant solution (2% TWEEN 80 in RO water) with 4 parts of dry biochar. Dry biochar (100 g) was weighed into the mixing bowl (Part No.9) of a KitchenAid Blender followed by the slow addition (-120 sec) of surfactant solution (50 g) while mixing on the lowest setting (i.e., No. 1) with the flex edge beater (Part No. 14). The product was mixed for a further 5 min prior oven drying at 60°C for 24h prior to use. This biochar was used to prepare surfactant treated formulations (i.e., F28, F31, F34, F37).4) Haloform in oil (HFO) solutions were prepared as follows: a) Add 1.0 g of bromoform to 99.0 g of canola oil to get 10 mg / g BFO. b) Add 50 g of 10 mg / g BFO to 50 g of canola oil to get 5 mg / g BFO. c) Add 1.0 g of iodoform to 99.0g of canola oil to get 10 mg / g IFO. d) Add 50 g of lOmg / g IFO to 50g of canola oil to get 5 mg / g IFO.5) F26 - F37 were prepared as per Table 6 in a ratio of 4BC: 1HF0 by weighing 20 g of pretreated biochar (Step 1 - 3) into a 500 mL beaker with an 80 mm magnetic stirrer bar. For each product 5 g of haloform in oil (HFO) was added dropwise over ~60 sec with stirring at lOOrpm on a magnetic stirrer plate. On completion of the addition of HFO, products were stirred for a further 4 minutes. Samples were then stored in 250 mL screw top vials and allowed to equilibrate for 24 h prior to use.6) Bromoform release kinetics were determined in water at 39°C and quantified via GCMS analysis. To do this, triplicate samples (~1 g) of the 4BCTHFO formulations were weighed into separate 50 mL Falcon tubes prior to the addition of 40 mL of pre-heated 39°C reverse osmosis water. The Falcon tubes were then placed in racks on their side in an orbital incubator shaker (New Brunswick Scientific Innova 44 Incubator) and extracted over 4 h at 39°C and 100 rpm prior to sampling and GCMS analysis (as described below).

[0244] GCMS analysis: Samples (100 pL) were subjected to a 1 in 10 dilution with methanol containing 5 pg / rnL naphthalene used as an internal standard. All analytical samples were subjected to centrifugation (5 min at 13,000 rpm) prior to analysis. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Stabilwax capillary column (30 m x 0.25 mm i.d., 0.25 pm film thickness) using helium as a carrier at a column flow rate of 1.25 mL / min. The GC was used in splitless mode where 1 pL of sample was injected. The GC oven program was as follows, 1 min at 40°C, ramping at 16°C / min to 250°C, followed by a 2 min hold at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 180°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software.Table 6: Biochar Formulation Descriptions for Formulations.Biochar Activated™ from Soft Agriculture (softwood)Pinechar from Biogrow (softwood)Pretreatments include pre-wetting (W), drying (D) and surfactant (S). These formulations were prepared with 5 mg haloform / g oil to deliver 1 mg haloform / g formulation.

[0245] Biochar type and surfactant had a surprising effect on the release kinetics of haloforms from the HFO formulations. We used this information to inform the choice of the HFO formulation to test in an in vitro rumen fluid fermentation assay.

[0246] It was found was that haloforms were more labile from formulations with Biochar Activated™ (e.g., 4SC: 1BFO; SC = Biochar Activated™ (no BET provided); F29-31; F35-37) than equivalent formulations with pinechar (e.g., 4PC: 1BFO; PC = Pine Biochar (High BET pinechar from Biogrow); F26-28; F32-34) releasing 2.8 - lOOx more haloform over the 4-hour extraction period (Figure 12). The differential was greater for iodoform with 76 % iodoform released from pre-wet Biochar Activated™ (i.e., 4SCW: 1IFO; F35) and only 1 % from pre-wet pinechar (i.e., 4PCW: 1IFO; F32). Iodoform formulated in Biochar Activated™ (4SCW: 1IFO (F35), 4SCD: 1IFO (F36) and 4SCS: 1IFO (F37)) was between 1.15-1.8 times more labile than bromoform formulated in Biochar Activated™ (4SCW: 1BFO (F29), 4SCD: 1BFO (F30) and 4SCS: 1BFO (F31)). Pretreatment of biochar with surfactant increased haloform release over untreated biochar by 1.2-40.0 times, with the greatest improvements recorded for iodoform release from pinechar formulations (e.g., 4PCW: 1IFO (F32)) vs 4PCS: 1IFO (F34)). Finally, milled pinechar (-150-400 pm) improved bromoform release by 58 % over unmilled pinechar (-1-10 mm) (data not shown).Example 6: Retention of Bromoform in Biochar Formulations Over a 7-day Exposure toAmbient Conditions

[0247] Using samples F29 and F30 from Example 5, the retention of bromoform in Biochar Activated™ formulations over a 7-day exposure to ambient conditions was investigated.Method:

[0248] 1) F29 and F30 were prepared as per Example 5 methods.2) Triplicate samples (1.000g) of F29 and F30 for day 0, day 3, and day 7 were accurately weight and transferred to Supelco 15 mL vials prior to either capping and storing (day 0 = DO) or placement in a Panasonic climate-controlled cabinet set at a constant temperature of 25 °C with no light (D3 & D7). Samples were capped at each time point then stored at -20°C until analysis by GCMS.

[0249] Samples for GCMS analysis were extracted into 40mL of methanol over a 20h on an orbital shaker table prior to a 1 in 10 dilution with methanol containing 5 pg / mL naphthalene as internal standard. All analytical samples were subjected to centrifugation (5 min at 13,000 rpm) prior to analysis. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Stabilwax capillary column (30m x 0.25mm i.d., 0.25 pm film thickness) using helium as carrier at column flow rate of 1.25mL / min. The GC was used in splitless mode where 1 pL of sample was injected. The GC oven program was as follows, 1 min at 40°C, ramping at 16°C / min to 250°C, followed by a 2min hold at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 180°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software.Results:

[0250] The retention of bromoform in Biochar Activated™ biochar formulations over a 7-day exposure to ambient conditions (i.e., 25°C exposed to atmosphere) was improved by over 20 % in dry (“D”) Biochar Activated™ (i.e., 4SCDTBFO; (F30)) compared to pre-wet (“W”) Biochar Activated™ (i.e., 4SCW: 1BFO) (F29) (Figure 13). Whilst this improvement over BFO was modest compared similar 4BC: 1BFO formulations prepared with Feedchar® (F3) or Pinechar (F7), which improved bromoform retention by over 100 % relative to BFO formulations, the improvement in bromoform release kinetics in Biochar Activated™ combined with some capacity to stabilise bromoform is compelling.Example 7: Bioavailability of Bromoform and Iodoform from Biochar Activated™ (softwood) Formulations Using an In vitro Rumen Fluid Fermentation Assay

[0251] The bioavailability of bromoform versus iodoform from Biochar Activated™ (from Soft Agriculture) formulations using an in vitro rumen fluid fermentation assay was investigated. Given the improvements in bromoform release kinetics from pinechar (from Biogrow) pre-treatedwith surfactant (Tween 80) the bioavailability of bromoform and iodoform from surfactant treated pinechar formulations was also investigated. This work also investigated the correlation between in vitro bioavailability in the rumen fluid fermentation assay with the aqueous extraction conditions used as a surrogate for rumen fluid in an analytical setting.Materials:

[0252] Preparation and analysis of the HFO formulations (BFO and IFO formulations) was conducted at the University of Waikato, Tauranga, New Zealand. In vitro rumen fluid fermentation assays were conducted at the Ellinbank SmartFarm, Victoria, Australia (38°14’S, 145°56’E) and all animal procedures were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Approval to conduct the experiment was obtained from the DJPR Agricultural Research and Extension Animal Ethics Committee.

[0253] Bromoform (SKU: 241032, Sigma Aldrich), iodoform (SKU: 109452, Sigma Aldrich), naphthalene (SKU: 40053, Sigma Aldrich), canola oil (Australian, Countdown), Supelco 15 mb vials (SKU: 27161, Sigma Aldrich), 50 mb falcon tubes, 1.5 mb Eppendorf tubes, 2 mb amber Shimadzu GCMS vials (Cat#. 220-97331-31, Shimadzu), methanol (SKU: 34860, Sigma Aldrich), pine biochar with BET of 386 m2 / g (Biogrow Ltd, NZ; softwood) and Biochar Activated™ (Soft Agriculture; softwood).Methods:

[0254] To test the effect of the HFO formulation infused biochar (pine biochar from Biogrow; softwood) and Biochar Activated™ (Soft Agriculture; softwood)) on the bioavailability of bromoform, and therefore the efficacy of the HFO formulation, feed supplements that deliver either 40 mg or 80 mg of bromoform and / or 20 mg or 40 mg of iodoform per kilogram of dry matter intake (DMI) were targeted. These doses of bromoform versus iodoform were selected to target >80 % reduction in methane production. The HFO formulations were designed to deliver their bioactive payload as a 4.0 % inclusion of 4BC: 1BFO formulations (Table 7 below).

[0255] The haloform content of the HFO formulations were determined by GCMS analysis of methanolic extracts of these products.Table 7: Formulation Constituents and Contents of Treatments in In vitro Rumen Fluid Fermentation Assays.Biochar Activated™ from Soft Agriculture (softwood)Pinechar from Biogrow (softwood)

[0256] All biochar formulations in Table 7 are formulated as 4 parts biochar to 1-part haloform in oil (bromoform “BFO” or iodoform “IFO” or a mixture of these “MIX”).

[0257] Canola oil was used as the negative control (CON-) and formulations of BFO at 1.0 mg, 2.0 mg and 6.5 mg BF / g formulation act as positive controls (i.e., CON+, CON++ and CON+++). Measured in vitro haloform concentrations are based on the addition of 50 mg of controls (CON-, CON+, CON++, CON+++) or Carriers (A1-A5 and B1-B5) to in vitro fermentation bottles containing 1 g of vetch hay.

[0258] The experiment was conducted using a single in vitro run as outlined in Table 8 below.Table 8: Experimental Design of the In vitro Fermentation Assay Assessed against Positive (BFO) and Negative (Canola Oil) Controls.Biochar Activated™ from Soft Agriculture (softwood)Pinechar from Biogrow (softwood)

[0259] Product preparation and analysis'.

[0260] Carriers A1-A5 (F42-F45; F56) were prepared in a 4: 1 ratio of Biochar Activated™ biochar from Soft Agriculture (softwood) with haloform in canola oil (either bromoform (F42- F43), iodoform (F44-F45), or both bromoform and iodoform (F56)) formulations by weighing 20 g of oven-dried Biochar Activated™ (dried at 60°C for 48 h) into a 500 mb beaker with an 80 mm magnetic stirrer bar. For each product 5 g of haloform in oil (HFO) was added dropwise over ~60 sec with stirring at 100 rpm. On completion of the addition of HFO, products were stirred for a further 4 minutes. HFO formulations align with carriers as follows:(a) 5 mg / g BFO to give Carrier Al (F42)(b) 10 mg / g BFO to give Carrier A2 (F43)(c) 2.5 mg / g IFO to give Carrier A3 (F44)(d) 5.0 mg / g IFO to give Carrier A4 (F45)(e) HFO mixture with 5.0 mg / g bromoform and 2.5 mg / g iodoform to give Carrier A5 (F56).

[0261] The sample was prepared in the fume hood to avoid inhalation of biochar dust.

[0262] Carriers B1-B5 (F46-F49; F57) were prepared in a 4: 1 ratio of Pinechar biochar from Biogrow (softwood) with haloform in canola oil (either bromoform (F46-F47) or iodoform (F48- F49), or both bromoform and iodoform (F57)) in two steps. In the first step, 100 g of oven dried milled pinechar (<500pm) was weighed into the mixing bowl (part No.9) of a KitchenAid Blender followed by the slow addition (-120 sec) of 50 g of a 2 % aqueous solution of Tween 80 whilemixing on the lowest setting (i.e., no.l) with the flex edge beater (part no. 14). The product was mixed for a further 5 min prior oven drying at 60°C for 24 h. In the second step, 20 g of oven- dried surfactant treated pinechar was weighed into a 500 mL beaker with an 80 mm magnetic stirrer bar. For each product 5 g of haloform in oil (HFO) was added dropwise over ~60 sec with stirring at 100 rpm. On completion of the addition of HFO, products were stirred for a further 4 minutes. HFO formulations align with carriers as follows:(a) 5 mg / g BFO to give Carrier Bl (F46)(b) 10 mg / g BFO to give Carrier B2 (F47)(c) 2.5 mg / g IFO to give Carrier B3 (F48)(d) 5.0 mg / g IFO to give Carrier B4 (F49)(e) HFO mixture with 5.0 mg / g bromoform and 2.5 mg / g iodoform to give Carrier B5 (F57).

[0263] Controls and HFO solutions were prepared as follow.(a) Add 1.0 g of bromoform to 99.0 g of canola oil to get 10 mg BFO(b) Add 50 g of 10 mg / g BFO to 50 g of canola oil to get 5 mg BFO(c) Add 4.0 g of 5mg / g BFO to 16.0g of canola oil to get CON+ (i.e., 1 mg / g BFO) (F39)(d) Add 4.0 g of 10 mg / g BFO to 16.0g of canola oil to get CON++ (i.e., 2 mg / g BFO) (F40)(e) Add 13.0 g of 10 mg / g BFO to 7g of canola oil to get CON+++ (i.e., 6.5 mg / g BFO) (F41)(f) Add 1.0 g of iodoform to 99.0 g of canola oil to get 10 mg / g IFO(g) Add 50 g of 10 mg / g IFO to 50g of canola oil to get 5 mg / g IFO(h) Add 50 g of 5 mg / g IFO to 50 g of canola oil to get 2.5 mg / g IFO(i) HFO mixture was prepared by addition of 10g of lOmg / g BFO to 10g of 5mg / g IFO to get a 5mg of bromoform plus 2.5mg of iodoform per gram of HFO.

[0264] Carriers A1-A5 and B1-B5 were analysed by GCMS. Samples (0.1 g or 0.5 g for HFO or biochar formulations, respectively) for GCMS analysis were extracted with 20 mL of n-hexane at 21 °C with stirring (400 rpm) over 20 h. A 1 mL aliquot of the extract was clarified via centrifugation (5 min at 13,000 rpm) prior to diluting 1 in 5 with a 5 pg naphthalene / mL n-hexanesolution. All analytical samples were subjected to centrifugation (5 min at 13,000 rpm) prior to analysis. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Rxi-5Sil MS silica capillary column (30 m x 0.25 mm i.d., 0.25 pm film thickness) using helium at column flow rate of 1 mL / min as the carrier gas. The GC was used in splitless mode where 1 pL of sample was injected. The GC oven program was as follows, 1 min at 40°C, ramping at 15°C / min to 150°C to 8.3 min, then ramping at 35°C / min to 320°C at 13.2 min, followed by a 3 min hold into the injection port at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 250°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software.

[0265] Experimental procedure for in vitro fermentation assay and analysis'.The in vitro experiment was conducted by a published method using the automated ANK0MRFGP system (ANKOM GP; ANKOM Technology, Macedon, NY, USA) over a 24-h period (Alvarez-Hess et al., Animal Feed Science and Technology 256, 2019). The ANKOMRhGP system is comprised of 310 ml ANKOM incubation bottles with associated radio frequency (RF) modules, which sit in oscillating water baths maintained at 39°C (RATEK SWB20D, Ratek Instruments Pty Ltd, Victoria, Australia).Briefly, ruminal fluid (-650 ml per cow) was collected from cannulated (www.rumencannula.com), Holstein-Friesian cows and filtered, under a constant flow of CO2, through four layers of cloth to remove suspended particles. Except for blank treatments, 1 g of dry milled vetch hay (Vida sativa L.) and 50 mg of treatment carrier (see Table 8) was added to each 310 mb ANKOM incubation bottle. These bottles were prewarmed to 39°C in a water bath prior to the addition of 25 mb of ruminal fluid and 75 ml of Kansas State buffer solution (pH 6.8), leaving 210 ml of headspace volume. After addition of ruminal fluid and buffer solution, each bottle was capped with an ANKOM GP module, and the headspace flushed with CO2. The in vitro incubation was run over 24 h with measurement of total gas production (TGP, mb) achieved by the automated GP system.At the end the incubation run, separate gas samples were collected from the headspace of each bottle with an air-tight glass syringe (SGE International Pty Ltd, Ringwood, Vic, Australia), and transferred into separate Exetainers® (12 ml soda glass vial, Labco Ltd, Buckinghamshire, UK) that had previously been evacuated. Methane proportions in the samples were determined by gas chromatography (GC). Samples were analysed using Agilent 7890A equipped with 3 detectors (TCD, pECD, FID), and GILSON GX-271 auto sampler for transferring the pressurised sample from Labco Exetainers® to the GC loops (1 ml x 2). The GC loops and sample inlet were flushedusing helium between samples to avoid carryover. Columns used were: HayeSep® N 80 / 100 mesh, 0.5 m x 1 / 8 in. SST (precolumn for both channels); Porapak® QS 80 / 100 mesh, 2 m x 1 / 8 in. SST (analytical on TCD - FID channel); HayeSep® D, 80 / 100 mesh, 2 m x 1 / 8 in. SST (analytical to uECD). Unless stated otherwise, methane production has been expressed on a mL / g DM incubated basis. Gas production calculations were carried out as per Alvarez-Hess et al (2019).Results and Discussion'.GCMS verification of bromoform activity in formulations tested in vitro.

[0266] Carriers A1-A5 and B1-B5 tested in vitro were analysed for haloform content by GCMS. Total gas production between treatments is shown in Figure 14.

[0267] Results shown in Table 9 below represent the amount of haloform in mg / kg dry matter (DM) delivered in a 50 mg dose to the fermentation bottles with 1 g of vetch hay.

[0268] The term bioavailability and magnitude of bioavailability is inferred from the magnitude of methane mitigation detected in the in vitro fermentation assays. The term lability and magnitude of lability of haloforms from the HFO formulations was measured via an aqueous extraction / GCMS analysis methodology intended to be used as a surrogate for inferring bioavailability of bromoform in rumen fluid.

[0269] Stabilisation of the HFO formulations by infusion into porous biochar is bioavailable: Mitigation of methane production in ferments with Carriers Al, A2, A4 A5, B2 and B5 (delivering 35.0-69.5 mg of haloform / kg DM) were equivalent to fermentations with positive controls CON+, CON++ and CON+++ (delivering 40.0-260 mg of haloform / kg DM) (Figure 15).

[0270] Dose dependent mitigation of methane production: See Carriers A3 vs A4, Bl vs B2, and B3 vs B4 (Figure 15).

[0271] Biochar type effects the in vitro bioavailability of haloforms: CarrierAl (4SC: 1BFO) (F42) delivering 36.5 mg BF / kg DM performed better than Carrier Bl (4PCS: 1BFO) (F46) delivering 38.0 mg BF / kg DM. The latter reflects the lower in vitro bioavailability of bromoform in the HFO formulation with pinechar (softwood) biochar (e.g., Carrier Bl, 4PCS: 1BFO) (F46) than those with Biochar Activated® (softwood) biochar (e.g., Carrier Al, 4SC: 1BFO) (F42). These results are consistent with release rated kinetics data for these products (Figure 12).

[0272] Bromoform has similar bioavailability to iodoform in equivalent formulations: Bioavailability of bromoform infused into either Biochar Activated® (softwood) or pinechar (softwood) (Carriers Al (F42) and Bl (F46), respectively) were similar to the bioavailability’s of iodoform absorbed on either Biochar Activated® (softwood) or pinechar (softwood) (Carriers A4(F45) and B4 (F49), respectively). These results are consistent with the release kinetics study of these specific products (Figure 12; 4SCD: 1BFO (F30) vs 4SCD: 1IFO (F36) and 4PCS: 1BFO (F28) vs 4PCS: 1IFO (F34)).

[0273] In vitro fermentation results confirm that the aqueous extraction / GCMS analysis methodology used as a surrogate for inferring bioavailability of haloforms in rumen fluid is effective.

[0274] The addition of surfactant (tween 80) improves the bioavailability of haloforms from biochar: Using the above aqueous extraction / GCMS surrogate methodology, it was demonstrated that treatment of biochar with surfactant improved the release rate kinetics / bioavailability of haloforms absorbed on biochar compare with those formulated without surfactant (Figure 12).

[0275] Note that the GCMS results indicated that the carriers contained lower levels of haloform than the levels targeted in their manufacture. To attain the intended doses in the in vitro fermentations, the addition of oil controls and carriers was increased from 4 % to 5 % (i.e. 50 mg / g).Table 9: Experimental Design of the In Vitro Fermentation Assay Assessed against 5% Positive (BFO) and Negative (Canola Oil) Controls.Biochar Activated™ from Soft Agriculture (softwood).Pinechar from Biogrow (softwood).

[0276] In vitro assessment of the bioavailability of haloforms in 4BC.1HFO formulations.

[0277] Figure 14 shows that total gas production (TGP) ranged 75.4-95.2 mL over the 20 h fermentation period, with the highest gas production occurring for the negative control (CON-) (F38) with TGP decreasing numerically as bromoform addition increased across the positive controls, CON+ (50 mg BF / g DM; 83.1 mL) (F39), CON++ (100 mg BF / g DM; 79.2 mL) (F40) and CON+++ (325 mg BF / g DM; 75.4 mL) (F41). However, due to high variability between replicates within treatments and low replication the effect failed to reach significance at the confidence interval of p < 0.05 (PERMANOVA, p = 0.0573). It is worth noting that previous studies consistently detected significant reductions in total gas production when methanogenesis was inhibited with anti-methanogenic substances (e.g., haloforms, 3-NOP and Coenzyme M analogues) (data not shown).

[0278] Methane production ranged 0.07-6.12 mL over the 20 h fermentation period, with the highest methane production occurring for the negative control (CON-, 6.12 mL CH4) (F38), carrier A3 (i.e. Biochar Activated® (softwood) biochar delivering 21.5 mg IF / kg DM, 5.93 mL CH4) (F44) and carrier B3 (i.e. pinechar (softwood) biochar delivering 20.5 mg IF / kg DM, 5.77 mL CH4) (F48). Significant reductions in methane production were detected in all positive controls and remaining carrier formulations (Figure 15, PERMANOVA, p < 0.0001).

[0279] Positive controls reduced methane production by >98% at all levels of bromoform tested (i.e. 50-325 mg / kg DM). Carrier Al (Biochar Activated® (softwood); (F42)) delivering 36.5 mg BF / kg DM performed better than Carrier Bl (pinechar (softwood); (F46)) delivering 38.0 mg BF / kg DM. The latter reflects the lower lability of bromoform in the HFO formulation with pinechar (softwood) biochar (e.g., 4PCS: 1BFO; (F42); Figure 12) than those with Biochar Activated® (softwood) biochar (e.g., 4SC: 1BFO; Figure 12).

[0280] Carriers formulated with iodoform at the higher level (i.e. Carriers A4 (F45) and B4 (F49) formulated to deliver ~40 mg IF / kg DM) significantly reduced methane production by 89.9 % (Carrier A4) and 74.2% (Carrier B4).

[0281] The HFO formulation, formulated with a mixture of bromoform and iodoform (i.e., Carrier A5 and Carrier B5, delivering ~40 mg BF / kg DM and iodoform at ~20 mg / kg DM) inhibited methane production by >98%. There was a complete inhibition of methane production in fermentations with Carrier Al (one of the positive controls for Carrier A5). A clear synergistic effect was detected between bromoform and iodoform in Carrier B5 which reduced methane production by 97.5%, performing 39.5% better than the expected 69.9% reduction in methane production from the additive effects of its constituent haloforms, i.e., Carrier B 1 (64.1 % reduction in methane production) and Carrier B3 (5.8% reduction in methane production).

[0282] The in vitro results indicate that haloforms absorbed on Biochar Activated® (softwood) biochar (Carrier Al (F42) and Carrier A4 (F45)) are more labile than haloforms absorbed on pinechar (softwood) biochar (Carrier Bl (F46) and B4 (F49)). These results are consistent with the release kinetics study that used an aqueous extraction method combined with GCMS analysis of haloforms as a surrogate for in vitro fermentations. Furthermore, the apparent bioavailability’s of bromoform absorbed on either Biochar Activated® (softwood) or pinechar (softwood) biochar (Carriers Al (F42) and Bl (F46), respectively) were similar to the bioavailability’s of iodoform absorbed on either Biochar Activated® (softwood) or pinechar (softwood) biochar (Carriers A4 (F45) and B4 (F49), respectively). These results are also consistent with the release kinetics study (Figure 12. 4SCD: 1BFO (F30) vs 4SCD: 1IFO (F36) and 4PCS: 1BFO (F28) vs 4PCS: 1IFO (F34)). This information is useful because it indicates that the aqueous extraction method combined with GCMS analysis of haloforms is a viable surrogate for in vitro fermentations for testing the formulations of the invention.

[0283] Using the above-mentioned aqueous extraction / GCMS surrogate methodology, it was demonstrated that treatment of biochar with surfactant improved the release rate kinetics / bioavailability of haloforms absorbed on biochar compare with those formulated without surfactant (Figure 12). It was also demonstrated that biochar particle size influences haloform release rate kinetics / bioavailability.Conclusions

[0284] The studies showed that infused bromoform-in-oil (BFO) in microporous biochar (BC) reduced bromoform losses to <13 % over a 7-day exposure to ambient conditions (i.e., 25°C exposed to atmosphere) compared to unabsorbed BFO with losses of ~60 %. The studies allowed the subsequent optimisation of the formulation of the HFO formulations and it was found that high ratios of BC:BFO (i.e., 4: 1 and 9: 1) retained higher bromoform activity than lower ratios of BC:BFO (i.e., 1: 1 and 3:2) over all time points with 9: 1 formulations performing best out to 28 days of exposure to ambient conditions (Figure 2 and Figure 3). Stabilising bromoform to exposure to ambient conditions reduced bioavailability in vitro such that application rates equivalent to 80 mg of bromoform per kg of dry matter intake (DMI), typically inhibiting methanogenesis by >95 %, had no effect on methane production in vitro (Figure 16). The bromoform release kinetics in Kansas buffer (pH 6.8) at 39°C was studied, confirming that the bromoform release from 9BC: 1BFO formulations in pinechar (softwood) was indeed impaired over a timescale relevant for in vitro fermentation assays (Figure 11). The studies also showedthat in vitro bioavailability of bromoform from the HFO formulation with pinechar is slightly lower than the HFO formulation with Biochar Activated®.Example 8: Correlation of Physical Properties of Mesoporous and Microporous Carriers with Release into the Surrogate Rumen Fluid, and to the Atmosphere.Aim:

[0285] To correlate the physicochemical properties of various charcoals, biochars and activated carbons with haloform release kinetics in aqueous media (e.g., rumen fluid) and stability exposed to ambient conditions.Materials'.

[0286] The HFO formulation (Batch: #006, SEAFOREST Australia), Bromoform (SKU: 241032, Sigma Aldrich), canola oil (Australian, Countdown), Supelco 15 mb vials (SKU: 27161, Sigma Aldrich), 50 mb falcon tubes, 1.5 mb Eppendorf tubes, 2 mb amber Shimadzu GCMS vials (Cat#. 220-97331-31, Shimadzu), methanol (SKU: 34860, Sigma Aldrich). Ten commercial samples of charred mesoporous and microporous material (ie biochars, charcoals and activated carbons) were sourced from Australian and New Zealand suppliers and the surface area, size and volume of the pores were measured (Table 10).

[0287] Methods:1) The mesoporous and microporous materials from Table 10 were milled to <0.5 mm (i.e <500 pm) fine particles (Pulverisette 15 cutting mill, Fritsch GmbH, Germany).2) The milled mesoporous and microporous materials were dried at 60°C for a 48- hour period prior to analysis and preparation of 4BC: 1BFO and 9BC: 1BFO products (“BC” represents the milled mesoporous or microporous material).3) The HFO formulation 15 (HFO-15) and the HFO formulation 30 (HFO-30) were prepared by adding 1.0 g and 2.5 g of bromoform to 99.0 g and 97.5 g of the HFO formulation 5 (HFO-5), respectively.4) 4BC: 1BFO formulations were prepared to deliver 3mg of bromoform per gram of formulation. To do this 8 g of HFO-15 (i.e., 15mg of bromoform per gram of the HFO formulation) was added over ~60 sec to 32 g of dry milled mesoporous or microporous material in a 500 mb beaker with an 80 mm magnetic stirrer bar and stirred at 100 rpm on a stirrer plate. On completion of the addition of HFO-15, formulations were stirred for a further 4 minutes.Table 10: Correlation of Physical Properties Mesoporous and Microporous Carriers with Release into the Surrogate Rumen Fluid, and into the Atmosphere.) 9BC: 1BFO formulations were prepared to deliver 3 mg of bromoform per gram of formulation. To do this 4 g of HFO-30 (i.e., 30 mg of bromoform per gram of the HFO formulation) was added over ~60 sec to 36 g of dry milled mesoporous or microporous material in a 500 mb beaker with an 80 mm magnetic stirrer bar and stirred at 100 rpm on a stirrer plate. On completion of the addition of HFO-30, formulations were stirred for a further 4 minutes. ) Bromoform release kinetics were determined in water at 39°C and quantified via GCMS analysis. To do this triplicate samples (~1.000 g) of 4BC: 1BFO or 9BC: 1BFO formulations were weighed into separate 50 mb Falcon tubes prior to the addition of 40 mb of preheated 39°C RO water. The Falcon tubes were then place on them side in racks in an incubator shaker (New Brunswick Scientific Innova 44 Incubator) and extracted for 4 h at 39°C with orbital shaking at 100 rpm. Samples were extracted for 4 h prior to sampling and GCMS analysis (as described below). ) BC:BFO stability testing was assessed by bromoform content in formulations exposed to atmospheric conditions at 25°C in the dark. Duplicate samples (-1.000 g) of each formulation were weighed into 50 mb Falcon tubes at days 0, 7, 14 and 21. Samples were then extracted at room temperature in 40 mb of methanol over 20 h on an orbital shaker table prior to GCMS analysis (as described below). ) GCMS analysis: Samples (100 pL) were subject to a 1 in 10 dilution with methanol containing 5 pg / mL naphthalene as internal standard. All analytical samples were subjected to centrifugation (5 min at 13,000 rpm) prior to analysis. GCMS analyses were run on a Shimadzu GCMS-QP2020 NX fitted with a Shimadzu SH-Stabilwax capillary column (30 m x 0.25 mm i.d., 0.25 pm film thickness) using helium as carrier at column flow rate of 1.25 mL / min. The GC was used in splitless mode where 1 pL of sample was injected. The GC oven program was as follows, 1 min at 40°C, ramping at 16°C / min to 250°C, followed by a 2 min hold at 250°C. The injection port temperature, interface temperature and ion source temperature were set to 180°C, 200°C and 230°C, respectively. Data was analysed with Shimadzu LabSolutions GCMS software. ) Brunauer-Emmett-Teller (BET) surface area analysis, pore volume and pore size of the mesoporous or microporous materials were determined commercially with nitrogen (ISO 15901-2) by Particle and Surface Science Pty Ltd (Australia) using a Micromeritics® TriStar 3000 V6.08 A.10) Elemental analysis and ash content were determined commercially by OEA Laboratories Ltd in the UK.11) MIR spectra were collected on a Fourier-transform MIR (FT-IR) spectrometer (Shimadzu, IRSpirit, QATR-S, Japan), and intensity was measured as absorbance using single reflection attenuated total reflectance (ATR).12) SEM images were collected at the Electron Microscope Facility at the University of Waikato.13) Data analysis: The 'mgcv' package in R (v. 4.1.3; R Core Team 2013) was used to perform beta regressions ('betar' family with a 'logit' link) to determine whether there were correlations between (1) BET surface area and bromoform retention; and (2) BET surface area and bromoform release for the 4BC: 1BFO and 9BC: 1BFO formulations. Note that for the purposes of generating the beta regression models, which requires response values to be between 0 - 100 %, bromoform retention values that were > 100 % were replaced with a value of 99.9999 %. The 'stats' package in R was used to perform a linear regression to determine whether there was a significant correlation between the content of carbon and the content of ash in the biochar samples used in this study. Differences were considered significant if p < 0.001. Normality was tested using Q-Q plots and the Shapiro-Wilks test; homogeneity was tested by visual examination of the residuals; and goodness of fit was demonstrated using adjusted r2values and the percent deviance explained.Results and Discussion'.

[0288] The percentage of carbon, hydrogen, nitrogen, oxygen and ash were determined (Table 11). The content of carbon ranged 55.3 - 89.78 % and was negatively correlated with content of ash (ash = 361.87 - 8.45 carbon + 0.05 carbon2,' adjusted r2= 0.831; p < 0.001), which ranged 2.02 - 49.07 %. The combined contents of H, N, and O ranged 0.69 - 22.01 % and is indicative the degree of carbonisation occurring to the pyrolysis, with high contents typical of lower pyrolysis temperatures and lower contents typical of higher pyrolysis temperatures. For mesoporous or microporous materials produced with lower pyrolysis temperatures, more functionality remains in the form of aromatics and carbonyl groups.Table 11. Proximate analysis oxygen, hydrogen, nitrogen, carbon and ash content of the ten mesoporous and microporous samples used in this study.

[0289] The Van Krevelen diagram, plots H / C atomic ratio against O / C atomic ratio, demonstrates the development of the biochar's aromaticity and its associated reactions (Figure 18). In the current study the decrease in the H / C and O / C atomic ratios may correspond with increasing pyrolysis temperature. For example, BC 002 (biochar), BC 004 (high BET pinechar), and BC 010 (charcoal) are similar to biochars produced from pine and oak at maximum pyrolysis temperatures below 600°C.

[0290] In general, carbon structure is dependent on the pyrolysis temperature more than feedstock type. Typically increasing pyrolysis temperature leads to decreasing functionality and convergence on a graphite like structure. FTIR spectra were collected on all charcoal, biochar and activated carbon samples indicating significant structural differences between samples (Figure 19). Samples BC 002 (biochar, hardwood), BC 004 (high BET pinechar, softwood), BC 005 (Biochar Activated™, softwood) and BC_010 (charcoal, hardwood) exhibit much greater functionality, specifically with contributions from oxygenated groups, indicating that complete dehydration of the feedstock biomass has not been accomplished. These latter samples have structural features that are reminiscent of charcoals and biochars produced at temperatures below 600°C. The latter is consistent with the Van Krevelen plot. In comparison spectra collected for samples BC_003(activated carbon, coconut), BC 006 (Feedchar®, hardwood) and BC 007 (activated carbon, coconut) were reminiscent of graphite and is indicative of higher pyrolysis temperatures (>700°C).

[0291] SEM images capture the porous nature of these samples and to varying degrees intact structural features of the feedstock materials that the charcoals, biochars and activated carbons were manufactured from (Figure 20).

[0292] Feedstock material and pyrolysis parameters (peak temperature, heating rate and residence time) influence surface area and pore development. In this study, surface area (BET), ranged from 1.86 - 1285.37 m2 / g, and pore volumes of 0.045 - 1.088 cm3 / g (Table 10). Average pore size ranges from 1.498 - 10.532 nm, with most samples bridging the classification threshold between microporous (BC 003 (activated carbon), BC 004 (high BET pinechar), BC 007 (activated carbon), BC 010 (charcoal)) and mesoporous (BC 001 (activated charcoal), BC 002 (biochar), BC_006 (Feedchar®), BC_008 (biochar) and BC_009 (artisan biochar)) materials. Notably BC_005 (Biochar Activated™) sits firmly within the mesoporous classification.

[0293] The surface area and surface chemistry of the mesoporous or microporous material influences the absorption / desorption characteristics of the material. High stability of bromoform absorbed in a carrier such as charcoal, biochar or activated carbon (collectively designated “BC”) is desirable for manufacture and storage of feed formulations (e.g., pellets and lick blocks). In the current study the stability of bromoform in oil in such carriers at a 4: 1 or 9: 1 ratio (i.e., 4BC: 1BFO and 9BC: 1BFO) demonstrated a relationship between BET surface area and bromoform retention / stability (Figure 21A). Bromoform retention increased up to a BET surface area of 118.97 m2 / g where retention over 7 days of exposure to atmosphere reached -100 %. Notably, 9BC: 1BFO formulations generally retained more bromoform than 4BCTBFO formulations prepared with the same mesoporous or microporous material, except in formulations with BC 005 (Biochar Activated™; Table 12).

[0294] Figure 21B also shows a strong relationship between pore volume and bromoform retention.Table 12: Bromoform release and stability kinetics.

[0295] From a practical standpoint, charcoals, biochars and activated carbons with BET surface areas of 6.95 - 1285.37 m2 / g performed well. Note that bromoform retention as a function of pore volume follows the same trend as that with BET surface area (Figure 22B). Microporous and mesoporous materials with pore sizes between 1.498 - 3.387 nm retained >87 % of the bromoform payload over 7 days of exposure to atmosphere while formulations with BC_005 (Biochar Activated™) with an average pore size of 10.532 nm retained only 31.32 % and 22.44 % of their bromoform payloads in 4BC: 1BFO and 9BC: 1BFO formulations, respectively (Figure 21C).

[0296] Microporous and mesoporous materials with pore sizes between 1.498 - 3.387 nm retained >79.71 % of the bromoform payload over 14 days of exposure to atmosphere (Figure 23 A) while formulations with BC_005 (Biochar Activated™) with an average pore size of 10.532 nm retained only 18.05 % and 18.13 % of their bromoform payloads in 4BCTBFO and 9BC: 1BFO formulations, respectively (Figure 23B).

[0297] Stabilising bromoform in mesoporous and microporous carriers such as charcoals, biochars and activated carbons benefits feed manufacture and storage, however bromoform release is critical for methane mitigation in vivo. It was demonstrated that treating mesoporous and microporous carriers with surfactant increased bromoform release in 4: 1 (4BC: 1BFO) formulations by 67 % compared to untreated mesoporous and microporous carriers. Here we investigate the effect of BET surface area on the release of bromoform using a surrogate rumen extraction (i.e., water at 39°C). There were significant negative correlations between BET surface area and bromoform release for both 4BC: 1BFO and 9BC: 1BFO formulations (Figure 22A). Bromoform release as a percentage of the bromoform payload ranged between 2.48 % - 48.25 % and 0.67 % - 47.69 % for 4BCTBFO and 9BC: 1BFO formulations, respectively and higher bromoform release correlated with lower BET surface areas. Bromoform release as a function of pore volume (Figure 22B) followed the same trend as that of BET surface area (Figure 22A).

[0298] Mesoporous and microporous carriers that had pore sizes ranging from 1.498 nm - 2.351 nm (BC 002 (biochar), BC 004 (high BET pinechar) and BC 010 (charcoal)) released between 15.24 % and 26.2 % of their bromoform payloads, while formulations with BC_005 (biochar activated) released up to 48.25 % of its bromoform payload (Figure 22C). Formulations with samples BC_001 (activated charcoal), BC_003 (activated carbon), BC 006 (Feedchar®), BC 007 (activated carbon) and BC 009 (artisan biochar)released between 0.67 % - 8.92 % of their bromoform payloads. These low release samples correspond with mesoporous and microporous carriers that are hypothesized to be produced using higher pyrolysis temperatures (i.e. BC_001 (activated charcoal), BC_003 (activated carbon), BC 006 (Feedchar®), BC 007 (activated carbon) and BC 009 (artisan biochar). Notably, activated carbon samples had the highest BET surface areas (i.e., BC_001 = 1,285.37 m2 / g; BC_003 = 941.26 m2 / g; and BC_007 = 874.86 m2 / g) and the lowest bromoform release (i.e., 0.67 % - 2.83 %). These low release rates in the aqueous extraction may limit bioavailability in vivo.(Model 1) There was a significant, positive correlation between BET surface area and bromoform retention for the 4BC: 1BFO formulations (solid black line in Figure 8) and bromoform retention can be predicted using the following model (deviance explained = 38.5%; adjusted r2= 0.463):

[0299] There was a significant, negative correlation between BET surface area and bromoform release for the 4BC: 1BFO formulations (dashed black line in Figure 24A) and bromoform release can be predicted using the following model (deviance explained = 93.5 %; adjusted r = 0.915):(Model 2)

[0300] There was a significant, positive correlation between BET surface area and bromoform retention for the 9BC: 1BFO formulations (solid black line in Figure 24B) and bromoform retention can be predicted using the following model (deviance explained = 66.5%; adjusted r2= 0.670):(Model 3)

[0301] There was a significant, negative correlation between BET surface area and bromoform release for the 9BC: 1BFO formulations (dashed black line in Figure 24B) and bromoform release can be predicted using the following model (deviance explained = 95.6 %; adjusted i2= 0.945):(Model 4)(o,1349+(- 0.4829 xln(BET))+(-1.051e-06 X BBT2)) 9BC-. 1BFO release = _ X 100(1 +e(0 1349 + ( -0-4829xln(BBT)) + (-1.051e-06 X BBT2)))Preferred mesoporous and microporous materials parameters

[0302] Except for formulations with BC_005, the retention of bromoform was acceptable across the entire range of formulations prepared in this study. However, the release of bromoform from these formulations varied significantly (Figure 22) and will likely influence the bioavailability of bromoform in vivo. Without a surfactant, the range of BET surface areas that can be used in vivo is reduced to 0 - 600 m2 / g based on the following rationale:1. The rumen is a large fermentation vessel in which the material being digested remains for 20 to 48 hours. In the current experiment, we investigated the release of bromoform from 4BC: 1BFO and 9BC: 1BFO formulations, which ranged between 0.67 % - 48.4 % over a 4 hour period into surrogate rumen conditions (i.e., water at 39°C). Assuming a linear release rate and a minimum rumen residence time of 20 hours, 4BC: 1BFO formulations that are predicted to release > 50 % of their bromoform payload include BC_002 (99.5 %), BC_004 (96.75 %), BC_005 (100 %), BC_008 (57.0 %) and BC_010 (100 %). Under these same conditions 9BC: 1BFO formulations that are predicted to release > 50 % of theirbromoform payloads include BC_002 (76.2 %), BC_004 (79.2 %), BC_005 (100 %) and BC_010 (100 %).2. The HFO formulations produced using biochars treated with surfactants or wetting agents (e.g., F28) improve bromoform release by up to 67% compared to formulations with dry biochar (e.g., F27) (see Figure 12). Assuming such improvements can be made with the current selection of mesoporous and microporous materials, 4BC: 1BFO formulations prepared with BC 006 are also predicted to release > 50% of their bromoform payload (i.e., 74.5%) after a 20 hour residence time in the rumen.3. For longer rumen residence times (i.e., up to 48 hours), all 4BC: 1BFO formulations prepared with surfactant-treated biochar are predicted to release > 50 % of their bromoform payloads. Based on the above preamble, formulations that released > 50 % of their bromoform payloads were prepared from samples with BET surface areas < 362. 1 m2 / g (see dotted boxes in Figure 24).4. Furthermore, these results assume that there is no competitive binding between bromoform absorbed on the mesoporous and microporous materials and other organic / inorganic constituents in the rumen fluid, which would facilitate an increased release of bromoform. Therefore, it is proposed that mesoporous and microporous materials suitable for the manufacture of compositions of the invention, without a surfactant, will cover BET surface areas approximately greater than 362.1 m2 / g and recommend an inclusionary range of up to about 600 m2 / g.Synergy of Anti-Methanogenic AgentsExample 8 - stock solutions comprising bromoform and iodoform

[0303] Stock solutions (0.6, 1.2, 2.5, 5.0 mM) of anti-methanogenic agents bromoform (Sigma- Aldrich, USA) and iodoform (Sigma- Aldrich, USA) were prepared in food grade canola oil (Farmers Harvest™; 100% Bottling Company, Cheltenham, Victoria, Australia) by serial dilution. Initially, 0.63 g of bromoform or 0.98 g of iodoform was added to 500 mb of canola oil and then diluted to a standard concentration.Example 9 - Measurement of methane production following incubation of compositionsMethodology

[0304] The bromoform and iodoform standards of Example 8 were used to prepare compositions ASP1-ASP20 treatment compositions, which resulted in haloform concentrations as outlined in Table 13. Notably, five compositions contained only bromoform (ASP5, ASP 10, ASP 15, ASP 16 and ASP20) and four compositions contained only iodoform (ASP1, ASP2, ASP3 and ASP4).Table 13. Anti -methanogenic compositions.DM = Dry matter

[0305] Gas production was measured in vitro using the automated ANKOM RF Gas Production (GP) system (RFS; ANKOM Technology, Macedon, NY, USA) over a 24-h period. This system was comprised of 46 incubation bottles (310 m ) sealed with ANKOM radio frequency (RF) pressure modules and incubated in an oscillating water bath maintained at about 39°C (RATEK SWB20D, Ratek Instruments Pty Ltd, Victoria, Australia). Each of six consecutive 24 h in vitro runs included two replicates of each of the 20 haloform compositions (n = 12 for each haloform composition over the entireexperiment), two replicates of the control composition (canola oil without any haloforms; n = 12 over the entire experiment) and four blank replicates (empty bottles; n = 24 over the entire experiment). Each incubation bottle (except for the blanks) received 0.1 g of the respective treatment compositions and 1 g DM of vetch hay (Vicia sativa L.) as the feed substrate (Table 14). Immediately prior to an in vitro run and the addition of ruminal fluid, half of the replicate incubation bottles were placed randomly into water baths 1, 2 and 3 (block 1) and the other half were placed randomly into water baths 4, 5 and 6 (block 2) and were allowed to come to a temperature of about 39°C.Table 14. Nutrient composition of vetch hay used as a feed substrate in the incubation bottles.

[0306] The nutritive characteristics of the vetch hay feed substrate was analysed by wet chemistry (AOAC International, 2000) at Dairy One, NY, USA via Feed Central, Queensland.

[0307] For each experimental run, approximately 650 m of ruminal fluid was collected from each of three lactating, rumen cannulated (www.rumencannula.com), Holstein- Friesian cows. The cows were consuming a perennial ryegrass (Lolium perenne L.) dominant pasture and this was supplemented with a grain mix (rolled barley grain, 296 g / kg DM; rolled com grain, 280 g / kg DM; rolled wheat grain, 148 g / kg DM; solvent extracted canola meal, 206 g / kg DM; limestone, 10.0 g / kg DM; molasses, 15.0 g / kg DM; sodium bicarbonate, 15.0 g / kg DM; and minerals, 28.6 g / kg DM) and no additional rumen modifiers.

[0308] Immediately after morning milking, the ruminal fluid was collected from multiple sites in the rumen using a copper pipe and a 100-mL syringe and was subsequently mixedin 2 L glass botles that were prewarmed to 39°C. The samples were transported 700 m to the laboratory in an incubator that had been prewarmed to 39°C (Thermoline laboratory incubator, Thermo Fisher Scientific Inc., Scoresby, Vic, Australia). Prior to incubation, the ruminal fluid was filtered under constant flow of CO2 through a 500 pm mesh copper filter to remove any suspended particles. The incubations botles (with treatment compositions and feed substrate) were then filled with 25 mb of ruminal fluid and 75 mb of Kansas State buffer solution (pH 6.8), leaving 210 mb of headspace volume. The Kansas State buffer consisted of solution A, which contained (per litre of distilled water) 10 g of KH2PO4, 0.5 g of MgSO47H2O, 0.5 g ofNaCl and 0.1 g of CaCl2H2O. Solution A was then brought to a pH of 6.8 by adding solution B, which contained (per 100 mb of distilled water) 15 g of Na2CO3 and 1 g ofNa2S9H2O. After the addition of ruminal fluid and buffer solution, each incubation botle was capped with an ANKOM GP module, and the headspace flushed with CO2. The pH of the ruminal fluid was measured using a Metler-Toledo FG2 pH meter (Schwerzenbach, Switzerland) before and after each run.

[0309] At the end of each 24 h incubation run, separate gas samples were collected from the headspace of each botle with an air-tight glass syringe (SGE International Pty Ltd, Ringwood, Vic, Australia) and transferred into separate Exetainers® (12 mL soda glass vial, Labco Ltd, Buckinghamshire, UK) that had previously been evacuated. The proportion of methane in the samples were determined by gas chromatography (GC) system (Agilent 7890A) equipped with three detectors (TCD, pECD, FID) and a GILSON GX-271 auto sampler for transferring the pressurised sample from Labco Exetainers® to the GC loops (1 mL x 2). The GC loops and sample inlet were flushed with helium between samples to avoid any carryover. The columns used were (1) HayeSep® N 80 / 100 mesh, 0.5 m x 1 / 8 in. SST (precolumn for both channels); (2) Porapak® QS 80 / 100 mesh, 2 m x 1 / 8 in. SST (analytical on TCD - FID channel); and (3) HayeSep® D, 80 / 100 mesh, 2 m x 1 / 8 in. SST (analytical to uECD). Unless stated otherwise, methane production has been expressed on a mL / g DM basis.

[0310] Total gas production was measured continuously during each 24 h run (there being six runs over the course of the experiment). The ANKOM GP parameter setings were kept constant with a maximum pressure in the fermentation botle of 6.9 kPa (1 psi). When this pressure was exceeded, the pressure module would vent for 250 ms and the change in pressure was accounted for in the cumulative pressure recording. Gas pressure wasmeasured every 5 s and the cumulative pressure was recorded at 5-min intervals. Cumulative pressure measurements of the gas that was vented were converted to moles using the ideal gas law: / V \ n= p

[0311] where n is the gas produced in moles; p is the pressure in kPa; V is the headspace volume in L; R is the universal gas constant (8.314472 L kPa K-l mol-1); and T is the temperature in Kelvin. These results were subsequently converted to mL of gas produced using Avogadro’s law:Gas production (mL) = n x 22.4 x 1,000

[0312] The resulting data was averaged for each treatment over the course of the six runs and plotted against time to produce cumulative gas production curves for each composition. The final sum of cumulative gas production was taken as total gas production. The methane proportions from the gas sample analysis were then used to calculate total methane production from total gas production. For each run, the four blank replicates, which consisted of ruminal fluid and buffer solution only with no feed substrate, were used to correct for gas production resulting from residual feed particle fermentation in the ruminal fluid. The mean gas production data from the blanks was subtracted from the gas production data of the treatment composition data within each block.

[0313] Immediately after fermentation, the pH of the ruminal fluid mixture in each incubation bottle was measured using a Mettler-Toledo FiveGo FG2 pH meter (Schwerzenbach, Switzerland). Samples were then taken from the bottles to quantify volatile fatty acids (VFAs), in vitro digestibility and ammonia as described below.

[0314] For each incubation bottle, a 5 mL subsample was transferred to a 10 mL plastic test tube and frozen at -18°C with no additional preservatives. The sample was analysed for VFA concentrations by capillary GC according to Supelco Bulletin 749D on an Olympus AU400 autoanalyser after deproteinization with perchloric acid following the procedure in Erwin et al. (1961). Sample VFA peaks were identified by comparing retention times with those of a standard mixture of known VFAs and quantified using Agilent Chemstations software and Microsoft Excel using 4-methlyvaleric acid as theinternal standard. All results were calculated as ppm and converted to mg / L for subsequent analyses.

[0315] Apparent in vitro digestibility of substrate dry matter (IVDDM), defined as the difference between the substrate added before fermentation and the substrate remaining after fermentation, was calculated. Following incubation, the contents of each incubation bottle were gradually poured through pre-weighed 50 mb borosilicate Gooch crucibles (Duran #1; DWK Life Sciences, Mainz, Germany) with a 5 mm sand filtration aid layer under vacuum, which filtered out the buffered ruminal fluid and left only the incubated substrate. The crucibles plus filtrate were then oven dried at 60°C until constant weight and reweighed to determine the IVDDM.

[0316] A 4.8 mL subsample was transferred to a 10 mL plastic test tube to which 0.2 mL of HC1 had been added, then frozen at -18°C. This sample was analysed for Ammonia-N concentration by flow injection analysis (QuickChem 8500 Series 2 Flow Injection Analyser; Lachat Instruments, Milwaukee, WI, USA) with reference to NH4C1 dissolved in 0.1 M HC1 standards.

[0317] The raw data that was used to quantify the effects of the treatment compositions on the production of methane was prepared as follows: (1) replicates with missing data were deleted; (2) replicates were deleted if total gas production was less than 75 mL / g DMD; and (3) the median methane production values for replicate feeds within the same run were calculated from the remaining data to produce the raw data set that was used for the analyses. The mean methane produced by each feed was then calculated for Figure 25.

[0318] A one-factor permutational analysis of variance (PERMANOVA) was used to test for significant differences in the methane mitigation between treatment compositions (fixed factor). All PERMANOVA analyses were performed using PRIMER 6 (v. 6.1.13 (Clarke and Gorley 2006) and PERMANOV A+ (v. 1.0.3. (Anderson et al. 2008)). For PERMANOVA, Euclidean similarity matrices were produced using the untransformed raw data and dummy variables (0.0001) were used to account for zero values. The p values were calculated from 9,999 random permutations. Pairwise a posteriori comparisons were used to determine significant groupings and differences were considered significant if p < 0.05.

[0319] To determine whether there was any synergistic effect of using bromoform and iodoform on the production of methane, the first step was to model how each of the haloforms performed without the other. Therefore, beta regressions, which are appropriate for use with bounded percentage data, were fitted to: ( 1) the methane mitigation data of the treatment compositions that contained only bromoform (ASP5, ASP 10, ASP 15, ASP 16, ASP20; Figure 26) and (2) the methane mitigation data of the treatment compositions that contained only iodoform (ASP1, ASP2, ASP3, ASP4; Figure 27). The beta regressions were produced in R (v. 3.0.1 (R Core Team 2013)) using a generalized additive model in the ‘mgcv’ package with the ‘betar’ family and the ‘logit’ link function. The models were considered significant if p < 0.001. The control data from each run was combined to obtain a single mean value for methane production (7.76 ± 1.99 mL / g DDM). This value was then used to establish the “percent methane mitigation compared to the control” for each treatment composition containing bromoform and / or iodoform (ASP1-ASP20). The two models were used to determine the expected effect of any concentration of bromoform and iodoform on methane mitigation. Subsequently, the expected additive effect was calculated using the following formula:Expected additive effect (%) = (1 — (1 — bromoexp) x (1 — iodoexpf) x 100 where bromoexpis the expected methane mitigation of bromoform (%) and iodoexpis the expected methane mitigation of iodoform (%). The expected additive effect on methane mitigation was then subtracted from the actual methane mitigation of each composition. The difference between the two demonstrated whether there was a positive synergistic effect of using combinations of bromoform and iodoform on the mitigation of methane.

[0320] An incubation bottle with its associated ANKOM GP module was treated as the experimental unit. All 20 compositions were present in both ANKOM GP systems in each run, yielding 2 (systems) x 20 (treatment compositions) = 40 total statistical treatments per run. The data obtained was then analysed using a randomised, incomplete block design.

[0321] The kinetic parameters of gas production were computed by Genstat 22 (VSN International, Hemel Hempstead, UK) using the Gompertz model:where A is the y intercept; C is the maximum gas produced (mL / g DM); B is the rate of gas production (mL / h); x is the time (h) of incubation; and M is the time (h) at which the maximum rate of gas production is reached.

[0322] PERMANOVA was also used to test for significant differences in in vitro gas production parameters (total gas production, methane production, methane mitigation, time at max rate, rate constant and y intercept) and fermentation parameters (IVDDM, total VFAs, acetic :propionic (A:P), acetic+butyric: propionic (AB:P) and ammonia-N) between treatment compositions for bromoform alone (ASP5, ASP10, ASP15, ASP16 and ASP20), iodoform alone (ASP1, ASP2, ASP3 and ASP4) and compositions with both bromoform and iodoform (ASP6-ASP9; ASP11-ASP14; ASP17-ASP19).ResultsBromoform dose response

[0323] There was a significant positive relationship between the concentration of bromoform in the compositions without iodoform (ASP5, ASP10, ASP15, ASP16 and ASP20) and methane mitigation (PERMANOVA, pseudo-f(4,37) = 30.81, p < 0.001; Figure 26). Methane production was effectively eliminated when using the ASP20 (46.6 mg bromoform / kg DM) and ASP5 (85.3 mg bromoform / kg DM) compositions compared with ASP10, ASP15 and ASP16. These results were supported by the beta regression, which was significant (p < 0.001), exhibited an adjusted-r2 of 0.794 and explained 77.3% of the deviance (Figure 26). Based on the predictions of the model, the largest mitigation of methane in vitro occurs when the concentration of bromoform is > 63 mg / kg DM. Note that the model predicts a decrease in the mitigation of methane beyond a bromoform concentration of 63 mg / kg DM in the compositions. However, it can be assumed that beyond this point, methane mitigation will remain constant or continue to increase and that the power of the model could be improved by testing more data points.

[0324] Neither total gas production (mL / g IVDDM) nor gas production kinetics were affected by the inclusion of bromoform in the compositions (Table 15). However, total gas production of the ASP5 composition, which contained the greatest concentration of bromoform, was numerically lower than that of the control and showed a slower rate of gas production than the other bromoform treatments (Figure 28). There are two possible explanations for this observation. Firstly, methane is a component of gas production fromruminal fluid fermentation, and it is possible that an impact on methane production will also affect gas productionError! Bookmark not defined. (Brooke 2021) . Secondly, a decrease in gas production could suggest that, at such a high rate of bromoform, there was a negative feedback effect on in vitro fermentation. However, there was no effect of composition on IVDDM or total VFA production (Table 16). Both IVDDM and production of VFA are indicators of fermentation efficiency (Bergman, 1990). Thus, the absence of significant detrimental effects on in vitro fermentation parameters, in particular IVDDM and total VFA, at the rates of bromoform used in our experiment supports the potential for bromoform to decrease the production of methane with minimal adverse effects on ruminal fermentation.

[0325] There were significant (p < 0.001) decreases in A:P and AB:P with increasing concentrations of bromoform in the compositions (Table 16). The reduction in methane emissions is consistent with the decrease in A:P and AB:P since halogenated compounds can promote propionate production, which is considered an alternative hydrogen sink (Roque 2021). Similar results have been observed in vitro where the incubation of an assayconcentration of bromoform at 5 pM resulted in lower A:P. Similar shifts in the production of acetate and propionate occur for other methane inhibitors such as bromochloromethane (Goel 2009, Tomkins 2009 and Mitsumori 2012). This mechanism is driven by the reduction of methane produced, which results in the increase of hydrogen. The hydrogen control mechanism operates by increasing the flux of carbon towards propionate, while the flux towards the reaction that produces only acetate decreases (Munoz-Tamayo 2021).Table 15. Effect of bromoform on mean (± SE) in vitro gas production parameters.Notes:1total gas production (mL / g in vitro dry matter disappearance);2superscript letters represent significant groupings;3methane production (mL / g in vitro dry matter disappearance);4percent relative to the control.Table 16. Effect of bromoform on mean (± SE) in vitro fermentation parameters.Notes:1in vitro dry matter disappearance;2volatile fatty acids;3superscript letters represent significant groupings;4acetate to propionate ratio;5acetate plus butyrate to propionate ratio.

[0326] A previous meta-analysis of 15 published articles reported that with each increase of 10 g / kg DM in dietary lipid concentration, enteric methane emissions are reduced by approximately 3.5% (Moate 2011). However, since the control composition in this experiment contained the same oil without haloforms, the methane mitigations reported here are greater than those that could have been caused by the oil alone. The findings reported above suggest that bromoform has potent anti-methanogenic properties, and that this anti-methanogenic effect is dose dependent with no negative effects on in vitro ruminal fermentation (Table 16).Iodoform dose response

[0327] There was a significant positive relationship between the concentration of iodoform in the compositions without bromoform (ASP1, ASP2, ASP3 and ASP4) and methane mitigation (PERMANOVA, pseudo-f(3,29) = 199.8, p < 0.001; Figure 27). Methane production was effectively eliminated when using the ASP3 (17.3 mg iodoform / kg DM) and ASP4 (37.3 mg iodoform / kg DM) compositions compared with ASP1 and ASP2.

[0328] These results were supported by the beta regression, which was significant (p < 0.001), exhibited an adjusted-r2 of 0.917 and explained 91. 1% of the deviance (Figure 27). Based on the predictions of the model, the largest mitigation of methane in vitro occurs when the concentration of iodoform is > 32 mg / kg. Note that the model predicts a decrease in the mitigation of methane beyond an iodoform concentration of 32 mg / kg in the compositions. However, it can be assumed that beyond this point, methane mitigation will remain constant or continue to increase and that the power of the model could be improved by testing more data points.

[0329] Neither total gas production (mL / g IVDDM) nor gas production kinetics were affected by the inclusion of iodoform in the compositions (Table 17, Figure 29).Table 17. Effect of iodoform on mean (± SE) in vitro gas production parameters.Notes:1total gas production (mL / g in vitro dry matter disappearance);2superscript letters represent significant groupings;3methane production (mL / g in vitro dry matter disappearance);3percent mitigation relative to the control.Table 18. Effect of iodoform on mean (± SE) in vitro fermentation parameters.Notes:1in vitro dry matter disappearance;2volatile fatty acids;3superscript letters represent significant groupings;4acetate to propionate ratio;5acetate plus butyrate to propionate ratio.

[0330] These results follow a similar trend to that of bromoform and to other haloforms in the literatureError! Bookmark not defined.. It is postulated that most haloforms act in a similar manner, acting as a methane analogue, interrupting the cobamide-dependent methyltransferase reaction, the terminal step in enteric methane synthesis. The findings reported above suggest that iodoform has potent anti-methanogenic properties, and this anti-methanogenic effect is dose dependent with no negative effects on in vitro ruminal fermentation (Table 18).

[0331] As with bromoform, there were significant (p < 0.001) decreases in A:P and AB:P with increasing concentrations of bromoform in the compositions (Table 18).Example 10 - Synergistic effects of bromoform and iodoform combinations

[0332] Feed compositions were prepared comprising combinations of bromoform and iodoform. For the first time, it has been surprisingly found that such combinations can act synergistically to reduce methane production in vitro. In eight of the eleven treatment compositions that were tested (in Table 19), the actual methane mitigation of compositions with both haloforms was greater than what was expected based on the methane mitigation capacity of each haloform by itself. The regression analyses resulted in the models underestimating the expected mitigation of methane in many of the compositions (Figures 26 and 27). Therefore, compositions with smaller synergistic effects (ASP6, ASP8 and ASP9) were identifiable. More notable was the identification of compositions with much larger synergistic effects (ASP7, ASP11, ASP12, ASP13 and ASP17). Further, the data shows that there is a plateau that is reached where, above a certain concentration of haloform, methane will always be completely mitigated.Table 19. Expected versus actual effects of bromoform and iodoform on the mitigation of methane in vitro.Notes:1The beta regression in Figure 26 was used to predict the methane mitigation from bromoform;2the beta regression in Figure 27 was used to predict the methane mitigation from iodoform;3these values were edited to reflect to represent the largest mitigation predicted by the model.

[0333] When considering the feeding of Asparagopsis to ruminants, although Machado et al . (2016b) found that multiple haloforms present in Asparagopsis have methane mitigating properties, only bromoform was present in Asparagopsis in sufficient quantities to reduce methane.

[0334] Regardless of the concentration of bromoform, increasing the iodoform concentration resulted in greater methane mitigation, with no effects on total gas production or gas production kinetics (Table 20). To the inventors’ knowledge, there are no studies evaluating the individual and synergistic effect of different haloforms.

[0335] It should be noted that no treatment in the compositions tested had any significant effect on total gas production (mL / g IVDDM), other gas production parameters, or the cumulative gas production curves (Figure 30). As with bromoform and iodoform, there were significant (p < 0.001) decreases in A:P and AB:P with increasing concentrations of haloforms in the combination compositions (Table 21). These decreases were most evident in the higher haloform inclusion rates for A:P and in every treatment apart from ASP16 for AB:P. Moreover, there were no significant differences in the IVDDM or total VFA production between the compositions. It can be concluded that haloform combinations demonstrated signs of a synergistic methane mitigation effect in vitro, with no negative effects on in vitro fermentation.

[0336] In an alternative analysis, modelling of the positive synergistic results of Table 19 (last column) revealed that the concentration of iodoform had no effect on the system overall (p = 0.510; Figure 31). However, iodoform concentrations of ~5 mg / kg DM still resulted in the largest synergistic effects when used in combination with bromoform (see dashed outline in Figure 31). Conversely, there was a significant, positive correlation between the concentration of bromoform and synergistic effects (p = 0.002; adjusted r- squared = 0.851; Figure 32).Table 20. Combined effects of bromoform and iodoform on mean (± SE) in vitro gas production parameters.p value - - 0.588 < 9,001 < 9901 9.778 9,774 069Notes:1total gas production (mL / g in vitro dry matter disappearance);2superscript letters represent significant groupings;3methane production (mL / g in vitro dry matter disappearance); 4 percent mitigation relative to the control.Bibliography

[0337] Anderson M., Gorley R. & Clarke K. 2008. PERMANOVA+ for Primer: guide to software and statistical methods. Plymouth: PRIMER-E pp. 214.

[0338] Bergman, E. 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiological reviews, 70, 567-590.

[0339] Brooke, C. G., Roque, B. M., Shaw, C., Najafi, N., Gonzalez, M., Pfefferlen, A., De Anda, V., Ginsburg, D. W., Harden, M. C., Nuzhdin, S. V., Salwen, J. K., Kebreab, E. & Hess, M. 2020. Methane Reduction Potential of Two Pacific Coast Macroalgae During in vitro Ruminant Fermentation. Frontiers in Marine Science, Vol. 7; pg 1-7.

[0340] Chagas, J. C., Ramin, M. & Krizsan, S. J. 2019. In vitro Evaluation of Different Dietary Methane Mitigation Strategies. Animals (Basel), 9: 1120; 1-17

[0341] Clarke K.R. & Gorley R. 2006. PRIMER v6: user manual / tutorial. Plymouth: PRIMER-E; p. 190.

[0342] Goel, G., Makkar, H. P. & Becker, K. 2009. Inhibition of methanogens by bromochloromethane: effects on microbial communities and rumen fermentation using batch and continuous fermentations. British journal of nutrition, 101, 1484-1492.

[0343] Machado, L., Magnusson, M., Paul, N. A., Kinley, R., de Nys, R. & Tomkins, N. 2016b. Identification of bioactives from the red seaweed Asparagopsis taxiformis that promote antimethanogenic activity in vitro. J. Appl. Phycol, 28, 3117-3126.

[0344] Mitsumori, M., Shinkai, T., Takenaka, A., Enishi, O., Higuchi, K., Kobayashi, Y., Nonaka, I., Asanuma, N., Denman, S. E. & Mcsweeney, C. S. 2012. Responses in digestion, rumen fermentation and microbial populations to inhibition of methane formation by a halogenated methane analogue. Br J Nutr, 108, 482-91.

[0345] Moate, P. J., Williams, S. R. O., Grainger, C., Hannah, M. C., Ponnampalam, E. N. & Eckard, R. J. 2011. Influence of cold-pressed canola, brewers grains and hominy meal as dietary supplements suitable for reducing enteric methane emissions from lactating dairy cows. Animal Feed Science and Technology, 166-167, 254-264.

[0346] Munoz-Tamayo, R., Chagas, J. C., Ramin, M. & Krizsan, S. J. 2021. Modelling the impact of the macroalgae Asparagopsis taxiformis on rumen microbial fermentation and methane production. Peer Community Journal, 1,1-16.

[0347] Roque, B. M., Venegas, M., Kinley, R. D., de Nys, R., Duarte, T. L., Yang, X. & Kebreab, E. 2021. Red seaweed (Asparagopsis taxiformis) supplementation reduces enteric methane by over 80 percent in beef steers. PLoS One, 16, e0247820.

[0348] Tomkins, N. W., Colegate, S. M. & Hunter, R. A. 2009. A bromochloromethane formulation reduces enteric methanogenesis in cattle fed grain-based diets. Animal Production Science, 49, 1053-1058.

Claims

CLAIMS1. A composition for reducing methane production in a ruminant animal comprising a meso- or micro- porous material infused with at least one anti-methanogenic agent, wherein the meso- or micro- porous material is charcoal, biochar or activated carbon, wherein each anti-methanogenic agent is a halogenated methane analogue, a halogenated alkane or a halogenated organic acid, wherein the anti-methanogenic agent(s) is / are dissolved in a carrier to produce an anti- methanogenic agent-carrier (AC) solution, and wherein infusion of the AC solution into the meso- or micro- porous material protects the anti- methanogenic agent against volatilisation and / or degradation.

2. The composition of claim 1, wherein the infused meso- or micro- porous material comprises pores with an average surface area, as calculated by the Brunauer-Emmett-Teller (BET) method, of at least about 1 m2 / g.

3. The composition of any one of the previous claims, wherein the meso- or micro- porous material (MM) is infused with the AC solution at a ratio of MM: AC of at least about 1 : 1 to about 20: 1, preferably from about 4: 1 to about 9: 1.

4. The composition of any one of the previous claims, wherein the infused meso- or micro- porous material comprises pores with an average pore volume of from about 0.001 cm3 / g to about 1.3 cm3 / g.

5. The composition of any one of the previous claims, wherein the meso- or micro- porous material is derived from hardwood, soft wood, or coconut.

6. The composition of any one of the previous claims, wherein the meso- or micro- porous material has a carbon content of no less than 50 %.

7. The composition of any one of the previous claims, wherein the meso- or micro- porous material has an oxygen content of no more than 20 %.

8. The composition of any one of the previous claims, wherein the anti -methanogenic agent comprises one or more compounds selected from the group consisting of: 3-NOP (3- nitrooxypropanol), dichloromethane, dibromomethane, bromochloromethane, bromodichloromethane, bromodiiodomethane, dibromoiodomethane, bromoiodomethane,chloroiodomethane, 2-bromoethanesulfonic acid, chloral hydrate, chloroform, iodoform, chloroethane, dichloroethane, tetrachloroethane, hexachloroethane, bromoethane, dibromoethane, tetrabromoethane, 1,2-dibromotetrachloroethane, iodoethane, diiodoethane, iodopropane, bromoform, carbon tetrachloride, carbon tetrabromide, carbon tetraiodide, and dibromochloromethane .

9. The composition of any one of the previous claims, wherein the anti -methanogenic agent is bromoform or iodoform, or a combination thereof.

10. The composition of any one of the preceding claims, wherein the carrier is an oil.

11. The composition of claim 10, wherein the oil is a vegetable oil or an edible oil blend, preferably canola oil.

12. The composition of any one of the previous claims, wherein the meso- or micro- porous material is further infused with a surfactant.

13. The composition of claim 12, wherein the surfactant is selected from the group consisting of: triton X-100; poloxamers; glycerol monostearate; glycerol monolaurate; sorbitan monolaurate; sorbitan monostearate; sorbitan tristearate; polysorbate 20; polysorbate 40; polysorbate 60; polysorbate 80; ammonium lauryl sulfate; sodium lauryl sulfate; sodium laureth sulfate; sodium myreth sulfate; sodium stearate; phospholipids; phosphatidylserine; phosphatidylethanolamine; phosphatidylcholine; sphingomyelins; lauryldimethylamine oxide; and myristamine oxide; or combinations thereof.

14. The composition of any one of the preceding claims, wherein the composition is in the form of a powder, liquid suspension, solution, emulsion, tablet, capsule, pellet, bolus, or lick block, or is incorporated into animal feed.

15. The composition of any one of the preceding claims, wherein the composition is formulated to deliver a sustained release of the infused meso- or micro- porous material into a ruminant animal’s rumen.

16. The composition of any one of the preceding claims, wherein the composition is incorporated into animal feed and is formulated to deliver at least about 5 mg of the anti- methanogenic agent per kg of animal feed intake.

17. The composition of any one of the preceding claims comprising a combination of a first anti-methanogenic agent and second anti-methanogenic agent.

18. A composition for reducing methane production in a ruminant animal, said composition comprising a combination of a first anti-methanogenic agent and second anti-methanogenic agent, wherein the first anti-methanogenic agent and second anti-methanogenic agent are present in the composition in amounts which, when in use, act synergistically to reduce methane production in a ruminant animal when compared to an additive methane reduction produced by corresponding controls of the first anti-methanogenic agent or second anti-methanogenic agent used independently, and wherein the first and second anti -methanogenic agents are dissolved in a carrier to produce an anti- methanogenic agent-carrier (AC) solution.

19. The composition of claim 18, wherein the first anti-methanogenic agent is iodoform and the second anti-methanogenic agent is bromoform.

20. A method of mitigating methane production in a ruminant animal, comprising administering to the ruminant animal an effective amount of the composition of any one of the preceding claims.

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