Cell-based vaccines

Abstract

The present invention relates to cell-based vaccine compositions and methods that reduce methane and / or hydrogen production in animals. The present invention relates to the treatment of diseases that are associated with methanogens. The present invention also relates to methods of growing hydrogenotrophs in safe anaerobic conditions.

Description

[0001] CELL-BASED VACCINES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 608,497, filed on December 11, 2023; and U.S. Provisional Application No. 63 / 645,276, filed on May 10, 2024, the entire contents of each of said applications are incorporated herein in their entirety by this reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Methane (CH4) is the world’s second most abundant greenhouse gas after carbon dioxide (CO2), accounting for -16% of total greenhouse gas emissions. Livestock emissions, in particular, account for -32% of all anthropogenic CH4 emissions - equating to -6% of all CO2 equivalents (CO2e) of greenhouse gas emissions and -3 billion Tonnes / yr of CChc. CH4 is a powerful greenhouse gas with a potential global warming effect -28-fold higher than that of CO2 over a 100 year period and -80-fold higher than that of CO2 over a 20 year period. Furthermore, CH4 has an atmospheric half-life of -10 years, thus reducing enteric CH4 emissions could have an immediate and dramatic effect on limiting the rate of global warming, which would be of great significance to efforts to reduce global greenhouse gas emissions. Furthermore, CH4 emissions also represent energy losses during ruminant production. On average, approximately 2-12% of the energy consumed in feed is lost in the form of CH4 emissions.

[0006] For ruminants, CH4 is predominantly formed in the ruminant fore-stomach (rumen) by methanogens, a subgroup of the Archaea. During normal rumen function, plant material is broken down by fiber-degrading microorganisms and fermented mainly to volatile fatty acids, ammonia, H2 and CO2. Ruminal methanogens principally use H2 to reduce CO2 to CH4 in a series of reactions that are coupled to ATP synthesis. Additional biochemical pathways to produce CH4 from acetate and methanol / methylamine substrates are also present, but represent a lower fraction of the total CH4 biosynthesis in the rumen.

[0007] Attempts have been made to inhibit the action of methanogens in the rumen using a variety of interventions but most have failed, or were met with only limited success, due to low efficacy, poor selectivity, toxicity of compounds against the host, and / or build-up of resistance to anti-methanogen compounds. Further most solutions, e.g., feed additives and antibiotics, are only applicable to intensive production environments where the animal’s diet can be effectively controlled to ensure precise daily dosages of the intervention. These solutions are, thus, not effective in extensive, grass-fed production environments where the animals a rarely handled and / or operations where feed cannot readily be mixed daily.

[0008] Accordingly, there is a great need in the art for effective, selective, and safe compositions and methods for inhibiting methanogens in ruminant populations.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention is based, at least in part, on the discovery that vaccines of the present disclosure (e.g., cell-based vaccines comprising a cell and / or a cell part) against at least one cell surface antigen or a fragment thereof (e.g., antigenic fragment, epitope) of at least one methanogen, when administered to a subject (e.g., animal, ruminant), are surprisingly effective in inducing immune response and antibody production against the methanogen, and reducing the CH4 production in ruminants.

[0011] Previous attempts to vaccinate ruminants and reduce CH4 production have been largely unsuccessful. Research on a vaccine targeting methanogen(s) has cost between $4 million to $5 million a year for more than 20 years. However, it has been very challenging because of the mechanism used (see World Wide Web at dairyherd.com / news / dairy- production / vaccine-could-provide-cattle-ghg-solution). Specifically, the vaccine must elicit an immune response that results in the production of antibodies. At least a portion of the produced antibodies are present in the ruminant’s saliva, which then pass to the ruminant’s rumen and bind with the methanogens, e.g., those that convert H2 and CO2 into CH4. The antibody must then impair the methanogen thereby reducing total CH4 production in the rumen and subsequent emission. As of current knowledge, the rumen is a relatively isolated organ lacking an adaptive immune response similar to that found systemically, thereby requiring the produced antibodies to have a direct effect on methanogens in the rumen.

[0012] While previous attempts at vaccination were able to induce an immune response in sheep resulting in production of antibodies that bind to methanogens, it was unfortunately not possible to induce production of a consistently large amount of antibodies introduced to the rumen via saliva; and to produce effective antibodies that can neutralize the growth of the methanogen and / or the production of CH4.

[0013] Wright et al (2004) Vaccine 22:29-30 was able to immunize sheep with a whole-cell preparation from a mixture of 3 methanogens and tentatively reduce CH4 production (per kg / DMI) by 7.7%. However, when the study was repeated with a mixture of 5 methanogens, vaccination failed to demonstrate any CH4 abatement, although it changed the microbial fauna in the rumen (Williams et al. (2009) Appl Environ Microb 75(7): 1860-1866). p

[0014] Accordingly, there has been a long-felt need that could not be resolved due to failure of others.

[0015] Compounding the failed attempts to reproducibly reduce CH4 emissions in sheep following vaccination, there has yet to be any successful demonstration of reduced CH4 emission in other ruminants following vaccination. This is even more important in cattle, which contribute the majority of ruminant greenhouse gas emissions.

[0016] In contrast to the failures of others, the vaccines of the present disclosure yielded at least 17% reduction in the emission of CH4 by ruminants following treatment. Furthermore, the ruminants vaccinated with a vaccine of the present disclosure resulted in continued and sustained reductions of CH4 emissions, and over the course of 5 weeks have mitigated ~1.1 kg of CH4 emissions per treated ruminant, which over the course of a year equates to -0.3-0.4 tonnes (1000 kg) of mitigated CH4 per ruminant per year. These results are unprecedented in the field, even more so in cattle. Treated ruminants also showed continued in sustained reduction of H2 emissions. Accordingly, the vaccines of the present disclosure provide a surprising and unexpected means of reducing CH4 and / or H2 emission in ruminants, which could not have been achieved by others despite diligent efforts.

[0017] The vaccine compositions and methods of the present disclosure are useful beyond reducing the CH4 emission in ruminants.

[0018] In addition to CH4 emission reductions, vaccine compositions and methods of the present disclosure have shown surprising and unexpected reductions in emitted H2 following treatment. These results are both surprising and unexpected as reductions in CH4 following treatment with small molecule inhibitors and feed additives have contrarily shown increases in H2 emissions. The reduction in both CH4 and H2 emissions suggest that the vaccine compositions and methods of the present disclosure have utility in improving the feed conversion efficiency and thereby the productivity of treated animals, e.g., increasing the production and / or ruminal concentration of one or more volatile fatty acids (e.g., propionate, butyrate, acetate) in the rumen of the animal, increasing the average daily gain of the animal, reducing the dry matter intake of the animal, reducing the feed requirements of the animal during lactation, and / or increasing the milk production of the animal. Increasing productivity of treated animals can further reduce the carbon intensity of resultant animal products (e.g., milk and meat) as treated animals are not only emit less CH4 but also produce more product per animal. Thus, the total reduction in carbon intensity of animal-derived products from animals treated with a vaccine composition of the present disclosure may can be calculated as the composite of the CH4 reduction of the animal following treatment in combination with the reduced carbon footprint associated with growing and maintaining those animals (e.g., less total feed, manure, urine, etc.).

[0019] Additionally, it is well documented that methanogens are associated with various diseases, including periodontal disease, inflammatory bowel disease (IBD), irritable bowel syndrome (ISB), e.g., IBS-C, SIBO, colorectal cancer, obesity and metabolic syndrome, diverticulosis and diverticulitis, gingivitis, and bloat. Thus, the vaccine compositions and methods of the present disclosure have utility in treating these diseases in animals including humans. The vaccines of the present disclosure also provide a surprising and unexpected effect on lactic acidosis (e.g., reducing rumen lactate, increasing pH, or combination thereof). Therefore, the vaccine compositions methods of the present disclosure have utility in treating diseases associated with elevated, increased, or severe lactic acidosis, e.g., liver abscess.

[0020] Further provided herein are compositions, systems, and methods of growing hydrogenotrophs (e.g., methanogens) without explosive and flammable concentrations of H2 under high pressure.

[0021] BRIEF DESCRIPTION OF FIGURES

[0022] Fig. lA-Fig. IB show schematic diagrams representing the relative abundance of methanogen species across geographies and herds. The diagrams indicate that methanogen populations are relatively stable across geographies and herds. For example, Methanobrevibacter gottschalkii comprises -30-40% of ruminal methanogens globally; thus, a vaccine comprising cells and / or cell parts of at least one methanogen could provide an effective global solution for reducing the amount of CH4 generated by ruminants.

[0023] Fig. 2 shows a representative graph demonstrating the reduction of CH4 emissions by cows vaccinated with a cell-based vaccine of the present disclosure. The graph represents data from the first 40 days of vaccination and indicates a significant reduction (-17%) in emitted CH4 after boosting.

[0024] Fig. 3 shows an increased antigen-specific antibody titer in the sera of vaccinated ruminants in Fig. 2.

[0025] Fig. 4 shows a schematic diagram that illustrates vaccination and post-vaccination testing of a ruminant (e.g., a cow).

[0026] Fig. 5A-Fig. 5C show schematic diagrams that illustrate exemplary tests performed for a ruminant vaccinated with a cell-based vaccine of the present disclosure. Fig. 5A shows quantifying Ig antibody in serum and saliva of a vaccinated ruminant. The antibody is evaluated for methanogen binding to methanogens (e.g., affinity, specificity, etc.) and / or its effect on the fitness of methanogens (e.g., proliferation, CH4 production, etc.) upon binding. Fig. 5B shows measuring the amount of CH4 produced by a vaccinated ruminant using the GreenFeed system (C-lock Inc., Rapid City, South Dakota). Fig. 5C shows evaluation of the changes in the rumen microbiome in a vaccinated ruminant. The 16S rRNA sequencing identifies the presence and the amount of specific methanogens.

[0027] Fig. 6A-Fig. 6B show an exemplary vaccine preparation workflow. Fig. 6A shows growth of methanogens, subsequent fixation (e.g., with formaldehyde) and, washing (using e.g., Phosphate-Buffered Saline (PBS)). Fig. 6B shows how cells are counted and cell titer is normalized. Adjuvant is added to the mixture of cells to prepare cell-based methanogen vaccines.

[0028] Fig. 7 shows a schematic diagram of an exemplary instrument (e.g., GreenFeed) that measures the CH4 produced from the rumen of a ruminant.

[0029] Fig. 8 shows an exemplary vaccination schedule. The bottom panel shows the measurements of animal temperature taken during the vaccinated period. No adverse events were reported for the vaccinated ruminants.

[0030] Fig. 9-Fig. 10 show CH4 emissions analysis performed using the GreenFeed system. Fig. 9 and Fig. 10 demonstrate statistically significant reductions in CH4 emitted by vaccinated cows after boosting.

[0031] Fig. 11 shows a representative analysis of sera from vaccinated cows by whole-cell methanogen ELISA. The analysis showed increased binding following consecutive vaccinations, a specific binding response that correlates to the vaccinated strain of methanogen, and binding to both strains in the blended vaccine.

[0032] Fig. 12 shows the CH4 produced by the cows vaccinated with M. gottschalkii. A significant reduction (-17% reduction) in emitted CH4 was observed for 4 out of 5 ruminants vaccinated with M. gottschalkii.

[0033] Fig. 13 shows a representative H2 emissions analysis (using GreenFeed system). A statistically significant reduction in emitted H2 was observed after boosting, especially in cows vaccinated with M. gottschalkii.

[0034] Fig. 14 is a schematic of a biochemical pathway and enzymes for the production of CH4 from acetate (i.e., the acetoclastic pathway), H2 and CO2 (i.e., the hydrogenotrophic pathway), and methanol and derivatives thereof (i.e., the methylotrophic pathway).

[0035] Fig. 15 is a schematic describing the reduction of methyl-CoM and coenzyme B into CH4 by methyl-coenzyme M reductase (MCR), a key enzyme present in CH4 production via the acetoclastic, hydrogenotrophic, and methylotrophic pathways. 4

[0036] Fig. 16 is a schematic describing the application of small molecules that affect MCR activity to modulate CH4 production.

[0037] Fig. 17 is a cross-sectional diagram of the application of a composition for reduction of deleterious atmospheric gases and / or precursors thereof to a water source, such as a trough or a pond. The water source (401) comprises the composition (402). While the composition (402) is shown to be at the bottom of the water source (401), the composition (402) can be at any suitable position, for example floating at the top, admixed within, or dissolved within the water source (401).

[0038] Fig. 18 shows aerial delivery of a composition for reduction of deleterious atmospheric gases and / or precursors thereof to a water source. A aerial delivery device (501) delivers (502) a composition (within the device) to a water source (503), such as a trough or a pond.

[0039] Fig. 19 shows 3NOP concentration (mM) v. adsorbent (~20 mM stock solution).

[0040] Fig. 20 shows 3NOP concentration (mM) v. adsorbent (~8 mM stock solution).

[0041] Fig. 21 shows exemplary multilayer polyelectrolyte coatings of 15% activated carbon tablets.

[0042] Fig. 22 shows exemplary multilayer polyelectrolyte coatings of 25% activated carbon tablets.

[0043] Fig. 23 shows exemplary multilayer polyelectrolyte coatings of 15% activated carbon tablets with 5% sodium lignosulfonate.

[0044] Fig. 24 shows exemplary multilayer polyelectrolyte coatings of 15% activated carbon with 5% hydroxypropyl cellulose.

[0045] Fig. 25 is a graph showing the release profiled of a silica v. activated carbon adsorbent.

[0046] Fig. 26 is a table showing the composition of various polycaprolactone-based formulations according to some embodiments of the invention.

[0047] Fig. 27 is a bar graph showing 3NOP release in mM for exemplary polycaprolactone- based formulations.

[0048] Fig. 28 is a graph showing normalized 3NOP concentration v. release time in days of exemplary polybutylene succinate-based formulations.

[0049] Fig. 29 is a bar graph showing 3NOP release (%) from exemplary PEC microcapsules. Samples are prepared with a 3NOP concentration of 100 pM. Final pH solution ~7. K, L, F, and E refer to lysine, leucine, phenylalanine, and glutamic acid, respectively. PSS refers to polystyrene sulfonate. Fig. 30 is bar graph showing 3NOP release (%) from exemplary PEC microcapsules. Samples are prepared with a 3NOP concentration of 100 pM. Final pH solution ~7. PLR refers to poly (L-arginine), and PLK refers to poly (L-lysine). SLS and PSS refer to sodium lignosulfonate and polystyrene sulfonate.

[0050] Fig. 31 shows the sera antibody binding to M. gottschalkii in the ELISA assay.

[0051] Fig. 32 shows the sera antibody binding to M. ruminantium in the ELISA assay.

[0052] Fig. 33 shows the sera antibody (from sera collected on d49) binding to M. gottschalkii in the FACS analysis.

[0053] Fig. 34 shows the binding of antibodies in clarified sera, collected from Animal 432K, to M. ruminantium and M. gottschalkii, observed using Western blot.

[0054] Fig. 35 shows the mitigated CH4 emissions per treatment group.

[0055] Fig. 36 shows the mitigated H2 emissions per treatment group.

[0056] Fig. 37A shows the mitigated CCL-normalized CH4 emissions per treatment group.

[0057] Fig. 37B shows the increased CO2 emissions per treatment group.

[0058] Fig. 38 shows the results of in vitro CH4 production using the d63 sera from ruminants vaccinated with cell-based vaccines.

[0059] Fig. 39 shows mandatory components of system for growing hydrogenotrophic organisms.

[0060] Fig. 40 shows a process flow diagram of the continuous flow methanogen (e.g., M. gottschalkii ) culturing system. Gas is sparged through anaerobic water for humidification prior to entering the methanogen culture bottle, to prevent evaporation. The methanogen converts H2 and CO2 into CH4 during growth. The H2, CO2, N2, CH4 mixture then passes through a trap before entering a flow meter to minimize fouling risk to the flow meter.

[0061] Fig. 41 shows an example of a gas diffusion system for the improvement of gas transfer within the system. In this example, a sintered stainless steel gas diffuser is used. However, other instruments, e.g., spargers, baffles, impellers, etc., that are used in culturing microorganisms may be used. Optionally, a humidifying water bottle can be placed upstream to minimize volume loss of the culture. Alternatively, a condenser or other volume capture or addition system can be used.

[0062] Fig. 42 shows a labeled picture of the water bottle used in the water bath. The culture bottle followed the same setup.

[0063] Fig. 43 shows cell titer within an M. gottschalkii culture grown with continuously flowing 4% H2, 10% CO2, and 86% N2 (“continuous”) compared to an M. gottschalkii culture grown with over pressurization of the headspace at multiple time points throughout growth 4 with 80% H2, 20% CO2 to 15 psi overpressure. Cell titering was performed on a SONY SH800 FACS using the protocol described in Example 1.

[0064] Fig. 44 shows an exemplary process flow diagram for manufacturing of a whole cell vaccine. A vial of the Master Cell Bank is thawed and expanded to produce a Working Cell Bank (1), which is then used to inoculate the seed train (2-3) and production fermenter (4). Cells are then killed and buffer exchanged (5), formulated (6), and then filled into vials for distribution (7).

[0065] Fig. 45 shows CH4 produced from a M. gottschalkii culture grown in BY medium (bottom curve) or semi-defined media SD0001 (top curve) in Hungate tubes with ~25 psi 80% H2 / 20% CO2 initial batched headspace. CH4 measurements were performed using a Gazomat Gazoscan using a custom jig for consistent positioning of the tubes relative to the measurement device. Data shown is the average of 2 duplicate tubes with error bars representing 1 standard deviation.

[0066] Fig. 46 shows reduction in CH4 production in vitro following treatment with sera before and after vaccination with a vaccine as disclosed herein.

[0067] Fig. 47A shows reduction in methanogen growth and Fig. 47B shows reduction in CH4 production in vitro following treatment with sera before and after vaccination.

[0068] DETAILED DESCRIPTION OF THE INVENTION

[0069] Provided herein are vaccines (e.g., cell-based vaccines comprising a cell and / or a cell part) against at least one cell surface antigen or a fragment thereof (e.g., antigenic fragment, epitope) of at least one methanogen, which are effective in inducing immune response and antibody production against the methanogen antigen, and reducing the CH4 production in subjects. The vaccines of the present disclosure are also useful in treating diseases in subjects (e.g., animals, mammals, ruminants, humans) that are associated with methanogens (e.g., periodontal disease, Inflammatory Bowel Disease (IBD), gingivitis, and / or bloat). The vaccines of the present disclosure are also useful in treating diseases in subjects (e.g., animals, mammals, ruminants, humans) that are associated with elevated, increased, or severe lactic acidosis, e.g., liver abscess.

[0070] Further provided herein are compositions, systems, and methods of growing hydrogenotrophs (e.g., methanogens), e.g., without explosive and flammable concentrations of H2 under high pressure. 4

[0071] DEFINITIONS

[0072] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0073] As used herein, the term "about" when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by (+) or (-) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0074] The term “administering” is intended to include routes of administration which allow an agent (e.g., a vaccine composition, an agent that reduces CH4 production in a subject) to perform its intended function. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, intradermal, intramuscular, etc.), oral, inhalation, and transdermal routes. The injections can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent (e.g., a vaccine composition, an agent that reduces CH4 production in a subject) can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier or adjuvant. The agent (e.g., a vaccine composition, an agent that reduces CH4 production in a subject) also may be administered as a prodrug, which is converted to its active form in vivo.

[0075] As used herein, “anaerobic conditions” are conditions with reduced levels of oxygen compared to normal atmospheric conditions. For example, in some embodiments anaerobic conditions are conditions wherein the oxygen levels are partial pressure of oxygen (pCh) no more than 8%. In some instances, anaerobic conditions are conditions wherein the pCh is no more than 2%. In some instances, anaerobic conditions are conditions wherein the pCh is no more than 0.5%. In certain embodiments, anaerobic conditions may be achieved by purging a growth chamber and / or a bioreactor with a gas other than oxygen such as, for example, N2, H2, and / or CO2.

[0076] The term “cell parts,” as used herein encompasses any and all that is less than a whole cell. In some embodiments, cell parts of the present disclosure comprise the cell membrane with the membrane -bound proteins. In some embodiments, the cell parts comprise an antigenic part of the cell. Such antigenic part may comprise at least one epitope that binds to the antibody. In preferred embodiments, the cell parts of the present disclosure are effective in eliciting immune response and / or inducing antibody production when administered to a subject. In some embodiments, the vaccine composition of the present disclosure comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or 100% whole cells. In some embodiments, the vaccine composition of the present disclosure comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or 100% cell parts.

[0077] The term “conjoint” or “combination” administration, as used herein, refers to the administration of two or more agents that aid in reducing CH4 production in a subject. The different agents comprising the combination may be administered concomitant with, prior to, or following the administration of one or more agents.

[0078] The term “fragment,” as used herein encompasses any and all that is less than the full length. In some embodiments, a fragment of a polypeptide of the present disclosure is an antigenic fragment of the polypeptide. Such antigenic fragment may comprise at least one epitope that binds to the antibody. In preferred embodiments, a fragment of a polypeptide of the present disclosure is a fragment of the polypeptide that is effective in eliciting immune response and / or inducing antibody production when administered to a subject.

[0079] The term “methanogen,” as used herein, refers to a microorganism that produces CH4 as a metabolic byproduct. Methanogens belong to the domain Archaea, and include, but are not limited to those of a family Methanobacteriaceae, e.g., those of genera Methanobrevibacter, Methanosphaera, Methanomassiliicoccaceae, Methanomicrobium, Methanobacterium, Methanocorpusulum, Methanosaeta, Methanoculleus, Methanosarcina, and Thermoplasmatales. Specific methanogens include, but are not limited to, Methanobrevibacter ruminantium (e.g., the Ml strain or strain DSM 1093 (see e.g., World Wide Web at dsmz.de / microorganisms / html / strains / strain.dsm001093.htm) and Methanobrevibacter gottschalkii. Additional relevant species are further described below.

[0080] The term “ruminant” refers to a hoofed herbivorous grazing or browsing mammal that is able to acquire nutrients from plant-based food by fermenting it in a specialized stomach prior to digestion, principally through microbial actions. The process, which takes place in the front part of the digestive system and therefore is called foregut fermentation, typically requires the fermented ingesta (known as cud) to be regurgitated and chewed again. The roughly 200 species of ruminants include both domestic and wild species. Ruminants include, but are not limited to, cattle (e.g., large domesticated ruminant animals, e.g., cows (including dairy cattle), bulls), all domesticated and wild bovines (i.e., those belonged to the family Bovidae; e.g., cows, bulls, bisons, yaks, African buffalos, water buffalos, antelopes), goats, sheep, giraffes, deer, caribou, and gazelles. In preferred embodiments, ruminants are domesticated. As used herein, the term “ruminant” includes ruminant-like animals or pseudoruminant animals such as macropods, llamas, camels, and alpacas. In some embodiments, a ruminant has not been administered with an agent that reduces CH4. In other embodiments, a ruminant has been administered or is being administered with an agent that reduces CH4.

[0081] The term “subject” refers to any healthy or diseased animal, including any mammal, ruminant, canine, feline, or human.

[0082] METHANOGENS

[0083] The diversity of ruminal methanogens is much lower than that of rumen bacteria, with archaeal SSU rRNA only accounting for 6.8% of rumen total SSU rRNA. Archaea in the rumen is represented by <3.3% of the total rRNA (both 16S and 18S) therein. Representative family of methanogens includes Methanobacteriaceae. Rumen methanogens typically comprises 2-3% of the total microbial biomass in the rumen.

[0084] Representative genera of methanogens include Methanobrevibacter, Methanosphaera, Methanomassiliicoccaceae, Methanomicrobium, Methanobacterium, Methanocorpusulum, Methanosaeta, Methanoculleus, Methanosarcina, and Thermoplasmatales.

[0085] Certain species of ruminal methanogens have been isolated into pure cultures: Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacter gottschalkii, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleus olentangyi, and Methanosarcina barkeri. Additional species have been recently isolated, including Methanobrevibacter boviskoreani (isolated from the rumen of Korean native cattle), Methanobacterium beijingense (isolated from the rumen of goat), Methanoculleus marisnigri (isolated from the rumen of Indian crossbred cattle), Methanoculleus hour gensis (isolated from the rumen of Holstein cattle), and Methanosarcina mazei (isolated from the rumen of Korean Hanwoo cattle) (based on the RDP database). A Thermoplasmatales-like pyrrolysine- dependent archaeon BRNA1 was also isolated from bovine (GenBank access number: CP002916).

[0086] Collectively, 16S rRNA gene sequences from cultured methanogens only accounted for approximately 0.7% of the total archaeal sequences of rumen origin, and several taxa do not have a single cultured representative. Most of the isolates are members of the family Methanobacteriaceae. Compared to other anaerobic habitats where >100 species of methanogens of 28 genera have been isolated, the diversity and species richness of ruminal methanogens are quite low, reflecting the highly selective ruminal environment for methanogens. In addition, sequenced ruminal 16S rRNA gene clones shared >95% sequence similarity with that of Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii and Methanosphaera stadtmanae, indicating that these species may be common ruminal methanogens.

[0087] Much of the ruminal methanogen diversity was characterized by 16S rRNA gene sequences. The RDP Release 11 (Update 3) contains 8,623 archaeal 16S rRNA gene sequences of rumen origin. These sequences were generated using the Sanger sequencing technology, which produces higher sequence accuracy than NGS technologies, in 96 separate studies including 48 unpublished studies. About 90% of these sequences were assigned to methanogens. These sequences were classified to 10 known genera, with Methanobrevibacter being represented by 63.2% of all the sequences followed by Methanosphaera (9.8%), Methanomicrobium (7.7%), and Methanobacterium (1.2%). The order Thermoplasmatales, which was previously referred to as the rumen cluster C (RCC) group, is represented by 7.4% of the total archaeal sequences.

[0088] CELL SURFACE PROTEINS OF METHANOGENS

[0089] At least one cell surface protein or a fragment thereof that is present on the cell surface of at least one methanogen may be effective in eliciting immune response, antibody production, and antibody-mediated neutralization of the growth of methanogens and / or production of CH4. The representative cell surface antigens of a methanogen, Methanobrevibacter ruminantium, are listed in Table 1. The representative cell surface antigens of various methanogens, and their nucleic acid sequences and amino acid sequences are provided in the US Application No. 18 / 350526 (e.g., Table 2A, Table 2B, Table 3, Table 19, Table 20, and Table 21), US Application No. 63 / 359,978, or US Application No. 63 / 524,513, the entire contents of each of which are incorporated herein by reference in their entirety.

[0090] Table 1: Representative cell surface antigens of a methanogen, Methanobrevibacter ruminantium (Ml (DSM 1093))

[0091] GROWING HYDROGENOTROPHS (e.g., METHANOGENS)

[0092] HYDROGENOTROPHIC ORGANISMS

[0093] In certain aspects provided herein are compositions, systems, and methods of growing at least one hydrogenotrophic organism or hydrogenotroph (e.g., bacteria, e.g., methanogen), which are able to metabolize molecular hydrogen as a source of energy. Additional information regarding hydrogenotrophs or their growth conditions can be found in He et al. (2019) Chapter 3.09 Biogas, Comprehensive Biotechnology (Third Edition), pages 110-127 (ISBN 9780444640475; World Wide Web at doi.org / 10.1016 / B978-0-444-64046-8.00154-3); 4 and Kim and Whitman (2014) Methanogens, Encyclopedia of Food Microbiology (Second Edition), pages 602-606 (ISBN 9780123847331; World Wide Web at doi.org / 10.1016 / B978- 0-12-384730-0.00204-4), each of which is incorporated herein by reference. These composition, systems, and methods for growing hydrogenotrophs, e.g., methanogens, have broad utility included but not limited to production of antigenic material for vaccines as disclosed herein.

[0094] In some embodiments, the at least one hydrogenotroph comprises at least one methanogen selected from those known in the art or described herein.

[0095] In some embodiments, the at least one hydrogenotroph comprises a methanogen of a genus of Methanobrevibacter.

[0096] In some embodiments, the at least one hydrogenotroph comprises Methanobrevibacter ruminantium.

[0097] In some embodiments, the at least one hydrogenotroph comprises Methanobrevibacter gottschalkii.

[0098] The compositions and methods disclosed herein are suitable for coculturing hydrogenotrophs. In some embodiments, the at least one hydrogenotroph comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different hydrogenotrophs. In some embodiments, at least one of the hydrogenotrophs comprises Methanobrevibacter ruminantium. In some embodiments, at least one of the hydrogenotrophs comprises Methanobrevibacter gottschalkii. In some embodiments, the compositions, systems, and methods disclosed herein are suitable for coculturing Methanobrevibacter ruminatium and Methanobrevibacter gottschalkii, optionally with one or more additional hydrogenotrophs.

[0099] In some embodiments, the at least one hydrogenotroph comprises an organism selected from Table A. Table A lists organisms that contain the 5,10- methenyltetrahydromethanopterin hydrogenase gene, which catalyzes the addition of hydrogen with 5,10-methenyl-5,6,7,8-tetrahydromethanopterin to form 5,10- methylenetetrahydromethanopterin. Source: UniProt (World Wide Web at uniprot.org). However, a skilled artisan would understand that this list is not meant to be limiting and that any suitable hydrogenotroph can be used.

[0100] In some embodiments, the at least one hydrogenotroph comprises an organism selected from Table B. Table B lists organisms that have been reported to utilize H2 according to NCBI Taxonomy (World Wide Web at ncbi.nlm.nih.gov / taxonomy). However, a skilled artisan would understand that this list is not meant to be limiting and any suitable hydrogenotroph can be used. Table A: Exemplary hydrogenotrophs

[0101]

[0102] Table B: Additional exemplary hydrogenotrophs 4

[0103] SYSTEMS, COMPOSITIONS, AND METHODS FOR GROWING HYDROGENOTROPHS

[0104] Hydrogenotrophs are typically strict anaerobes - they are sensitive to oxygen and oxidants and thus cannot survive with exposure to oxygen or air. Sensitivity to oxygen and oxidants does vary between hydrogenotrophs. Some hydrogenotrophs may require addition of medium additives such as methanol, ethanol, or other non-hydrogenotrophic substrates. Importantly, traditional techniques for culturing hydrogenotrophs require highly explosive gas mixes consisting of 80% H2 and 20% CO2 under high pressure (e.g., a range of 180 kPa to 276 kPa) for optimal growth. Thus, growing hydrogenotrophs, especially on a large scale, are difficult, pose significant risks of explosion, and require high capital investment in systems that can both withstand high pressure and operate anaerobically.

[0105] In certain aspects, provided herein are systems, compositions, and methods for growing at least one hydrogenotroph that are different from those understood to be required for growing hydrogenotrophs. In some embodiments, a system comprises lower pressure (less than 180 kPa), which is less than the pressure commonly used in the art. In some embodiments, the H2 concentration in the growth chamber is lower than 80%, which is the concentration commonly used in the art. Such a lower H2 concentration in the growth chamber allows the use of a lower H2 concentration in a supply tank. For example, the H2 concentration in the supply tank may be non-flammable, e.g., less than 4% (as anything higher than 4% is typically flammable). The systems of the present disclosure have not been previously utilized for growing hydrogenotrophs and were considered to provide an inadequate condition for growing hydrogenotrophs, especially at commercial scale.

[0106] Systems

[0107] The systems of the present disclosure may comprise any container(s) or growth chamber(s) (e.g., flask, bottle, bioreactor, etc.) that may be adequate for growing hydrogenotrophs.

[0108] In certain aspects, provided herein are systems comprising at least one growth chamber. In certain aspects, provided herein are growth chambers of various sizes. In some embodiments, the growth chambers are at least 0.1 L in volume, at least 0.2 L in volume, at least 0.3 L in volume, at least 0.4 L in volume, at least 0.5 L in volume, at least 0.6 L in volume, at least 0.7 L in volume, at least 0.8 L in volume, at least 0.9 L in volume, at least 1 L in volume, at least 5 L in volume, at least 10 L in volume, at least 15 L in volume, at least 20 L in volume, at least 30 L in volume, at least 40 L in volume, at least 50 L in volume, at least 100 L in volume, at least 200 L in volume, at least 250 L in volume, at least 500 L in volume, at least 750 L in volume, at least 1000 L in volume, at least 1500 L in volume, at least 2000 L in volume, at least 2500 L in volume, at least 3000 L in volume, at least 3500 L in volume, at least 4000 L in volume, at least 5000 L in volume, at least 7500 L in volume, at least 10,000 L in volume, at least 15,000 L in volume, or at least 20,000 L in volume. In some embodiments, the bioreactors are about 1 L in volume, about 5 L in volume, about 10 L in volume, about 15 L in volume, about 20 L in volume, about 30 L in volume, about 40 L in volume, about 50 L in volume, about 100 L in volume, about 200 L in volume, about 250 L in volume, about 500 L in volume, about 750 L in volume, about 1000 L in volume, about 1500 L in volume, about 2000 L in volume, about 2500 L in volume, about 3000 L in volume, about 3500 L in volume, about 4000 L in volume, about 5000 L in volume, about 7500 L in volume, about 10,000 L in volume, about 15,000 L in volume, or about 20,000 L in volume.

[0109] In some embodiments, the at least one growth chamber comprises a flask, a bottle, or a suitable alternative. In some embodiments, the at least one growth chamber comprises a bioreactor, e.g., a chemostat, a turbidostat. A skilled artisan would understand how to select suitable alternatives.

[0110] Culturing methods

[0111] In certain aspects, provided herein are methods and / or compositions that facilitate the growth of at least one hydrogenotroph. The methods may comprise maintaining the temperature, substrate concentration, cell density, and pH of the growth media. The culturing may begin in a relatively small volume of growth media (e.g., 1 L) where the at least one hydrogenotroph is allowed to reach the log phase of growth. Such culture may be transferred to a larger volume of growth media (e.g., 20 L) for further growth to reach a larger biomass. Depending on the need of the final amount of biomass, such transfer may be repeated more than once.

[0112] In some embodiments, a culturing method of growing at least one hydrogenotroph comprises a batch process, in which no extra feeding (e.g., supply of a substrate for growth, e.g., mineral salt, sugar, etc.) of the hydrogenotrophs from beginning to end of the process.

[0113] In some embodiments, the culturing method comprises a fed-batch process, in which feeding with substrate and supplements can extend the duration of culture for higher cell densities or switch metabolism. The fed-batch process can comprise any suitable number of feeds, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, or 19 feeds 4 and / or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, or 20 feed, for example 2-20 feeds.

[0114] In some embodiments, the culturing method comprises a continuous process, where either the feed rate of a growth-limiting substance keeps cell density constant or cell density determines the feed rate of the substrate. Cell retention can offer an option of perfusion, which is a process that uses a method to keep cells in a bioreactor while continuously exchanging culture medium. Fresh medium replenishes nutrients and carbon sources, while cellular waste and medium depleted of nutrients are removed. The balanced nature of the feeding allows a steady state to be achieved which can last for days to months. This state is good for long-term production. In preferred embodiments, the continuous process comprises continuous harvesting of hydrogenotrophs (e.g., methanogens) through a liquid output while replacing new medium / substrate at the same rate. In some such embodiments, volume of the reactor may stay within a defined range.

[0115] Pressure & anaerobic conditions

[0116] Anaerobic conditions may be established and / or maintained by inflow of anaerobic gas(es). In a system comprising a growth medium, gas may be introduced into the headspace and / or sparged through the growth medium. In some embodiments, a system comprising a growth medium (a) is filled with a gas mixture in the headspace; and (b) the headspace gas is sparged through the medium.

[0117] In some embodiments where the system comprises at least one methanogen, CPU is produced as methanogens grow in number and utilize H2 / CO2. In some such embodiments, the headspace may be refreshed to provide inflow of new H2 / CO2 to support growth. In some embodiments, H2 / CO2 is supplied in batches periodically during culturing. In other embodiments, H2 / CO2 is supplied continuously during culturing. In other embodiments, H2 / CO2 is supplied to maintain a desired concentration in the growth vessel, for example using one or more sensors that measures one or more substrate or product of methanogenesis (e.g., H2, CO2, CH4, bicarbonate, etc.) or a functional measure thereof (e.g., turbidity, OD, absorbance, fluorescence, transmittance), transmitting that information to a controller, and then adjusting the flow of H2 / CO2 using an actuator (e.g., a valve).

[0118] In some embodiments, the system may comprise at least one auxiliary instrument that may facilitate the growth of at least one hydrogenotroph. Auxiliary instruments include but are not limited to analytical instruments (sensors, meters, detection devices), control instruments, and actuators. Exemplary auxiliary instruments are described herein and listed in Table C.

[0119] In some embodiments, the system may comprise at least one auxiliary (e.g., analytical) instrument that determines various parameters in the growth chamber (e.g., cell density, pH, level of any type of gas (e.g., CH4, CO2, H2, etc.). The auxiliary instruments may include, e.g., a pH meter, a spectrophotometer, a turbimeter, and / or an instrument analyzing the gas content. For example, an auxiliary instrument may determine the gas content in the system (e.g., the level of CH4, CO2, H2, or any other gas that is emitted or consumed by a particular hydrogenotroph). In some embodiments, a sampling valve may be connected to an auxiliary instrument to directly determine the gas composition in the headspace.

[0120] In some embodiments, the systems of the present disclosure further comprise at least one auxiliary (e.g., control or actuator) instrument, which triggers inflow of or controls the flow rate of (a) fresh medium, (b) any growth-limiting substance (e.g., nutrient(s) or additive(s)), (c) additional gas, (d) venting, or (e) any combination of two or more of (a)-(e). In some embodiments, the at least one auxiliary instrument alters the inflow or the flow rate of (a) fresh medium, (b) any growth-limiting substance (e.g., nutrient(s) or additive(s)), (c) additional gas, (d) venting, or (e) any combination of two or more of (a)-(e) based on the parameter(s) determined by the auxiliary instrument.

[0121] In some embodiments, the system comprises an auxostat, a culturing system in which while in operation, uses feedback from a measurement (e.g., cell density, pH, level of any type of gas (e.g., CH4, CO2, H2, etc.)) taken on the growth chamber to control the flow rate of fresh medium, any growth- limiting substance (e.g., nutrient(s) or additive(s)), and / or at least one type of gas that enter the growth chamber, thereby maintaining the measurement at a constant.

[0122] In some embodiments, the system comprises a turbidostat, which has feedback between the turbidity of the culture vessel and the dilution rate. A turbidostat dynamically adjusts the flow rate (and therefore the dilution rate) to make the turbidity constant.

[0123] In some embodiments, the system comprises a chemostat, a culturing system in which fresh medium, any growth-limiting substance (e.g., nutrient(s)), and / or at least one type of gas are continuously added, while culture liquid containing left over nutrients, metabolic end products and hydrogenotrophs is continuously removed at the same rate to keep the culture volume constant.

[0124] In some embodiments, the system may comprise continuous inflow of at least one type of gas (e.g., CH4, CO2, H2, or any other gas that is consumed by a particular 4 hydrogenotroph), preferably at a constant rate. In some embodiments, the continuous inflow is independent of any one of the parameters determined by an auxiliary instrument.

[0125] Inflow gas may first be passed through an apparatus configured to reduced and / or scavenge any trace oxidants (e.g., oxygen) in the inflow gas, e.g., catalytic converter, a palladium catalyst, or the like.

[0126] In certain aspects, the culturing methods of the present disclosure comprise incubating at least one hydrogenotroph under anaerobic atmosphere.

[0127] In some embodiments, the culturing method comprises incubating at least one hydrogenotroph under anaerobic atmosphere comprising H2.

[0128] In some embodiments, the anaerobic atmosphere comprises at least about, no more than about, less than about, or about 0.1%, 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%, 25%,

[0129] 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

[0130] 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

[0131] 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

[0132] 74%, 75%, 76%, 77%, 78%, 79%, or 80% H2. In preferred embodiments, the anaerobic atmosphere comprises no more than about 4% H2, which is non-flammable and nonexplosive.

[0133] In some embodiments, the anaerobic atmosphere comprises about 0.5%-79%, preferably about 0.5-50%, more preferably about 0.5-20%, even more preferably about 0.5- 4%, still more preferably about 1-4%, yet still more preferably about 2-4% H2.

[0134] In some embodiments, the culturing method comprises incubating at least one hydrogenotroph under anaerobic atmosphere comprising CO2.

[0135] In some embodiments, the anaerobic atmosphere comprises at least about, no more than about, less than about, or about 0.1%, 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%, 25%,

[0136] 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

[0137] 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

[0138] 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,

[0139] 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

[0140] 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% CO2.

[0141] In preferred embodiments, the anaerobic atmosphere comprises at least about 1 part of CO2 for every 4 parts of H2. In some embodiments, it is preferable that the CO2 concentration 4 is non-limiting, for example to ensure maximal utilization of the H2. Thus, some preferred embodiments have an anaerobic atmosphere comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts of CO2 for every 4 parts of H2.

[0142] In some embodiments, the culturing method comprises incubating at least one hydrogenotroph under anaerobic atmosphere comprising inert gas. Any inert gas can be used. An example of a cost-effective inert gas is N2. Accordingly, in some embodiments, the inert gas is N2.

[0143] In some embodiments, the anaerobic atmosphere comprises at least about, no more than about, less than about, or about 0.1%, 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%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% inert gas.

[0144] In some embodiments, the anaerobic atmosphere comprises at least about, no more than about, less than about, or about 0.1%, 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%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% N2.

[0145] Thus, a gas composition of 0.5-4% H2, 1-99.5% CO2, and the remainder balanced with an inert gas such as N2 is preferred. An exemplary gas composition is 1-4% H2, 1-25% CO2, and 71-98% N2. Another exemplary cost-effective gas composition is about 4% H2, about 10% CO2, and about 86% N2.

[0146] In some embodiments, the culturing method comprises incubating at least one hydrogenotroph under anaerobic atmosphere comprising a gaseous mixtures comprising H2, CO2, and inert gas.

[0147] In some embodiments, a combination of two or more anaerobic gases can be premixed as a blend (e.g., using a mixing device upstream of the system) before being 4 supplied into the growth chamber. In some embodiments, each gas can be supplied separately to the growth chamber independently.

[0148] In some embodiments, an anaerobic gaseous mixture is continuously added to the growth chamber (e.g., flask, bioreactor) during culturing. In some embodiments, the continuously added anaerobic gaseous mixture is added at a gas flow rate of at least about, no more than about, less than about, or about 0.01 vvm, 0.02 vvm, 0.03 vvm, 0.04 vvm, 0.05 vvm, 0.06 vvm, 0.07 vvm, 0.08 vvm, 0.09 vvm, 0.1 vvm, 0.11 vvm, 0.12 vvm, 0.13 vvm, 0.14 vvm, 0.15 vvm, 0.16 vvm, 0.17 vvm, 0.18 vvm, 0.19 vvm, 0.2 vvm, 0.21 vvm, 0.22 vvm, 0.23 vvm, 0.24 vvm, 0.25 vvm, 0.26 vvm, 0.27 vvm, 0.28 vvm, 0.29 vvm, 0.3 vvm, 0.31 vvm, 0.32 vvm, 0.33 vvm, 0.34 vvm, 0.35 vvm, 0.36 vvm, 0.37 vvm, 0.38 vvm, 0.39 vvm, 0.4 vvm, 0.41 vvm, 0.42 vvm, 0.43 vvm, 0.44 vvm, 0.45 vvm, 0.46 vvm, 0.47 vvm, 0.48 vvm, 0.49 vvm, 0.5 vvm, 0.51 vvm, 0.52 vvm, 0.53 vvm, 0.54 vvm, 0.55 vvm, 0.56 vvm, 0.57 vvm, 0.58 vvm, 0.59 vvm, 0.6 vvm, 0.61 vvm, 0.62 vvm, 0.63 vvm, 0.64 vvm, 0.65 vvm, 0.66 vvm, 0.67 vvm, 0.68 vvm, 0.69 vvm, 0.7 vvm, 0.71 vvm, 0.72 vvm, 0.73 vvm, 0.74 vvm, 0.75 vvm, 0.76 vvm, 0.77 vvm, 0.78 vvm, 0.79 vvm, 0.8 vvm, 0.81 vvm, 0.82 vvm, 0.83 vvm, 0.84 vvm, 0.85 vvm, 0.86 vvm, 0.87 vvm, 0.88 vvm, 0.89 vvm, 0.9 vvm, 0.91 vvm, 0.92 vvm, 0.93 vvm, 0.94 vvm, 0.95 vvm, 0.96 vvm, 0.97 vvm, 0.98 vvm, 0.99 vvm, 1 vvm, 2 vvm, 3 vvm, 4 vvm, 5 vvm, 6 vvm, 7 vvm, 8 vvm, 9 vvm, 10 vvm, 11 vvm, 12 vvm, 13 vvm, 14 vvm, 15 vvm, 16 vvm, 17 vvm, 18 vvm, 19 vvm, 20 vvm, 21 vvm, 22 vvm, 23 vvm, 24 vvm, 25 vvm, 26 vvm, 27 vvm, 28 vvm, 29 vvm, 30 vvm, 31 vvm, 32 vvm, 33 vvm, 34 vvm, 35 vvm, 36 vvm, 37 vvm, 38 vvm, 39 vvm, 40 vvm, 41 vvm, 42 vvm, 43 vvm, 44 vvm, 45 vvm, 46 vvm, 47 vvm, 48 vvm, 49 vvm, 50 vvm, 51 vvm, 52 vvm, 53 vvm, 54 vvm, 55 vvm, 56 vvm, 57 vvm, 58 vvm, 59 vvm, 60 vvm, 61 vvm, 62 vvm, 63 vvm, 64 vvm, 65 vvm, 66 vvm, 67 vvm, 68 vvm, 69 vvm, 70 vvm, 71 vvm, 72 vvm, 73 vvm, 74 vvm, 75 vvm, 76 vvm, 77 vvm, 78 vvm, 79 vvm, 80 vvm, 81 vvm, 82 vvm, 83 vvm, 84 vvm, 85 vvm, 86 vvm, 87 vvm, 88 vvm, 89 vvm, 90 vvm, 91 vvm, 92 vvm, 93 vvm, 94 vvm, 95 vvm, 96 vvm, 97 vvm, 98 vvm, 99 vvm, or 100 vvm. In some embodiments, the gas flow rate is about 0.01 to about 100 volume per volume per minute (vvm). In preferred embodiments, the gas flow rate is about 0.1 to about 10 vvm. In even more preferred embodiments, the gas flow rate is about 0.5 to about 3 vvm. In some embodiments, the gas flow rate is about 0.01 to about 0.1 vvm. In some embodiments the continuously added anaerobic gaseous mixture is added at a gas flow rate of about 0.02 vvm. In some embodiments, the continuously added anaerobic gaseous mixture comprises any one of the gases described above or mixtures thereof. An exemplary use of a continuously flowing gas apparatus to grow methanogens is 4 described in Bryant, et al. (1968) Hydrogen-oxidizing methane bacteria. Journal of Bacteriology, which is incorporated herein by reference in its entirety.

[0149] In certain aspects, the culturing methods of the present disclosure comprise incubating at least one hydrogenotroph under pressure.

[0150] In some embodiments, the system of growing at least one hydrogenotroph is under the pressure of at least about, no more than about, less than about, or about 100 kilopascal (kPa),

[0151] 101 kPa, 102 kPa, 103 kPa, 104 kPa, 105 kPa, 106 kPa, 107 kPa, 108 kPa, 109 kPa, 110 kPa,

[0152] 111 kPa, 112kPa, 113 kPa, 114 kPa, 115 kPa, 116 kPa, 117 kPa, 118 kPa, 119 kPa, 120 kPa, 121 kPa, 122 kPa, 123 kPa, 124 kPa, 125 kPa, 126 kPa, 127 kPa, 128 kPa, 129 kPa, 130 kPa,

[0153] 131 kPa, 132 kPa, 133 kPa, 134 kPa, 135 kPa, 136 kPa, 137 kPa, 138 kPa, 139 kPa, 140 kPa,

[0154] 141 kPa, 142 kPa, 143 kPa, 144 kPa, 145 kPa, 146 kPa, 147 kPa, 148 kPa, 149 kPa, 150 kPa,

[0155] 151kPa, 152 kPa, 153 kPa, 154 kPa, 155 kPa, 156 kPa, 157 kPa, 158 kPa, 159 kPa, 160 kPa,

[0156] 161kPa, 162 kPa, 163 kPa, 164 kPa, 165 kPa, 166 kPa, 167 kPa, 168 kPa, 169 kPa, 170 kPa,

[0157] 171kPa, 172 kPa, 173 kPa, 174 kPa, 175 kPa, 176 kPa, 177 kPa, 178 kPa, 179 kPa, or 180 kPa.

[0158] In some embodiments, the pressure within the growth chamber (e.g., flask, bioreactor) is no more than about 180 kPa. In some embodiments, the pressure is at least 100 kPa but no more than 180 kPa. In some embodiments, the pressure is about 1 atm (101.325 kPa). In preferred embodiments, the pressure is about 105 kPa, about 110 kPa, about 120 kPa, or about 125 kPa.

[0159] It may be beneficial to culture the hydrogenotroph under pressure to prevent trace introductions of air into the system through minor leaks given the anaerobic nature of methanogens. Thus, a pressure of at least about 1 atm to about 120 kPa, preferably about 1 atm to about 115 kPa, more preferably about 1 atm to about 110 kPa is beneficial.

[0160] In some embodiments, inoculum can be prepared in flasks or in smaller bioreactors where growth is monitored. For example, the inoculum size may be between about 0.1% v / v and about 5% v / v of the total growth chamber (e.g., bioreactor) volume. In some embodiments, the inoculum is about 0.1- about 3% v / v, about 0.1- about 1% v / v, about 0.1- about 0.5% v / v, or about 0.5- about 1% v / v of the total final culture volume. In some embodiments, the inoculum is about 0.1% v / v, about 0.2% v / v, about 0.3% v / v, about 0.4%, v / v, about 0.5% v / v, about 0.6% v / v, about 0.7% v / v, about 0.8% v / v, about 0.9% v / v, about 1% v / v, about 1.5% v / v, about 2% v / v, about 2.5% v / v, about 3% v / v, about 4%, v / v, or about 5% v / v of the total final culture volume. In some embodiments, the inoculum size of the hydrogenotrophs may be between approximately 0.5 and 3% of the total final culture volume. 4

[0161] In some embodiments, the inoculum size is at least about, no more than about, less than about, or about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the final culture volume. In some embodiments, the inoculum size is between about 0.001% to about 50% of the final volume. In preferred embodiments, the inoculum size is between about 0.1% to about 20% of the final volume. In even more preferred embodiments, the inoculum size is between about 1% to about 10% of the final volume.

[0162] In some embodiments, the culturing method comprises incubating at least one hydrogenotroph at a temperature of at least about, no more than about, less than about, or about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, or 65°C. In some embodiments, the temperature is between about 25 °C and about 65 °C. In some embodiments, the temperature is between about 25 °C and about 45 °C. In preferred embodiments, the temperature is between about 35 °C to about 50°C. In even more preferred embodiments, the temperature is between about 37°C to about 40°C. In yet other preferred embodiments, the temperature is at about 38 °C.

[0163] Table C: Exemplary auxiliary instrument

[0164] This Table provides exemplary auxiliary instruments (e.g., analytical, control, actuator) that may facilitate the growth of at least one hydrogenotroph. The at least one auxiliary instrument may be coupled with or be part of the systems of the present disclosure. The systems of the present disclosure may comprise any one or more of the instruments or devices described herein.

[0165] 1. Temperature control: In some embodiments, the temperature control system detects and maintains the temperature between about 25°C and about 65°C. In some embodiments, the temperature control system detects and maintains the temperature between about 25 °C and about 45°C. In preferred embodiments, the temperature is between about 35°C to about 50°C. In even more preferred embodiments, the temperature is between about 37°C to about 40°C. In yet other preferred embodiments, the temperature control system detects and 4 maintains the temperature at about 38 °C, which is the suitable temperature for growing many methanogens.

[0166] 2. Gas flow control: Gas flow sensor combined with one or more gas valves to control the input and / or output of gas into the growth chamber, which (a) controls flow rate of the gas, and / or (b) controls timing of gas input and / or output. A gas flow control may optionally further comprise or be coupled with gas supplies and / or a gas mixer that combines gases from one or more cylinders, wherein the flow rates determine the ratios of gases to be combined to achieve the desired gas mixture into the growth chamber. An exemplary use of a continuously flowing gas apparatus to grow methanogens is described in Bryant, et al. (1968) Hydrogen-oxidizing methane bacteria. Journal of Bacteriology, which is incorporated herein by reference in its entirety.

[0167] 3. H2 / CO2 / CH4 sensors: These sensors monitor the level of H2 / CO2 / CH4 in growth chambers. The sensors may optionally be coupled with gas flow control and actuate gas input (of gas(es) that are required for growth, e.g., H2, CO2) and / or output of the emitted gas (e.g., CH4), ensuring that the culture have sufficient gaseous substrate to grow. These sensors may also optionally be coupled with the gas supplies and / or a gas mixer (e.g., an impeller).

[0168] 4. Spectrophotometer / turbimeter / fluorometer: An instrument to measures cell quantity / concentration in the growth chamber (e.g., turbidity or light absorbance). Certain hydrogenotrophs, e.g., methanogens, are fluorescent, thus a fluorometer may be used to detect the level of methanogens in the growth chamber. These instruments may be coupled with gas flow control, gas supplies, a gas mixer, or any combination thereof, to (a) increase the gas flow rate as the cell density increases; and / or (b) trigger gas inputs and / or outputs during a fed batch process at certain cell densities. These instruments may also be coupled with a shaker or a mixer to increase the mixing rate as the cell density increases.

[0169] 5. pH sensor: It is well known in the art that the pH of the media changes in the course of cell growth, which indirectly indicates the cell density. The pH deviations may trigger addition of one or more buffering agents (e.g., acid, base) and / or reducing agents to maintain the correct solubility of CO2 in the media. The pH sensor may be coupled with liquid flow control described below.

[0170] 6. Oxygen sensor and potentially sacrificial electrode(s) in line with the gas supply

[0171] 7. Liquid flow control: At least one liquid flow control may be coupled with the systems of the present disclosure. The liquid flow control may hold and actuate the inflow of any liquid necessary for growth of hydrogenotrophs. The liquid includes but is not limited to fresh media, any solution comprising any one or more of nutrients (e.g., carbon source, minerals, vitamins, additives (e.g., methanol)), buffering agents, reducing agents, and any other liquid known in the art that aids growth of hydrogenotrophs.

[0172] 8. Vessel level control

[0173] 9. Others

[0174] Exemplary sensors

[0175] • pH meter: for example electrochemical, electrode, differential sensor, combination sensor

[0176] • O2 meter, liquid and / or gas phase: for example electrochemical, zirconia, titania, optical, dark, infrared, electro, ultrasonic, laser, paramagenetic, wideband, narrowband

[0177] • H2 meter, liquid and / or gas phase: for example FID, thermal conductivity, catalytic combustion, catalytic, electrochemical, thin film, thick film, microelectromechanical, chemochromic, diode, semiconductor, metal oxide semiconductor (MOS), non-resistive semiconductor, palladium based, optical, fiber Bragg grating (FBG) coated with a palladium layer, micromirror, tapered fibre coated with palladium, conductive, metallic Ea-Mg2-Ni, clark-type, any one of the above with or without Siloxane

[0178] • CO2 sensor, liquid and / or gas phase: for example catalytic, non-dispersive infrared, electrochemical, semiconductor, metal oxide semiconductor, optical, catalytic combustion

[0179] • CH4 meter, liquid and / or gas phase: for example laser, infrared, FID, catalytic, catalytic bead, optical, calorimetric, pyroelectric, semiconducting oxide, electrochemical, pellistor, non-dispersive infrared

[0180] • Acetate sensor: for example bioelectrochemical, biosensor, electrochemical

[0181] • Volatile fatty acid sensors: for example Isovaleric, Isobutyric, Valeric, 2- methylbutyric, 2-methylvaleric

[0182] • Metabolite sensors: for example metabolic precursors, products and / or intermediates of methanogenesis

[0183] • Cell density monitoring: for example absorbance, permittivity, in situ, biomass capacitance, optical

[0184] • Fluorometer: for example filter fluorometer, spectrofluorometer

[0185] • Temperature sensor: for example thermistor, thermocouple, semiconductor, RTD, contact temperature sensor, thermometer, infrared, NTC thermistor • Pressure sensor, gas and / or liquid phase: for example strain gauge, piezoelectric, capacitive, manometers, vacuum pressure, bourdon tube, aneroid barometer, resistive, optical, MEMS, potentiometric, inductive, variable reluctance

[0186] • Foam sensor: for example conductivity switch, ultrasonic gas switch, ultrasonic gas switch, RF admittance, ultrasonic, infrared, camera, contact sensors, contactless sensors

[0187] • H2S sensor, liquid and / or gas phase: for example electrochemical, metal oxide semiconductor, semiconductor, solid electrolyte

[0188] • Scale: for example spring scale, hydraulic, pneumatic, balance, torsion balance, roberval balance, strain gauge, beam balance, digital

[0189] • Gas composition analytical system

[0190] • Flow meter, liquid and / or gas phase: for example differential pressure, rotameter, variable area, orifice, venturi, pitot tube, positive displacement, reciprocating piston, oval-gear, nutating-disk, rotary-vane, volumetric, ultrasonic, mass flow, velocity, vortex, electromagnetic, turbine, paddlewheel, open channel, flow nozzle, sonic nozzle, thermal, target type, magnetic induction, scale

[0191] • Camera

[0192] • Eevel sensor: for example ultrasonic, float level, rotating paddle, diaphragm switch, radar, microwave, displacer switch, capacitance, optical, float switch, Ultrasound, baser profiler, Inductive displacement, tuning fork, hydrostatic level transmitter, magnetic, optical, buoy level, level switch, scale

[0193] • Redox sensor

[0194] • Contamination sensor (e.g., spores, biosensor) Exemplary actuators

[0195] • Motor

[0196] • Magnetic stirrer

[0197] • pH regulation (e.g., with acid-base reservoir and pump)

[0198] • Regulated recirculation of headspace (valve)

[0199] • Unregulated recirculation of headspace (valve)

[0200] • Recirculation of liquid

[0201] • Pressure regulator upstream of tank • Pressure regulator downstream of tank

[0202] • Condenser

[0203] • Cooling water

[0204] • Heating water

[0205] • Cooling jacket

[0206] • Heating jacket

[0207] • Water bath

[0208] • Hot plate

[0209] • Burner at outlet

[0210] • Overpressure release valve

[0211] • Flow controller, liquid phase

[0212] • Flow controller, gas phase

[0213] • Automatic sampling port(s), can be triggered by time or sensor readout

[0214] • Automated nutrient inlet (pump)

[0215] • Automated antifoam addition (pump), can be triggered by foam sensor

[0216] • Continuous / periodic culture removal (pump or valve), optionally triggered by sensor

[0217] • Continuous / periodic liquid feed (pump or valve), optionally triggered by sensor

[0218] Exemplary hardware

[0219] • Vessel type: stainless steel, glass, plastic, polycarbonate, others

[0220] • Various culture vessel (growth chamber) geometries / shapes: round, conical, cylindrical, spherical, long and horizontal culture system (to maximize gas exchange), others

[0221] • Impeller

[0222] • Stirring paddles

[0223] • Baffles

[0224] • Sparger

[0225] • Stainless steel gas diffuser

[0226] • Filtration system

[0227] • Humidifying tank upstream

[0228] • Gas mixer upstream of tank 4

[0229] • Multiple gas inlets to tank

[0230] • Single gas inlet to tank

[0231] • Sampling port(s), manual and / or automatic

[0232] • Antifoam addition, manual and / or automatic

[0233] • Injection port(s): septum, tubing with clamp / valve, triclamp

[0234] • Harvest port

[0235] • Sampling port, head space

[0236] • Sampling port, dip tube: quick connects, tubing, triclamp

[0237] • Viewing port

[0238] • Antifoam addition, manual and / or automatic

[0239] • Carbon filter

[0240] • Exhaust system

[0241] • Incubated room / environment

[0242] Other

[0243] • Electrolytic converter

[0244] • CH4 converter

[0245] • CH4 recovery

[0246] • H2S removal

[0247] • Three phase system: e.g., gas, liquid, oil

[0248] • CH4 conversion system (converts CH4 to CO2 and H2)

[0249] In some embodiments, a gas outlet comprises a CH4 conversion system that converts CH4 to CO2 and H2, which allows recycling of gas. An exemplary use of this system comprises a burner or catalyst that oxidizes the CH4 with a suitable amount of air, the CO2 and H2 is then reintroduced into the culturing system.

[0250] In some embodiments, systems of the present disclosure comprises a H2S scrubber, such as a catalytic scrubber, water scrubber, chemical scrubber, biological scrubber, biochemical scrubber and / or a carbon filter.

[0251] In some embodiments, the system of the present disclosure comprise an apparatus configured to oxidize any effluent CH4 and / or capture produced CH4 or oxidized derivatives thereof.

[0252] In some embodiments, the system may further comprise an apparatus configured to electrolyze water as a source of H2. It would be understand that an H2 / O2 separation system would be required as well to provide an anaerobic source of H2 to the culture. 4

[0253] In some embodiments, the system may further comprise an oxygen impermeable membrane.

[0254] In some embodiments, the system may further comprise an apparatus configured to collect and optionally liquidize collected CH4, thereby allowing transfer to suitable sites capable of using the collected CH4.

[0255] Media

[0256] Any culturing volume may be suitable for the culturing methods of the present disclosure. In some embodiments, the culturing volume or the amount of media is at least 0.01 L, at least 0.05 L, at least 0.1 L in volume, at least 0.2 L in volume, at least 0.3 L in volume, at least 0.4 L in volume, at least 0.5 L in volume, at least 0.6 L in volume, at least 0.7 L in volume, at least 0.8 L in volume, at least 0.9 L in volume, at least 1 L in volume, at least 5 L in volume, at least 10 L in volume, at least 15 L in volume, at least 20 L in volume, at least 30 L in volume, at least 40 L in volume, at least 50 L in volume, at least 100 L in volume, at least 200 L in volume, at least 250 L in volume, at least 500 L in volume, at least 750 L in volume, at least 1000 L in volume, at least 1500 L in volume, at least 2000 L in volume, at least 2500 L in volume, at least 3000 L in volume, at least 3500 L in volume, at least 4000 L in volume, at least 5000 L in volume, at least 7500 L in volume, at least 10,000 L in volume, at least 15,000 L in volume, or at least 20,000 L in volume.

[0257] In some embodiments, the culturing volume or the amount of media is about 0.01 L in volume, about 0.05 L in volume, about 0.1 L in volume, about 0.5 L in volume, about 1 L in volume, about 5 L in volume, about 10 L in volume, about 15 L in volume, about 20 L in volume, about 30 L in volume, about 40 L in volume, about 50 L in volume, about 100 L in volume, about 200 L in volume, about 250 L in volume, about 500 L in volume, about 750 L in volume, about 1000 L in volume, about 1500 L in volume, about 2000 L in volume, about 2500 L in volume, about 3000 L in volume, about 3500 L in volume, about 4000 L in volume, about 5000 L in volume, about 7500 L in volume, about 10,000 L in volume, about 15,000 L in volume, or about 20,000 L in volume.

[0258] In some embodiments, the culturing volume or the amount of media is about 100 L in volume, about 200 L in volume, about 300 L in volume, about 400 L in volume, about 500 L in volume, about 600 L in volume, about 700 L in volume, about 800 L in volume, about 900 L in volume, or about 1000 L in volume.

[0259] The systems, compositions, and / or methods of the present disclosure may use any media that are known in the art to facilitate growth of hydrogenotrophs may be used. In some embodiments, the media comprises BY medium (Joblin K.N. 2005. Methanogenic archaea. Methods in Gut Microbila Ecology for Ruminants, which is incorporated herein by reference), BCYT media, SAB media (Khelaifia et al. (2013) PloS One, 8(4):e61563, which is incorporated herein by reference), and / or DSMZ media (e.g., DSMZ 119 media, DSMZ 322 media, DSMZ 334c media). In some embodiments, the systems, compositions, and / or methods of the present disclosure may use any media that has been developed and disclosed herein (e.g., the media in Tables D and E, or the media in working Examples).

[0260] Table D: An Exemplary Media Composition

[0261] * In the preferred range of concentrations above, endpoints are included. Furthermore, the values of the preferred range of concentrations above can assume any specific value or subrange within the stated ranges, to the tenth of the unit of the lower limit of the range.

[0262] It should be appreciated that any suitable hydrated form of a listed component can be used, and the concentration should be adjusted to account for differences in the molecular weight of the alternative. Additionally, the rumen fluid may be dried or dehydrated. A skilled artisan would understand how to calculate the amount of powdered rumen fluid to add based on weight differences between the liquid and powdered preparations. Dried rumen fluid can be advantageous for consistent and reproducible productions of cell mass as it can be harvested, combined, checked for any transmissible elements, e.g., viruses, sterilized, and stored long term under suitable conditions. Thus, this disclosure provides compositions comprising dried, sterilized rumen fluid suitable for mass production of methanogens.

[0263] Table E: An Exemplary Media Composition values of the preferred range of concentrations above can assume any specific value or subrange within the stated ranges, to the tenth of the unit of the lower limit of the range.

[0264] It should be appreciated that any suitable hydrated form of a listed component can be used, and the concentration should be adjusted to account for differences in the molecular weight of the alternative.

[0265] In some embodiments, any culture medium that facilitates the growth of a hydrogenotroph may be used for the methods of the present disclosure. In some embodiments, a culture medium comprises at least one component selected from those listed in Table D or Table E. In some embodiments, a culture medium comprises any combination of two or more components listed in Table D or Table E. In some embodiments, a culture medium comprises all components listed in Table D, but without at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 components. In some embodiments, a culture medium comprises all components listed in Table E, but without at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 components. In some embodiments, the culture medium comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 components listed in Table D. In some embodiments, the culture medium comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 components listed in Table E. In preferred embodiments, a culture medium comprises all components listed in Table D. In preferred embodiments, a culture medium comprises all components listed in Table E. In preferred embodiments, where a culture medium comprises at least one component from Table D or Table E, the at least one component is present in the concentration listed in Table D or Table E; or in a range of concentrations, or any range in between, or any value in between, listed in Table D or Table E.

[0266] In some embodiments, a culture medium comprises one or more positively charged cations. In some embodiments, the positively charged cation is a transition metal, alkali metal, alkaline Earth metal, lanthanide, basic metal, semimetal, or nonmetal. Any suitable alkali metal could be used, for example lithium, cesium, rubidium, preferably potassium, more preferably sodium. Any suitable alkaline Earth metal could be used, for example beryllium, barium, strontium, preferably magnesium, more preferably calcium. Any suitable transition metal could be used, for example lead, palladium, gold, zirconium, chromium, rhenium, vanadium, or platinum, more preferably copper, cobalt, iron, manganese, nickel, or zinc. Any suitable lanthanide could be used, for example lanthanum, cerium. Any suitable semimetal could be used, such as antimony. Any suitable basic metal could be used, such as gallium. Alternatively or in combination, any suitable nonmetal can be used, for example H2. In some embodiments, the cation is polyatomic, for example dihydrogen, guanidium, more preferably ammonium.

[0267] Additionally or alternatively, the culture medium also comprises one or more negatively charged anions. In some embodiments, the anion is a halogen or a nonmetal. Any suitable anion can be used, for example sulfur or chloride. In some embodiments, the anion is polyatomic, for example arsenate or iodate, preferably permanganate, more preferably nitrate, sulfate, phosphate, acetate, formate, carbonate, selenite, tungstate, borate, molybdate, or selenium trioxide.

[0268] These cations and anions can be added to the medium in the form of one or more salts. Any suitable combination of cation and anion species can comprise the salt, such as those listed in Table D or E. Any suitable concentration range can be used, such as those listed in Table D or E for example. The concentration of these salts can be determined based on the measured quantity added during media preparation.

[0269] In some embodiments, a culture medium comprises cofactors. In preferred embodiments, a culture medium comprises coenzyme M. Other cofactors include but are not limited to nicotineamideadenine dinucleotide (NAD), nicotineamide adenine dinucelotide phosphate (NADP), flavin adenine dinucleotide (FAD), and coenzyme A (CoA). In some embodiments, a culture medium comprises an alcohol. In some embodiments, a culture medium comprises an alcohol selected from methanol, ethanol, propanol, butanol, and isopropanol.

[0270] In some embodiments, a culture medium comprises one or more sugars. In some embodiments, a culture medium comprises monosaccharide, disaccharide, polysaccharide, or any combination of two or more thereof.

[0271] In some embodiments, a culture medium comprises at least one monosaccharide. In some embodiments, a culture medium comprises at least one monosaccharide selected from glucose, mannose, fructose, ribose, galactose, xylose, and arabinose.

[0272] In some embodiments, a culture medium comprises at least one disaccharide. In some embodiments, a culture medium comprises at least one disaccharide selected from lactose, sucrose, and maltose.

[0273] In some embodiments, a culture medium comprises at least one polysaccharide. In some embodiments, a culture medium comprises at least one polysaccharide selected from starch, glucan, cellulose, dextran, and xanthan.

[0274] In some embodiments, it is preferable that the sugars are reducing sugars, such as glucose, fructose, galactose, mannose, ribose, xylose, arabinose, lactose, maltose, cellobiose, a suitable alternative, and / or a combination thereof. The reducing sugars may further act as scavengers of trace oxidizing agents, thereby improving hydrogenotroph culturing.

[0275] In some embodiments, a culture medium comprises one or more components which are themselves a combination of chemicals. In some embodiments, a culture medium comprises peptone, tryptone, casamino acids, trypticase, or any combination of two or more thereof. In some embodiments, the culture medium comprise yeast and / or yeast extract.

[0276] In some embodiments, a culture medium comprises at least one amino acid (natural or unnatural). Over 500 natural amino acids exist in nature. In some embodiments, a culture medium comprises at least one amino acid selected from 22 amino acids found in proteins (e.g., alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine). In preferred embodiments, a culture medium comprises at least one amino acid selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, selenocysteine, glycine, proline, alanine, valine, methionine, phenylalanine, tyrosine, tryptophan, citrulline, ornithine, pyroglutamate, 4-aminobutyrate, and P-alanine. In some embodiments, a culture medium comprises at least one organic acid. In some embodiments, a culture medium comprises at least one organic acid selected from 3- hydroxybutyrate, 3-HPA, 3-phenylpropionate, 4-hydroxybutyrate, acetate, acetoacetate, benzoate, butyrate, ferulate, formate, fumarate, isobutyrate, isovalerate, lactate, nicotinate, phenylacetate, propionate, succinate, valerate, 2-hydroxyvalerate, 2-hydroxyisovalerate, 3- hydroxyphenylacetate, citrate, pyruvate, formate, benzoate, and fumarate.

[0277] In some embodiments, a culture medium comprises at least one vitamin. In some embodiments, a culture medium comprises at least one vitamin selected from vitamin A, niacin, choline, calciferol, vitamin E, vitamin K, pyridoxine, ascorbic acid, pantothenate, lipoic acid, nicotinamide, 4-aminobenzoic acid, pyridoxal, riboflavin, thiamine, biotin, folic acid, cyanocobalamin.

[0278] In some embodiments, a culture medium comprises at least one mineral. In some embodiments, a culture medium comprises at least one mineral selected from fluoride and iodine.

[0279] In some embodiments, a culture medium comprises at least one material of animal origin. In some embodiments, a culture medium comprises at least one material of animal origin is selected from rumen fluid, fetal bovine serum, bovine serum albumin, blood, bone, and fat. In some embodiments, the material of animal origin, for example rumen fluid, is delivered to the media preparation as a liquid, or in a frozen state. In other embodiments, the material of animal origin, for example rumen fluid, may be dried to a solid, and in some cases powdered. Where possible, the dried form may offer storage advantages over liquid. In some embodiments, the material of animal origin is sterilized and free of detectable amount of transmissible elements, e.g., viruses. This can be especially important to prevent disease transmission when producing antigenic material for a similar subject to that of subject from which the material of animal origin was isolated.

[0280] Thus, provided herein are powdered rumen fluid compositions prepared from at least about 1, 2, 3, 4, 5, 10, 20, 25, 50, or 100 different animals. In certain embodiments, the powdered rumen fluid is free of detectable transmissible disease elements, e.g., viruses. Any suitable method can be used to test for the transmissible disease elements, for example qPCR.

[0281] In some embodiments, a culture medium comprises at least one metabolite found in a ruminant’s rumen. In some embodiments, a culture medium comprises at least one metabolite found in a bovine rumen. In some embodiments, a culture medium comprises at least one metabolite selected from cadaverine, nicotinate, acetone (or Propanone), 4-Hydroxy-3- methoxymandelate, imidazole, 1,3-DHA, cadaverine, caffeine, choline, dimethylamine, ethanolamine, glycerol, hypoxanthine, methylamine, N-nitrosodimethylamine, NADMA, PAG, thymine, uracil, xanthine, and endotoxin.

[0282] In some embodiments, a culture medium comprises at least one buffer. Any buffer known in the art can be used. In some embodiments, a culture medium comprises at least one buffer selected from phosphate buffer, HEPES, MOPS, MES, BES, MOPSO, ACES, TAPS, Bicine, and Tris.

[0283] In some embodiments, a culture medium comprises at least one trace element. In some embodiments, a culture medium comprises at least one trace element selected from copper, beryllium, boron, aluminum, thallium, zinc, chromium, molybdenum, cobalt, nickel, selenium, fluorine, iron, iodine, manganese, magnesium, rubidium, strontium, molybdenum, lead, arsenic, vanadium, and cadmium.

[0284] In some embodiments, a culture medium comprises at least one biogenic amine. In some embodiments, a culture medium comprises at least one biogenic amine selected from dimethylarginine, acetylornithine, carnosine, histamine, kynurenine, methioninesulfoxide, phenylethylamine, sarcosine, taurine, serotonin, and putrescine.

[0285] In some embodiments, a culture medium comprises at least one acylcarnitine. In some embodiments, a culture medium comprises at least one acylcarnitine selected from tetradecenoylcarnitine, tetradecadienylcarnitine, hydroxytetradecadienylcarnitine, hexadecanoylcarnitine, hydroxyhexadecanoylcarnitine, hexadecadienylcarnitine, hydroxyoctadecanoylcarnitine, octadecadienylcarnitine, propionylcarnitine, hydroxybutyrylcarnitine, hydroxypropionylcarnitine, butenylcarnitine, valerylcarnitinec, methylglutarylcarnitine, tiglylcarnitine, glutaconylcarnitine, hexenoylcarnitine, and pimelylcarnitine .

[0286] In some embodiments, a culture medium comprises at least one fatty acid. In some embodiments, a culture medium comprises at least one fatty acid selected from C6:0, C8:0, C10:0, Cll:0, C12:0, C13:0, C14:0, C14:ln5, C15:0, C16:0, C16:ln7, C17:0, C18:0, C18:ln9, C18:2n6, C18:3n6, C20:0, C20:ln9, C20:2n6, C20:4n6, C22:0, C22:ln9, C22:2n6, C24:ln9, FFA16:1(9), FFA18:l(isomer), FFA18: 1(9), FFA18:2(9,11), and FFA18:3(9,12,15).

[0287] In some embodiments, a culture medium comprises at least one cholesterol ester. In some embodiments, a culture medium comprises at least one cholesterol ester selected from CE12:0, CE14:0, CE15:0, CE16:0, CE16:1, CE18:0, CE18:l(isomer), CE18:2(9,11), CE20:0, CE22:0, CE22:1(13), and CE24:0. In some embodiments, a culture medium comprises at least one lysophosphatidylcholine. In some embodiments, a culture medium comprises at least one lysophosphatidylcholine selected from lysoPC a C16:l, lysoPC a C17:0, lysoPC a C18:0, lysoPC a C18:l, lysoPC a C18:2, lysoPC a C20:4, lysoPC a C26:0, and lysoPC a C16:0.

[0288] In some embodiments, a culture medium comprises at least one phosphatidylcholine. In some embodiments, a culture medium comprises at least one phosphatidylcholine selected from PC aa C28:l, PC aa C30:2, PC aa C32:0, PC aa C32:2, PC aa C32:3, PC aa C34:l, PC aa C34:2, PC aa C34:3, PC aa C34:4, PC aa C36:l, PC aa C36:2, PC aa C36:3, PC aa C36:4, PC aa C36:5, PC aa C36:6, PC aa C38:l, PC aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:3, PC aa C40:6, PC aa C42:l, PC aa C42:2, PC aa C42:4, PC aa C42:5, PC ae C30:2, PC ae C32:l, PC ae C32:2, PC ae C34:0, PC ae C34:l, PC ae C34:2, PC ae C34:3, PC ae C36:l, PC ae C36:2, PC ae C36:3, PC ae C36:4, PC ae C36:5, PC ae C38:l, PC ae C38:2, PC ae C38:3, PC ae C38:4, PC ae C38:5, PC ae C38:6, PC ae C40:2, PC ae C40:3, PC ae C42:l, PC ae C42:3, PC ae C44:3, PC ae C44:4, and PC ae C44:5.

[0289] In some embodiments, a culture medium comprises at least one sphingomyelin. In some embodiments, a culture medium comprises at least one sphingomyelin selected from SM (OH) (dl8:l / 14:l), SM (OH) (dl8:l / C16: 1), SM (OH) (dl8: 1 / 22: 1), SM (OH) (dl8:l / 22:2), SM (OH) (dl8: 1 / 24: 1), SM (dl8: 1 / 16:0), SM (dl8: 1 / 16: 1), SM (dl8:l / 20:2), SM (dl8:l / 22:3), and SM (dl8:l / 24:l).

[0290] In some embodiments, a culture medium comprises at least one reducing agent. In some embodiments, a culture medium comprises at least one reducing agent selected from sodium thioglycolate, iron sulfide, dithiothreitol, sodium dithionite, lithium aluminum hydride, sodium borohydride, diisobutyl aluminum hydride, palladium, and platinum, more preferably sodium sulfide or cysteine.

[0291] In some embodiments, a culture medium comprises at least one solid phase within a liquid culture. In some embodiments, a culture medium comprises at least one solid phase selected from sand and aluminum oxide.

[0292] In some embodiments, a culture medium comprises at least one oil or oil phase. In some embodiments, a culture medium comprises at least one oil or oil phase is selected from mineral oil and halogenated oil. In some embodiments, the halogenated oil comprises a fluorinated oil. Fluorinated oils typically readily solubilize gases and thus could increase the availability of H2 and CO2 to cells in the culture

[0293] In some embodiments, a culture medium comprises at least one component, ingredient, or compound described in Malheiros, et al. (2021) Comparative untargeted 4 metabolome analysis of ruminal fluid and feces of Nelore steers Bos indie us). Sci Rep', or Saleem, et al. (2013). The bovine ruminal fluid metabolome. Metabolomics, each of which is incorporated herein by reference.

[0294] Prior to inoculation, pH may be adjusted between pH 0-14, more preferably between 4-9, even more preferably between 5-8. During incubation or culturing, pH may be further controlled between pH 0-14, more preferably between 4-9, even more preferably between 5- 8.

[0295] The media compositions of the present disclosure may be grown in liquid in various vessel types made of glass, stainless steel, plastic, polycarbonate, or other material and in shapes that may be cylindrical, spherical, conical, a long horizontal system, or other shape or combination of shapes. It may beneficial to select materials that are relatively impermeable to O2. It may also be beneficial to select materials that are relatively impermeable to H2 to prevent substrate loss.

[0296] The media compositions of the present disclosure may optionally be converted to a solid medium by the addition of a gelling agent, such as agar, xantham gum, gellam gum, carrageenan, isubgol, and / or guar gum. Cells may be grown atop the solid medium or within the solid medium. Cells grown atop solid medium might improve gas substrate availability to the cells. Percentage of the gelling agent can range from 0.0001% to 99.9999%, or any range in between, or any value in between, preferably 0.1% to 10%, more preferably 0.5% to 2%. Biomass can be harvested from the plates by scraping, for example with a sterile “hockey stick.”

[0297] The biomass can then be prepared or processed using various downstream processes. In some embodiments, the biomass may be resuspended in a liquid solution. In some embodiments, the liquid solution comprises a cryoprotectant. In other embodiments, the biomass is converted to a powder by lyophilization or spray drying.

[0298] In some embodiments, the biomass of a single bacterial or archaeal strain may be prepared as a product (e.g., cell-based vaccine). In other embodiments, the biomass of multiple bacterial or archaeal strains may be combined before or after downstream processing, and be prepared as a product.

[0299] Culture may receive gas substrate (i.e., H2 and CO2) in a batched manner or via continuous flow.

[0300] CELL-BASED VACCINES 4

[0301] Vaccines of the present disclosure comprises at least one antigenic component comprising at least one cell surface antigen or a fragment thereof (e.g., cell and / or cell part) of at least one methanogen. In certain embodiments, the vaccine comprises at least one cell (e.g., whole cell) of at least one methanogen. In certain embodiments, the vaccine comprises cell part (e.g., fragment of a cell) of at least one methanogen. In certain embodiments, the vaccine is at least partially depleted of intracellular components. In some embodiments, the cell part or cell fragment is produced intentionally. In other embodiments, the cell part or cell fragment is produced during the preparation of a vaccine comprising a cell (e.g., whole cell).

[0302] Without being bound by theory, the vaccine presents one or more proteins, preferably cell surface proteins, that elicit an immune response. The vaccine of the present disclosure has the advantage of (a) presenting the native conformation of the cell surface protein that is recognized by an antibody in its natural context, (b) targeting multiple proteins present on a methanogen, and / or (c) targeting multiple methanogens, for example when a plurality of different methanogens have similar surface antigens and / or when a combination of methanogens is used.

[0303] In certain embodiments, the vaccine of the present disclosure comprises a cell and / or cell part of at least one hydrogenotroph, wherein the at least one hydrogenotroph comprises any one of the hydrogenotrophs listed in Table A or Table B.

[0304] In certain embodiments, the vaccine of the present disclosure comprises a cell and / or cell part of at least one hydrogenotroph, wherein the at least one hydrogenotroph comprises any two or more of the hydrogenotrophs listed in Table A or Table B.

[0305] In certain embodiments, the vaccine of the present disclosure comprises a cell and / or cell part at least one methanogen. In some embodiments, the at least one methanogen is of a genus Methanobrevibacter.

[0306] In some embodiments, the at least one methanogen comprises Methanobrevibacter ruminantium. In some embodiments, the at least one methanogen comprises Methanobrevibacter ruminantium Ml.

[0307] In some embodiments, the at least one methanogen comprises Methanobrevibacter gottschalkii. In some embodiments, the at least one methanogen comprises Methanobrevibacter gottschalkii DSM11977.

[0308] In some embodiments, the at least one methanogen comprises Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii. In some embodiments, the ratio of Methanobrevibacter ruminantium to Methanobrevibacter gottschalkii is at least about 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 2.5:1, 3: 1, 3.5:1, 4:1, 4.5: 1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 4

[0309] 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, or greater. In some embodiments, the ratio of Methanobrevibacter gottschalkii to Methanobrevibacter ruminantium is at least about 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 2:1, 2.5:1, 3: 1, 3.5:1, 4:1, 4.5: 1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, or greater.

[0310] In some such embodiments, the ratio of Methanobrevibacter ruminantium to Methanobrevibacter gottschalkii may be at least about 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1.

[0311] The relative dosage of cells and / or cell parts of each methanogen may be determined using any suitable technique, for example dry cell weight or a suitable cell counting technique. The relative dosage may be adjusted to account for batch differences, for example by using a potency assay, to ensure that consistency is maintained batch-to-batch.

[0312] In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells and / or cell parts of the vaccine are cells (e.g., whole cells and / or fractions thereof, such as an extracellular fraction depleted in the quantity of one or more intracellular components).

[0313] In some embodiments, the vaccine composition comprises at least about 106, 107, 108, 109, 1010, or 1011cells.

[0314] In some embodiments, the cell(s) and / or cell part(s) of the vaccine composition are killed, fixed, and / or irradiated. Any suitable method can be used to kill and / or fix the cell(s) and / or cell part(s) of the vaccine’s composition.

[0315] In some embodiments, the cell(s) and / or cell part(s) of the vaccine composition are killed by high temperature (heat), steam, or low temperature (freezing).

[0316] In some embodiments, the cell(s) and / or cell part(s) of the vaccine composition are fixed by formaldehyde or formalin.

[0317] In some embodiments, the cell(s) and / or cell part(s) of the vaccine composition are irradiated by UV or gamma irradiation.

[0318] In some embodiments, the cell(s) and / or cell part(s) of the vaccine composition are sonicated.

[0319] In some embodiments, the cell(s) and / or cell part(s) of the vaccine composition are crosslinked.

[0320] In some embodiments, the vaccine comprises cells and / or cell parts from at least 2, 3, 4, 5, 6, 7, 8, or 9 and / or not more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 different methanogen, for example cells and / or cell parts from 2-20 different methanogens, preferably cells and / or cell parts from 2-10 different methanogens. In p embodiments, the vaccine comprises cells and / or cell parts from M. gottschalkii and M. ruminantium optionally in combination with cells and / or cell parts from 1-18 additional different methanogens.

[0321] In some embodiments, the cells and / or cell parts of the methanogens are endotoxin- free (e.g., less than or equal to about 100 EU as determined using a suitable test). In some embodiments, the production of the antigenic material does not require an endotoxin purification step.

[0322] In some embodiments, the vaccine composition comprises at least one adjuvant. Any suitable adjuvant can be used, for example a veterinary approved adjuvant. One of skill in the art would understand how to select an appropriate adjuvant.

[0323] In some embodiments, the at least one adjuvant comprises: oil emulsions, e.g., comprising at least (a) mineral oil lipid and (b) aqueous phase, (e.g., Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide ISA series, e.g., ISA70, or Montanide ISA61, ISA206, ISA50), squaline-based emulsion, e.g., MF59 and AS03, saponins, (e.g., Quil-A, Spikoside, QS21, or ISCOMs, e.g., ISCOPREP 703), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate), also known to a skilled artisan as ‘alum’, e.g., Imject Alum), dextran sulfate, chitosan thermogel, (e.g., monophosphoryl lipid A), Carbol, PEGA, MDP, CpG ODN, cytokine-based adjuvants such as IL- 12 and / or GM-CSF, a lipid nanoparticle / cationic liposome adjuvant, an immune stimulating complex, or any combination of two or more thereof. In preferred embodiments, the at least one adjuvant comprises Freund’s complete adjuvant and / or Freund’s incomplete adjuvant.

[0324] In some embodiments, the at least one adjuvant comprises Emulsigen-D, Emulsigen, Emulsigen-P, and / or Polygen (MVP adjuvant, Omaha, NE). In some embodiments, the at least one adjuvant comprises ENABL 06 (HuvePharma, Peachtree City, GA). In some embodiments, the at least one adjuvant comprises Montainde ISA 201 and / or Montanide Gel 02 (Seppic Inc., New Jersey).

[0325] Without being bound by theory, the rumen may be considered a continuous flowing bioreactor with feed, water, saliva being introduced and gas, liquid, and solids exiting. Thus, any mechanism that yields a reduction in the activity and / or concentration of ruminal methanogens may be useful for a vaccine, for example (1) by binding to an impairing adhesion to the gastrointestinal tract and / or a component of feed, (2) by slowing growth rather and / or division, (3) by preventing adhesion to a microbial partner that provides an advantageous substrate thus lowering a thermodynamic benefit of the close proximity of the 4 methanogen with its partner, (4) reducing the mobility of the methanogen, and / or (5) binding to and inhibiting a key protein / enzyme involved in methanogenesis either directly or indirectly.

[0326] In preferred embodiments, the vaccine composition induces immune response against at least one cell surface protein or a fragment thereof of the at least one methanogen. In some embodiments, the at least one cell surface protein or a fragment thereof is selected from an adhesin-like protein, adhesin-like protein with cysteine protease domain, a siderophore, a substrate / cofactor importer, a protein directly involved in methanogenesis (e.g., a bicarbonate transporter, tetrahydromethanopterin S -methyltransferase subunit), ATP generating enzymes, a fragment thereof, and / or any combination thereof.

[0327] In preferred embodiments, the vaccine composition induces production of an antibody that effectively neutralizes at least one methanogen and / or reduces the amount of CH4 produced by the at least one methanogen.

[0328] In some embodiments, the vaccine composition is a pharmaceutical composition comprising at least one carrier and / or at least one excipient. Any suitable carrier and / or excipient can be used, for example a buffer (e.g., PBS, etc.), cryoprotectant (e.g., monosaccharides, polysaccharides, glycerol, etc.), etc.

[0329] For the cell-based vaccine composition of the present disclosure, methanogen cells (or cell parts derived from cells) can be administered at 1, 10, 1000, 10,000, O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, l.OxlO6, 5.0xl06, l.OxlO7, 5.0xl07, l.OxlO8, 5.0xl08, l.OxlO9, l.OxlO10, l.OxlO11, l.OxlO12or more, or any range in between or any value in between, cells per kilogram of a subject body weight. The number of cells transplanted or injected may be adjusted based on the desired level of engraftment in a given amount of time. Generally, lxl05to about lxl09cells / kg of body weight, from about lxl06to about lxl08cells / kg of body weight, or about IxlO7cells / kg of body weight, or more cells, as necessary, may be transplanted or injected. In some embodiments, transplantation or injection of at least about 100, 1000, 10,000, O.lxlO6, 0.5xl06, l.OxlO6, 2.0xl06, 3.0xl06, 4.0xl06, 5.0xl06, l.OxlO7, l.OxlO8, l.OxlO9, l.OxlO10, l.OxlO11, or l.OxlO12total cells per dose for an average size subject is effective.

[0330] Alternatively, dosage may be held constant regardless of the body weight. In some embodiments of the cell-based vaccine composition of the present disclosure, methanogen cells (or cell parts derived from cells) can be administered at 1, 10, 1000, 10,000, O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, l.OxlO6, 5.0xl06, l.OxlO7, 5.0xl07, l.OxlO8, 5.OxlO8, l.OxlO9, l.OxlO10, l.OxlO11, l.OxlO12or more, or any range in between or any value in between, cells per dose.

[0331] In certain embodiments, antigenic material as disclosed herein is produced at a first location (e.g., manufacturing facility), transferred to a second location (e.g., kitting facility) for finishing (e.g., fill, finish, and kitting), and then transferred to a third location (e.g., animal site) for administration to the subject. The antigenic material may be stabilized before transfer from the first location to the second location, for example by inactivating, crosslinking, freezing, freeze-drying, or any suitable alternative. The antigen material may be transferred from the first location to the second location at any suitable temperature, for example at about environmental temperature, refrigerated, or frozen.

[0332] PHARMACEUTICAL COMPOSITION

[0333] Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, etc.), or other compositions of the present disclosure may be in a pharmaceutical composition, and thus further comprise at least one excipient and / or carrier described herein or those known in the art.

[0334] The vaccine, antibody, milk, animal feed, or agent of the present disclosure (e.g., cellbased vaccines) may comprise at least one excipient that (1) increases stability; (2) permits the sustained or delayed release (e.g., from a depot formulation); and / or (3) alters the biodistribution (e.g., target to specific tissues or cell types). In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, and combinations thereof.

[0335] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and / or one or more other accessory ingredients.

[0336] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., the vaccine). The amount of the active ingredient may be generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0337] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.01% and 99% (w / w) of the active ingredient. By way of example, the composition may comprise between 0.01 % and 100%, e.g., between 0.05 and 50%, between 0.1-30%, between 5-80%, at least 80% (w / w) active ingredient.

[0338] Pharmaceutical compositions may comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0339] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and / or emulsifiers, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.

[0340] CONTROLLED / SUSTAINED RELEASE

[0341] In some embodiments, the compositions or agents of the present invention can be formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a specific outcome. 4

[0342] In some embodiments, the compositions or agents may be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and / or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.

[0343] In some embodiments, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No. W02012131104 and WO2012131106; the contents of each of which is herein incorporated by reference in its entirety).

[0344] EXCIPIENTS

[0345] Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, etc.), or other compositions of the present disclosure may comprise at least one excipient and / or carrier described herein or those known in the art (e.g., a pharmaceutically acceptable excipient and / or carrier).

[0346] A pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired.

[0347] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition. In some embodiments, the one or more excipients or accessory ingredients may make up at least about 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention.

[0348] Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).

[0349] In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0350] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents.

[0351] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0352] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and / or combinations thereof. 4

[0353] Exemplary surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRU®30]), poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof.

[0354] Exemplary binding agents include, but are not limited to, starch (e.g., cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly( vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and / or combinations thereof. Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Oxidation is a potential degradation pathway for many compounds. In order to prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxy anisole, EDTA, m-cresol, methionine, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®, NEOLONE™, KATHON™, and / or EUXYL®.

[0355] In some embodiments, the pH of the vaccine solutions is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and / or sodium malate. In another embodiment, the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions.

[0356] Exemplary buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, etc., and / or combinations thereof.

[0357] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and / or combinations thereof.

[0358] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0359] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition.

[0360] Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA- 4 bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA- bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents can be used.

[0361] CRYOPROTECTANTS

[0362] In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4°C or lower, such as a temperature between about -150°C and about 0°C or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, - 40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C). For example, the pharmaceutical composition comprising one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and / or shipment at, for example, about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In certain embodiments, the disclosure also relates to a method of increasing stability of the lipid nanoparticles and by storing the lipid nanoparticles and / or pharmaceutical compositions thereof at a temperature of 4°C or lower, such as a temperature between about -150°C and about 0°C or between about -80°C and about -20°C, e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, - 70°C, -80°C, -90°C, -130°C or -150°C).

[0363] In some embodiments, vaccine formulations may comprise cryoprotectants. As used herein, there term “cryoprotectant” refers to one or more agent that when combined with a given substance, helps to reduce or eliminate damage to that substance that occurs upon freezing. In some embodiments, cryoprotectants are combined with vaccines in order to stabilize them during freezing. Frozen storage of vaccines between -20°C and -80°C may be advantageous for long term (e.g., 36 months) storage. In some embodiments, cryoprotectants are included in vaccine formulations through freeze / thaw cycles and under frozen storage conditions. Cryoprotectants of the present invention may include, but are not limited to sucrose, trehalose, lactose, glycerol, dextrose, raffinose and / or mannitol. Trehalose is listed by the Food and Drug Administration as being generally regarded as safe (GRAS) and is commonly used in commercial pharmaceutical formulations.

[0364] INACTIVE INGREDIENTS

[0365] In some embodiments, vaccine formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more inactive agents included in formulations. Exemplary non-exhaustive lists of inactive ingredients and the routes of administration the inactive ingredients may be formulated in are described in Tables 2 and 3.

[0366] Table 2: Exemplary inactive ingredients

[0367] Table 3: Exemplary inactive ingredients

[0368] In Table 3, "AN" means anesthetic, "CNBLK" means cervical nerve block, "NBLK" means nerve block, and "IV" means intravenous.

[0369] NAKED DELIVERY

[0370] The compositions or agents of the present invention may be delivered to a subject naked or in saline. The naked compositions or agents may be administered to an animal using routes of administration known in the art and described herein.

[0371] ADMINISTRATION

[0372] Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure (e.g., those reducing CH4 production in a subject) may be administered to a subject by any route which results in a therapeutically effective outcome. These include, but are not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra- amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra- articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the auras media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique, ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. In specific embodiments, compositions may be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0373] Non-limiting routes of administration for the compositions or agents of the present disclosure are described below.

[0374] PARENTERAL AND INJECTABLE ADMINISTRATION

[0375] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0376] A pharmaceutical composition for parenteral administration may comprise at least one inactive ingredient. A non- exhaustive list of inactive ingredients for use in pharmaceutical compositions for parenteral administration includes hydrochloric acid, mannitol, N2, sodium acetate, sodium chloride and sodium hydroxide. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. The sterile formulation may also comprise adjuvants such as local anesthetics, preservatives and buffering agents.

[0377] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0378] Injectable formulations may be for direct injection into a region of a tissue, organ and / or subject. As a non-limiting example, a tissue, organ and / or subject may be directly injected a formulation by intramyocardial injection into the ischemic region. (See e.g., Zangi et al. Nature Biotechnology 2013; the contents of which are herein incorporated by reference in its entirety).

[0379] In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0380] RECTAL AND VAGINAL ADMINISTRATION

[0381] Compositions for rectal or vaginal (e.g., transvaginal) administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating 4 excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

[0382] As a non-limiting example, the formulations for rectal and / or vaginal administration may be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and / or vagina to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0383] A pharmaceutical composition for rectal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for rectal administration includes alcohol, alcohol, dehydrated, aluminum subacetate, anhydrous citric acid, aniseed oil, ascorbic acid, ascorbyl palmitate, balsam pern, benzoic acid, benzyl alcohol, bismuth subgallate, butylated hydroxy anisole, butylated hydroxytoluene, butylparaben, caramel, carbomer 934, carbomer 934p, carboxypolymethylene, cerasynt-se, cetyl alcohol, cocoa butter, coconut oil, hydrogenated, coconut oil / palm kernel oil glycerides, hydrogenated, cola nitida seed extract, d&c yellow no. 10, dichlorodifluoromethane, dichlorotetrafluoroethane, dimethyldioctadecylammonium bentonite, edetate calcium disodium, edetate disodium, edetic acid, epilactose, ethylenediamine, fat, edible, fat, hard, fd&c blue no. 1, fd&c green no. 3, fd&c yellow no. 6, flavor fig 827118, flavor raspberry pfc-8407, fructose, galactose, glycerin, glyceryl palmitate, glyceryl stearate, glyceryl stearate / peg stearate, glyceryl stearate / peg-40 stearate, glycine, hydrocarbon, hydrochloric acid, hydrogenated palm oil, hypromelloses, lactose, lanolin, lecithin, light mineral oil, magnesium aluminum silicate, magnesium aluminum silicate hydrate, methylparaben, N2, palm kernel oil, paraffin, petrolatum, white, polyethylene glycol 1000, polyethylene glycol 1540, polyethylene glycol 3350, polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polysorbate 60, polysorbate 80, potassium acetate, potassium metabisulfite, propylene glycol, propylparaben, saccharin sodium, saccharin sodium anhydrous, silicon dioxide, colloidal, simethicone, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium hydroxide, sodium metabisulfite, sorbitan monooleate, sorbitan sesquioleate, sorbitol, sorbitol solution, starch, steareth-10, steareth-40, sucrose, tagatose, d-, tartaric acid, dl-, trolamine, tromethamine, vegetable oil glyceride, hydrogenated, vegetable oil, hydrogenated, wax, emulsifying, white wax, xanthan gum and zinc oxide. 4

[0384] A pharmaceutical composition for vaginal administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for vaginal administration includes adipic acid, alcohol, denatured, allantoin, anhydrous lactose, apricot kernel oil peg-6 esters, barium sulfate, beeswax, bentonite, benzoic acid, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, calcium lactate, carbomer 934, carbomer 934p, cellulose, microcrystalline, ceteth-20, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, cetyl palmitate, cholesterol, choleth, citric acid, citric acid monohydrate, coconut oil / palm kernel oil glycerides, hydrogenated, crospovidone, edetate disodium, ethylcelluloses, ethylene-vinyl acetate copolymer (28% vinyl acetate), ethylene- vinyl acetate copolymer (9% vinylacetate), fatty alcohols, fd&c yellow no. 5, gelatin, glutamic acid, dl-, glycerin, glyceryl isostearate, glyceryl monostearate, glyceryl stearate, guar gum, high density polyethylene, hydrogel polymer, hydrogenated palm oil, hypromellose 2208 (15000 mpa.s), hypromelloses, isopropyl myristate, lactic acid, lactic acid, dl-, lactose, lactose monohydrate, lactose, hydrous, lanolin, lanolin anhydrous, lecithin, lecithin, soybean, light mineral oil, magnesium aluminum silicate, magnesium aluminum silicate hydrate, magnesium stearate, methyl stearate, methylparaben, microcrystalline wax, mineral oil, nitric acid, octyldodecanol, peanut oil, peg 6-32 stearate / glycol stearate, peg- 100 stearate, peg- 120 glyceryl stearate, peg-2 stearate, peg-5 oleate, pegoxol 7 stearate, petrolatum, white, phenylmercuric acetate, phospholipon 90g, phosphoric acid, piperazine hexahydrate, poly(dimethylsiloxane / methylvinylsiloxane / methylhydrogensiloxane) dimethylvinyl or dimethylhydroxy or trimethyl endblocked, polycarbophil, polyester, polyethylene glycol 1000, polyethylene glycol 3350, polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyglyceryl-3 oleate, polyglyceryl-4 oleate, polyoxyl palmitate, polysorbate 20, polysorbate 60, polysorbate 80, polyurethane, potassium alum, potassium hydroxide, povidone k29 / 32, povidones, promulgen d, propylene glycol, propylene glycol monopalmitostearate, propylparaben, quaternium-15 cis-form, silicon dioxide, silicon dioxide, colloidal, silicone, sodium bicarbonate, sodium citrate, sodium hydroxide, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate, dibasic, anhydrous, sodium phosphate, monobasic, anhydrous, sorbic acid, sorbitan monostearate, sorbitol, sorbitol solution, spermaceti, stannous 2-ethylhexanoate, starch, starch 1500, pregelatinized, starch, corn, stearamidoethyl diethylamine, stearic acid, stearyl alcohol, tartaric acid, dl-, tert- butylhydroquinone, tetrapropyl orthosilicate, trolamine, urea, vegetable oil, hydrogenated, wecobee fs, white ceresin wax and white wax.

[0385] ORAL ADMINISTRATION

[0386] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents and / or excipients commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0387] Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0388] Suspensions for oral dosage may contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be suspending agents, as a non-limiting example the suspending agents may be sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate; or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0389] Oily suspensions for oral dosage can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.

[0390] The oral dosage may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally- occurring phosphatides, for example soybean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[0391] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay), and lubricants (e.g., talc, calcium stearate, magnesium stearate, 4 solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents. The solid dosage forms may also dissolve once they come in contact with liquid such as, but not limited to, salvia and bile.

[0392] Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.

[0393] Solid dosage forms may be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

[0394] Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0395] Dosage forms for oral delivery may also be chewable. The chewable dosages forms may be sustained release formulations such as, but not limited to, the sustained release compositions described in International Publication No WO2013082470 and US Publication No US20130142876, each of which is herein incorporated by reference in its entirety. The chewable dosage forms may comprise amphipathic lipids such as, but not limited to, those described in International Publication No W02013082470 and US Publication No US20130142876, each of which is herein incorporated by reference in its entirety.

[0396] TOPICAL OR TRANSDERMAL ADMINISTRATION

[0397] As described herein, compositions of the present disclosure may be formulated for administration transdermally. The skin may be an ideal target site for delivery as it is readily accessible. Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.

[0398] The site of cutaneous expression of the delivered compositions will depend on the route of delivery. Two routes are commonly considered to deliver compositions to the skin: (ii) intradermal injection; and (iii) systemic delivery (e.g., for treatment of dermatologic 4 diseases that affect both cutaneous and extracutaneous regions). Compositions can be delivered to the skin by several different approaches known in the art.

[0399] In some embodiments, the invention provides for the compositions or agents to be delivered in more than one injection.

[0400] In some embodiments, before transdermal administration at least one area of tissue, such as skin, may be subjected to a device and / or solution which may increase permeability. In one embodiment, the tissue may be subjected to an abrasion device to increase the permeability of the skin (see U.S. Patent Publication No. 20080275468, herein incorporated by reference in its entirety). In another embodiment, the tissue may be subjected to an ultrasound enhancement device. An ultrasound enhancement device may include, but is not limited to, the devices described in U.S. Publication No. 20040236268 and U.S. Patent Nos. 6,491,657 and 6,234,990; each of which are herein incorporated by reference in their entireties. Methods of enhancing the permeability of tissue are described in U.S. Publication Nos. 20040171980 and 20040236268 and U.S. Pat. No. 6,190,315; each of which are herein incorporated by reference in their entireties.

[0401] In some embodiments, a device may be used to increase permeability of tissue before delivering formulations of compositions described herein. The permeability of skin may be measured by methods known in the art and / or described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety. As a non-limiting example, a formulation may be delivered by the drug delivery methods described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.

[0402] In another non-limiting example tissue may be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and / or after the tissue may be subjected to a device which may increase permeability. Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated by reference in its entirety) showed that using the EMLA cream in combination with a low energy, an onset of superficial cutaneous analgesia was seen as fast as 5 min. after a pretreatment with a low energy ultrasound.

[0403] In some embodiments, enhancers may be applied to the tissue before, during, and / or after the tissue has been treated to increase permeability. Enhancers include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers. Non-limiting examples of enhancers are described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.

[0404] In some embodiments, a device may be used to increase permeability of tissue before delivering formulations of compositions described herein, which may further contain a 4 substance that invokes an immune response. In another non-limiting example, a formulation containing a substance to invoke an immune response may be delivered by the methods described in U.S. Publication Nos. 20040171980 and 20040236268; each of which are herein incorporated by reference in their entireties.

[0405] Dosage forms for transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and / or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and / or any needed preservatives and / or buffers as may be required.

[0406] Additionally, compositions of the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and / or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer matrix and / or gel.

[0407] A pharmaceutical composition for transdermal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for transdermal administration includes acrylates copolymer, acrylic acid-isooctyl acrylate copolymer, acrylic adhesive 788, adcote 72al03, aerotex resin 3730, alcohol, alcohol, dehydrated, aluminum polyester, bentonite, butylated hydroxytoluene, butylene glycol, butyric acid, caprylic / capric triglyceride, carbomer 1342, carbomer 940, carbomer 980, carrageenan, cetylpyridinium chloride, citric acid, crospovidone, daubert 1-5 pestr (matte) 164z, diethylene glycol monoethyl ether, diethylhexyl phthalate, dimethicone copolyol, dimethicone mdx4-4210, dimethicone medical fluid 360, dimethylaminoethyl methacrylate - butyl methacrylate - methyl methacrylate copolymer, dipropylene glycol, duro-tak 280-2516, duro-tak 387-2516, duro-tak 80- 1196, duro-tak 87-2070, duro-tak 87- 2194, duro-tak 87-2287, duro-tak 87-2296, duro- tak 87-2888, duro-tak 87-2979, edetate disodium, ethyl acetate, ethyl oleate, ethylcelluloses, ethylene vinyl acetate copolymer, ethylene -propylene copolymer, fatty acid esters, gelva 737, glycerin, glyceryl laurate, glyceryl oleate, heptane, high density polyethylene, hydrochloric acid, hydrogenated polybutene 635-690, hydroxyethyl cellulose, hydroxypropyl cellulose, isopropyl myristate, isopropyl palmitate, lactose, lanolin anhydrous, lauryl lactate, lecithin, levulinic acid, light mineral oil, medical adhesive modified s-15, methyl alcohol, methyl laurate, mineral oil, N2, octisalate, octyldodecanol, oleic acid, oleyl alcohol, oleyl oleate, pentadecalactone, petrolatum, white, polacrilin, polyacrylic acid (250000 mw), polybutene (1400 mw), 4 polyester, polyester polyamine copolymer, polyester rayon, polyethylene terephthalates, polyisobutylene, polyisobutylene (1100000 mw), polyisobutylene (35000 mw), polyisobutylene 178-236, polyisobutylene 241-294, polyisobutylene 35-39, polyisobutylene low molecular weight, polyisobutylene medium molecular weight, polyisobutylene / polybutene adhesive, polypropylene, polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polyvinyl chloride -polyvinyl acetate copolymer, polyvinylpyridine, povidone k29 / 32, povidones, propylene glycol, propylene glycol monolaurate, ra-2397, ra- 3011, silicon, silicon dioxide, colloidal, silicone, silicone adhesive 4102, silicone adhesive 4502, silicone adhesive bio-psa q7- 4201, silicone adhesive bio-psa q7-4301, silicone / polyester film strip, sodium chloride, sodium citrate, sodium hydroxide, sorbitan monooleate, stearalkonium hectorite / propylene carbonate, titanium dioxide, triacetin, trolamine, tromethamine, union 76 amsco-res 6038 and viscose / cotton.

[0408] A pharmaceutical composition for intradermal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for intradermal administration includes benzalkonium chloride, benzyl alcohol, carboxymethylcellulose sodium, creatinine, edetate disodium, glycerin, hydrochloric acid, metacresol, methylparaben, phenol, polysorbate 80, protamine sulfate, sodium acetate, sodium bisulfite, sodium chloride, sodium hydroxide, sodium phosphate, sodium phosphate, dibasic, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, anhydrous and zinc chloride.

[0409] DEPOT ADMINISTRATION

[0410] As described herein, in some embodiments, the composition is formulated in depots for extended release. Generally, a specific organ or tissue (a "target tissue") is targeted for administration.

[0411] In some aspects, the compositions or agents are spatially retained within or proximal to a target tissue. Provided are method of providing a composition to a target tissue of a mammalian subject by contacting the target tissue (which contains one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition is retained in the target tissue.

[0412] Aspects of the invention are directed to methods of providing a composition to a target tissue of a subject, by contacting the target tissue (comprising one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue.

[0413] In some embodiments, the compositions may be retained near target tissue using a small disposable drug reservoir, patch pump or osmotic pump. Non-limiting examples of patch pumps include those manufactured and / or sold by BD® (Franklin Lakes, NJ), Insulet Corporation (Bedford, MA), SteadyMed Therapeutics (San Francisco, CA), Medtronic (Minneapolis, MN) (e.g., MiniMed), UniLife (York, PA), Valeritas (Bridgewater, NJ), and SpringLeaf Therapeutics (Boston, MA). A non- limiting example of an osmotic pump include those manufactured by DURECT® (Cupertino, CA) (e.g., DUROS® and ALZET ®).

[0414] PULMONARY ADMINISTRATION

[0415] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are suitably in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and / or using a self-propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0416] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally, the propellant may constitute 50% to 99.9% (w / w) of the composition, and active ingredient may constitute 0.1 % to 20% (w / w) of the composition. A propellant may further comprise additional ingredients such as a liquid nonionic and / or solid anionic surfactant and / or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient). As a non-limiting example, the compositions described herein may be formulated for pulmonary delivery by the methods described in U.S. Pat. No. 8,257,685; herein incorporated by reference in its entirety. 4

[0417] Pharmaceutical compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and / or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface- active agent, and / or a preservative such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.

[0418] The compositions and formulations provided herein which may be used for pulmonary delivery may further comprise one or more surfactants. Suitable surfactants or surfactant components for enhancing the uptake of the compositions of the invention include synthetic and natural as well as full and truncated forms of surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant Protein E, di-saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; phosphatidic acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3- phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate; as well as natural and artificial lamellar bodies which are the natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomelic and polymeric, poly( vinyl amine) with dextran and / or alkanoyl side chains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf, Survan and Atovaquone, among others. These surfactants can be used either as single or part of a multiple component surfactant in a formulation, or as covalently bound to a component of a pharmaceutical composition herein.

[0419] INTRANASAL, NASAL AND BUCCAL ADMINISTRATION

[0420] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0421] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w / w) and as much as 100% (w / w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods and may, for example, 0.1 % to 20% (w / w) active ingredient, the balance comprising an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0422] A pharmaceutical composition for inhalation (respiratory) administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for inhalation (respiratory) administration includes acetone sodium bisulfite, acetylcysteine, alcohol, alcohol, dehydrated, ammonia, apaflurane, ascorbic acid, benzalkonium chloride, calcium carbonate, CO2, cetylpyridinium chloride, chlorobutanol, citric acid, d&c yellow no. 10, dichlorodifluoromethane, dichlorotetrafluoroethane, edetate disodium, edetate sodium, fd&c yellow no. 6, fluorochlorohydrocarbons, gelatin, glycerin, glycine, hydrochloric acid, hydrochloric acid, diluted, lactose, lactose monohydrate, lecithin, lecithin, hydrogenated soy, lecithin, soybean, lysine monohydrate, mannitol, menthol, methylparaben, nitric acid, N2, norflurane, oleic acid, polyethylene glycol 1000, povidone k25, propylene glycol, propylparaben, saccharin, saccharin sodium, silicon dioxide, colloidal, sodium bisulfate, sodium bisulfite, sodium chloride, sodium citrate, sodium hydroxide, sodium lauryl sulfate, sodium metabisulfite, sodium sulfate anhydrous, sodium sulfite, sorbitan trioleate, sulfuric acid, thymol, titanium dioxide, trichloromonofluoromethane, tromethamine and zinc oxide.

[0423] A pharmaceutical composition for nasal administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been approved by 4 the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for nasal administration includes acetic acid, alcohol, dehydrated, allyl .alpha.-ionone, anhydrous dextrose, anhydrous trisodium citrate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, caffeine, CO2, carboxymethylcellulose sodium, cellulose, microcrystalline, chlorobutanol, citric acid, citric acid monohydrate, dextrose, dichlorodifluoromethane, dichlorotetrafluoroethane, edetate disodium, glycerin, glycerol ester of hydrogenated rosin, hydrochloric acid, hypromellose 2910 (15000 mpa.s), methylcelluloses, methylparaben, N2, norflurane, oleic acid, petrolatum, white, phenylethyl alcohol, polyethylene glycol 3350, polyethylene glycol 400, polyoxyl 400 stearate, polysorbate 20, polysorbate 80, potassium phosphate, monobasic, potassium sorbate, propylene glycol, propylparaben, sodium acetate, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium phosphate, dibasic, sodium phosphate, dibasic, anhydrous, sodium phosphate, dibasic, dihydrate, sodium phosphate, dibasic, dodecahydrate, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, anhydrous, sodium phosphate, monobasic, dihydrate, sorbitan trioleate, sorbitol, sorbitol solution, sucralose, sulfuric acid, trichloromonofluoromethane and trisodium citrate dihydrate.

[0424] OPHTHALMIC AND AURICULAR (OTIC) ADMINISTRATION

[0425] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for delivery to and / or around the eye and / or delivery to the ear (e.g., auricular (otic) administration). Non-limiting examples of route of administration for delivery to and / or around the eye include retrobulbar, conjuctival, intracorneal, intraocular, intravitreal, ophthlamic and subconjuctiva. Such formulations may, for example, be in the form of eye drops or ear drops including, for example, a 0.1 / 1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and / or one or more other of any additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are contemplated as being within the scope of this invention. A multilayer thin film device may be prepared to contain a pharmaceutical composition for delivery to the eye and / or surrounding tissue.

[0426] A pharmaceutical composition for ophthalmic administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been 4 approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for ophthalmic administration includes acetic acid, alcohol, alcohol, dehydrated, alginic acid, amerchol-cab, ammonium hydroxide, anhydrous trisodium citrate, antipyrine, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, boric acid, caffeine, calcium chloride, carbomer 1342, carbomer 934p, carbomer 940, carbomer homopolymer type b (allyl pentaerythritol crosslinked), carboxymethylcellulose sodium, castor oil, cetyl alcohol, chlorobutanol, chlorobutanol, anhydrous, cholesterol, citric acid, citric acid monohydrate, creatinine, diethanolamine, diethylhexyl phthalate, divinylbenzene styrene copolymer, edetate disodium, edetate disodium anhydrous, edetate sodium, ethylene vinyl acetate copolymer, gellan gum (low acyl), glycerin, glyceryl stearate, high density polyethylene, hydrocarbon gel, plasticized, hydrochloric acid, hydrochloric acid, diluted, hydroxyethyl cellulose, hydroxypropyl methylcellulose 2906, hypromellose 2910 (15000 mpa.s), hypromelloses, jelene, lanolin, lanolin alcohols, lanolin anhydrous, lanolin nonionic derivatives, lauralkonium chloride, lauroyl sarcosine, light mineral oil, magnesium chloride, mannitol, methylcellulose (4000 mpa.s), methylcelluloses, methylparaben, mineral oil, nitric acid, N2, nonoxynol-9, octoxynol-40, octylphenol polymethylene, petrolatum, petrolatum, white, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, phosphoric acid, polidronium chloride, poloxamer 188, poloxamer 407, polycarbophil, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 8000, polyoxyethylene - polyoxypropylene 1800, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, polypropylene glycol, polysorbate 20, polysorbate 60, polysorbate 80, polyvinyl alcohol, potassium acetate, potassium chloride, potassium phosphate, monobasic, potassium sorbate, povidone k29 / 32, povidone k30, povidone k90, povidones, propylene glycol, propylparaben, soda ash, sodium acetate, sodium bisulfate, sodium bisulfite, sodium borate, sodium borate decahydrate, sodium carbonate, sodium carbonate monohydrate, sodium chloride, sodium citrate, sodium hydroxide, sodium metabisulfite, sodium nitrate, sodium phosphate, sodium phosphate dihydrate, sodium phosphate, dibasic, sodium phosphate, dibasic, anhydrous, sodium phosphate, dibasic, dihydrate, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, sodium phosphate, monobasic, anhydrous, sodium phosphate, monobasic, dihydrate, sodium phosphate, monobasic, monohydrate, sodium sulfate, sodium sulfate anhydrous, sodium sulfate decahydrate, sodium sulfite, sodium thiosulfate, sorbic acid, sorbitan monolaurate, sorbitol, sorbitol solution, stabilized oxychloro complex, sulfuric p acid, thimerosal, titanium dioxide, tocophersolan, trisodium citrate dihydrate, triton 720, tromethamine, tyloxapol and zinc chloride.

[0427] A pharmaceutical composition for retrobulbar administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for retrobulbar administration includes hydrochloric acid and sodium hydroxide.

[0428] A pharmaceutical composition for intraocular administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for intraocular administration includes benzalkonium chloride, calcium chloride, citric acid monohydrate, hydrochloric acid, magnesium chloride, polyvinyl alcohol, potassium chloride, sodium acetate, sodium chloride, sodium citrate and sodium hydroxide.

[0429] A pharmaceutical composition for intravitreal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for intravitreal administration includes calcium chloride, carboxymethylcellulose sodium, cellulose, microcrystalline, hyaluronate sodium, hydrochloric acid, magnesium chloride, magnesium stearate, polysorbate 80, polyvinyl alcohol, potassium chloride, sodium acetate, sodium bicarbonate, sodium carbonate, sodium chloride, sodium hydroxide, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate and trisodium citrate dehydrate.

[0430] A pharmaceutical composition for subconjunctival administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for subconjunctival administration includes benzyl alcohol, hydrochloric acid and sodium hydroxide.

[0431] A pharmaceutical composition for auricular administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for auricular administration includes acetic acid, aluminum acetate, aluminum sulfate anhydrous, benzalkonium chloride, benzethonium chloride, benzyl alcohol, boric acid, calcium carbonate, cetyl alcohol, chlorobutanol, chloroxylenol, citric acid, creatinine, cupric sulfate, cupric sulfate anhydrous, edetate disodium, edetic acid, glycerin, glyceryl stearate, hydrochloric acid, hydrocortisone, hydroxyethyl cellulose, isopropyl myristate, lactic acid, lecithin, hydrogenated, methylparaben, mineral oil, petrolatum, petrolatum, white, phenylethyl alcohol, polyoxyl 40 stearate, polyoxyl stearate, polysorbate 20, polysorbate 80, polyvinyl alcohol, potassium metabisulfite, potassium phosphate, monobasic, povidone k90f, 4 povidones, propylene glycol, propylene glycol diacetate, propylparaben, sodium acetate, sodium bisulfite, sodium borate, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, dibasic, anhydrous, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, anhydrous, sodium sulfite, sulfuric acid and thimerosal.

[0432] DOSING

[0433] Provided herein are methods comprising administering a vaccine composition to a subject. The specific dose level for any particular subject will depend upon a variety of factors including the species, the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the arts. Compositions in accordance with the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending administrator within the scope of sound judgment.

[0434] In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No WO2013078199, herein incorporated by reference in its entirety).

[0435] For the cell-based vaccine composition of the present disclosure, methanogen cells (or cell parts derived from cells) can be administered at 1, 10, 1000, 10,000, O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, l.OxlO6, 5.0xl06, l.OxlO7, 5.0xl07, l.OxlO8, 5.0xl08, l.OxlO9, l.OxlO10, l.OxlO11, l.OxlO12or more, or any range in between or any value in between, cells per kilogram of a subject body weight. The number of cells transplanted or injected may be adjusted based on the desired level of engraftment in a given amount of time. Generally, lxl05to about lxl09cells / kg of body weight, from about 4 lxl06to about lxlO8cells / kg of body weight, or about IxlO7cells / kg of body weight, or more cells, as necessary, may be transplanted or injected. In some embodiment, transplantation or injection of at least about 100, 1000, 10,000, O.lxlO6, 0.5xl06, l.OxlO6, 2.0xl06, 3.0xl06, 4.0xl06, or 5.0xl06, l.OxlO7, 1.0x10s, l.OxlO9, l.OxlO10, l.OxlO11, or l.OxlO12total cells per dose for an average size subject is effective.

[0436] For the cell-based vaccine composition of the present disclosure, methanogen cells (or cell parts derived from cells) can be administered at 1, 10, 1000, 10,000, O.lxlO6, 0.2xl06, 0.3xl06, 0.4xl06, 0.5xl06, 0.6xl06, 0.7xl06, 0.8xl06, 0.9xl06, l.OxlO6, 5.0xl06, l.OxlO7, 5.0xl07, l.OxlO8, 5.0xl08, l.OxlO9, l.OxlO10, l.OxlO11, l.OxlO12or more, or any range in between or any value in between, cells per dose irrespective of body weight.

[0437] The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, twenty, thirty, forty, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. In some embodiments, an animal is administered with a prime (initial dose), followed by a boost (second dose) at least about 1, 1.5, 2, 2.5, 3, 3.5, or 4 weeks after the prime. In some embodiments, at least one additional dose is given after the boost, optionally after 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, 25, 26, 27, 28,

[0438] 29, 30, 31, 32, 33, 34, 35, or 36 months after the boost. In some embodiments, the at least one additional dose is repeated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

[0439] 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. In preferred embodiments, an animal is administered with a prime, followed by a boost about 2 or 3 weeks after the prime. In yet other preferred embodiments, an animal is administered with (a) a prime, (b) a boost about 2 or 3 weeks after the prime, and (c) at least one additional dose every 3, 4, 5, or 6 months. Each dose in the dosing schedule may comprise the same or different number of cells. According to the present disclosure, the compositions of the present disclosure may be administered in split- dose regimens. As used herein, a "split dose" is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a "single unit dose" is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event. As used herein, a "total daily dose" is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In some embodiments, the compositions of the present disclosure are administered to a subject in split doses. The compositions may be formulated in buffer only or in a formulation described herein.

[0440] DOSAGE FORMS

[0441] Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, etc.), or other compositions of the present disclosure may be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

[0442] LIQUID DOSAGE FORMS

[0443] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0444] INJECTABLE

[0445] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer’s solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0446] In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compositions then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compositions may be accomplished by dissolving or suspending the compositions in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compositions in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compositions to polymer and the nature of the particular polymer employed, the rate of a compound release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the compositions in liposomes or microemulsions which are compatible with body tissues.

[0447] PULMONARY FORMULATION

[0448] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0449] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w / w) and as much as 100% (w / w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w / w) active ingredient, where the balance may comprise an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0450] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21sted., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0451] COATINGS OR SHELLS

[0452] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0453] MULTI-DOSE AND REPEAT-DOSE ADMINISTRATION

[0454] In some embodiments, vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure may be administered in two or more doses (referred to herein as "multi-dose administration"). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and / or volume of components or an altered mass and / or volume of components in comparison to a previous dose. In some embodiments, multi-dose administration may comprise repeat-dose administration. As used herein, the term "repeatdose administration" refers to two or more doses administered consecutively or within a regimen of repeat doses comprising same or different components. In some embodiments, the repeat dose may comprise substantially the same components provided at substantially the same mass and / or volume. In other embodiments, the repeat dose may comprise different components (e.g., different adjuvant for a vaccine composition). 4

[0455] ADJUVANT

[0456] Adjuvants or immune potentiators, may also be administered with or in combination with one or more vaccine composition of the present disclosure.

[0457] The term "adjuvant" refers to an agent that when administered in conjunction with or as part of a composition described herein augments, enhances, and / or boosts the immune response to a methanogen, but when the agent is administered alone does not generate an immune response. In some embodiments, the adjuvant generates an immune response to a methanogen and does not produce an allergy or other adverse reaction. Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages.

[0458] In some embodiments, an adjuvant acts as a co-signal to prime T-cells and / or B- cells and / or NK cells as to the existence of the cell surface protein of a methanogen in a vaccine composition of the present disclosure.

[0459] Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccinees. These may be co-administered by any route, e.g., intramusculary, subcutaneous, IV or intradermal injections.

[0460] Adjuvants useful in the present invention may include, but are not limited to, natural or synthetic. They may be organic or inorganic.

[0461] When a vaccine or immunogenic composition of the invention comprises adjuvants or is administered together with one or more adjuvants, the adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (Glaxo SmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as International Publication No. W02007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as International Publication No. W02007 / 109813) and saponins, such as QS21 (see Kensil et al, 4 in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund’s adjuvant (complete or incomplete). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al, N. Engl. J. Med. 336, 86-91 (1997)).

[0462] Adjuvants may be selected from any of the classes (1) mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidised detergent stabilised oil-in-water emulsion, purified saponin, oil-in- water emulsion, stabilised water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and / or (6) inert vehicles, such as gold particles; (7) microorganism derived adjuvants; (8) tensoactive compounds; (9) carbohydrates; or combinations thereof.

[0463] Other adjuvants which may be utilized in the vaccines of the present disclosure include any of those listed on the web-based vaccine adjuvant database, Vaxjo; World Wide Web at violinet.org / vaxjo / and described in for example Sayers, et al., J. Biomedicine and Biotechnology, volume 2012 (2012), Article ID 831486, 13 pages, the content of which is incorporated herein by reference in its entirety.

[0464] Selection of appropriate adjuvants will be evident to one of ordinary skill in the art. Specific adjuvants may include, without limitation, cationic liposome-DNA complex JVRS- 100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM(s)™, Freund’s complete adjuvant, Freund’s incomplete adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Eiposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IE-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium-derb / ed P40 Vaccine Adjuvant, MPL™ Adjuvant, AS04, AS02, Lipopolysaccharide Vaccine Adjuvant, Muramyl Dipeptide Adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetella pertussis component Vaccine Adjuvant, Cationic Liposomal Vaccine Adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3 Adjuvant, Aluminum vaccine adjuvant, Polygen Vaccine Adjuvant, Adjumer™, Algal Glucan, Bay R1005, Theramide®, Stearyl 4

[0465] Tyrosine, Specol, Algammulin, Avridine®, Calcium Phosphate Gel, CTA1-DD gene fusion protein, DOC / Alum Complex, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Recombinant hlFN-gamma / Interferon-g, Interleukin-i'P, Interleukin-2, Interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE Liposomes, Murametide, Murapalmitine, D-Murapalmitine, NAGO, Non- Ionic Surfactant Vesicles, PMMA, Protein Cochleates, QS-21, SPT (Antigen Formulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G Vaccine Adjuvant, E. coli heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic Adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly(LC), ISCOMATRIX®, Abisco-100 vaccine adjuvant, Albumin- heparin microparticles vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes Containing Antibodies to Costimulatory Molecules, SAF-1, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Threonyl muramyl dipeptide (TMDP), Ty Particles vaccine adjuvant, Bupivacaine vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide)) vaccine adjuvant, IL- 15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant El 12K of Cholera Toxin mCT-El 12K, and / or Matrix-S.

[0466] In some embodiments, the at least one adjuvant comprises oil emulsions comprising at least (a) mineral oil lipid and (b) aqueous phase (e.g., Freund’s complete adjuvant, Freund’s incomplete adjuvant, Montanide ISA70, or Montanide ISA61), saponins, (e.g., Quil-A, Spikoside, QS21, or ISCOPREP 703), aluminum salts, also known to a skilled artisan as ‘alum’, e.g., Imject Alum), dextran sulfate, chitosan thermogel, (e.g., monophosphoryl lipid A), a lipid nanoparticle / cationic liposome adjuvant, an immune stimulating complex, or any combination of two or more thereof. In preferred embodiments, the at least one adjuvant is Freund’s complete adjuvant or Freund’s incomplete adjuvant. See Spickler and Roth (2003) J Vet Intern Med, 17:273-281, which is incorporated herein by reference.

[0467] Other adjuvants which may be co-administered with the vaccine compositions of the invention include, but are not limited to interferons, TNF-alpha, TNF-beta, chemokines such as CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD-40 L, CD28 agonists, PD-1, soluble PD1, LI or L2, or interleukins such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-21, IL-23, IL-15, IL-17, and IL-18. 4

[0468] In some embodiments, the adjuvant comprises Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(LC), aluminum hydroxide, or Pam3CSK4.

[0469] In some embodiments, the adjuvant comprises: (a) (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-l-propanaminium bromide (GAP-DMORIE) and a neutral lipid; (b) a cytokine; (c) mono-phosphoryl lipid A and trehalosedicorynomycolateAF (MPL+TDM); (d) a solubilized mono-phosphoryl lipid A formulation; and / or (e) CRL1005 / BAK.

[0470] In some embodiments, the neutral lipid in (a) comprises (a) l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE); (b) l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE); and / or (c) l,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE).

[0471] In some embodiments, the adjuvant comprises saponin, Montanide ISA61, a chitosan thermogel, a lipid nanoparticle / cationic liposome adjuvant, or any combination thereof. In preferred embodiments, the adjuvant comprises Montanide ISA61.

[0472] ANTIBODY

[0473] Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE); and recombinant antibodies, such as single-chain antibodies, chimeric antibodies, and multispecific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

[0474] The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., at least one cell surface protein or fragment thereof of at least one methanogen). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity 4 determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes (e.g., IgGA). VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

[0475] Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab’)2fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[0476] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., chimeric, etc.). Antibodies may also be fully specific to the subject, e.g., the antibodies may be fully ruminant or fully human. The terms “monoclonal antibodies” and “monoclonal antibody composition,” as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

[0477] The present disclosure includes a monoclonal antibody that works particularly well in binding and neutralizing at least one methanogen. Upon immunizing a subject population (e.g., of milk-producing subject), the milk comprising the antibody can be screened for antibodies with superior activity (e.g., specific binding, neutralizing at least one methanogen, etc.). The amino acid sequence of such antibodies can be determined (e.g., mass spec -based sequencing, Next Gen Sequencing, or other methods known in the art), their expressing DNA vectors can be synthesized, and monoclonal antibodies can be produced. One or a combination of at least two or more antibodies can be added to the drinking water and / or animal feed and be given to a subject population.

[0478] Alternatively, such monoclonal antibodies can be generated by immunizing a vehicle animal (e.g., mouse, rabbit, etc.), and hybridomas expressing the animals can be recovered. Standard hybridoma methods for producing antibodies are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5thEd., Garland Publishing, New York, NY (2001)). Antibodies produced by hybridomas can be screened and utilized according to the methods described above and herein.

[0479] In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an IgG or IgA. In some embodiments, the antibody is IgA. The IgA isoform, at least in cattle, may be more stable in the rumen. For example, IgA levels in cattle saliva were reduced by only 40% after 8h exposure to rumen contents while IgG levels were reduced by 80%.

[0480] In some embodiments, the antibody is lyophilized. In some embodiments, the antibody is in a pharmaceutical composition of the present disclosure or those known in the art. In some embodiments, a composition comprising an antibody further comprises at least one excipient and / or carrier. In some embodiments, the antibody is in the animal feed. In some embodiments, the antibody is in the solid animal feed. In other embodiments, the antibody is in the liquid animal feed. In some embodiments, the antibody is in the drinking 4 water or milk. In preferred embodiments, the antibody is orally consumed by a subject such that the antibody comes in contact with at least one methanogen present in the gut of the subject. In preferred embodiments, oral administration of the antibody reduces the number and / or type of at least one methanogen.

[0481] METHODS FOR DETECTION OF ANTIBODY

[0482] In certain embodiments, an efficacious vaccine produces an antibody titer of greater than 1:40, greater that 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, greater than 1:10000. In some embodiments, the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In some embodiments, the titer is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a repeat dose.).

[0483] In certain aspects, antigen-specific antibodies are measured in units of pg / ml or are measured in units of IU / L (International Units per liter) or mIU / ml (milli International Units per ml). In some embodiments of the invention, an efficacious vaccine produces >0.5 pg / ml, >0.1 pg / ml, >0.2 pg / ml, >0.35 pg / ml, >0.5 pg / ml, >1 pg / ml, >2 pg / ml, >5 pg / ml or >10 pg / ml. In some embodiments, an efficacious vaccine produces >10 mIU / ml, >20 mIU / ml, >50 mIU / ml, >100 mIU / ml, >200 mIU / ml, >500 mIU / ml or > 1000 mIU / ml.

[0484] Methods of detecting the presence of antibodies are well known in the art.

[0485] In some embodiments, antibody level or concentration is determined or measured by neutralization assay, e.g., neutralization of at least one methanogen.

[0486] Other exemplary methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs; including variants such as competitive ELISA, sandwich ELISA, etc.), immunofluorescent assays, Western blotting, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference).

[0487] For example, ELISA and RIA procedures may be conducted such that a desired protein standard (e.g., an extracellular domain of at least one cell surface protein or a fragment thereof of at least one methanogen) is labeled (with a radioisotope such as125I or35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and is brought into contact with a sample comprising the antibody, whereon the amount of the labeled protein standard bound to the antibody is measured.

[0488] Enzymatic and radiolabeling of a protein and / or the antibodies may be affected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzymes are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

[0489] It may be desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

[0490] It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.

[0491] Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

[0492] MILK

[0493] In certain aspects, provided herein are milk and derivatives thereof. Milk produced by vaccinated female subjects (e.g., dairy cows) comprises antibodies that bind at least one cell surface protein or a fragment thereof of at least one methanogen. Such milk can be orally consumed by subjects such that the antibodies therein can come in contact with at least one methanogen present in the gut of the subjects. Upon contact, the antibodies in the milk can 4 neutralize the at least one methanogen and contribute to reducing CH4 production by the subjects.

[0494] Thus, milk from vaccinated female subject can be used to treat breast-feeding animals, thereby reducing methane production and / or methanogen colonization in young animals. This can be especially important when vaccination of young animals occurs concurrently with weaning.

[0495] In some embodiments, the milk is pasteurized and / or homogenized. In some embodiments, the milk is lyophilized, filtered, concentrated, evaporated, or processed to form dry milk powder (e.g., boiling at low pressure at low temperature). In some embodiments, said processing may allow longer shelf life of the milk / milk product and the antibodies present therein. In some embodiments, the fat content is removed / reduced from the milk. Processing of milk and / or preparation of derivatives of milk are well known in the art.

[0496] Appropriate care is taken to preserve the structural and functional (e.g., binding a methanogen) aspects of the antibodies. For example, in some embodiments, high pressure (-200 MPa) and low temperature (-4°C) are used throughout the process as described at least by Kim et al. (2008) Journal of Dairy Science, 91 :4176-4182. In other embodiments, milk may be pasteurized at low-temperature of 60 °C for 10 min. at standard pressure. These conditions may pasteurize milk without significantly altering the antibody function.

[0497] Alternatively, milk can be filtered to remove microorganisms instead of pasteurizing. Microfiltration is a process that replaces pasteurization and produces milk with fewer microorganisms and longer shelf life without a change in the quality of the milk. In this process, cream is separated from the skimmed milk and the skimmed milk is forced through ceramic microfilters that trap 99.9% of microorganisms in the milk (as compared to 99.999% killing of microorganisms in standard high temperature short time pasteurization).

[0498] Ultrafiltration uses finer filters than microfiltration, which allow lactose and water to pass through while retaining fats, calcium and protein. As with microfiltration, the fat may be removed before filtration and added back in afterwards. Ultrafiltered milk is used widely in the industry in cheesemaking.

[0499] Colostrum may similarly used in the milk embodiments disclosed herein.

[0500] ANIMAL FEED

[0501] Provided herein are animal feeds that are useful in reducing CH4 production by a subject. Such animal feed may be used in combination with any one of vaccines, antibodies, milk, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial 4 strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure (e.g., those reducing CH4 production in a subject). Animal feed comprises at least one feed additive, which reduces the CH4 production in a subject.

[0502] In some embodiments, the animal feed comprises an antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen.

[0503] In some embodiments, the animal feed comprises a composition comprising an antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen. For example, the animal feed may comprise milk or derivatives thereof comprising said antibody.

[0504] In some embodiments, the animal feed is liquid (e.g., drinking water, milk). An antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen or a composition comprising same may be added to the liquid animal feed (e.g., drinking water) before being given to the subject. In some embodiments, said antibody or a composition comprising same may be added to the drinking water. In other embodiments, the milk or derivatives thereof comprising said antibody may be given directly to the subject, or added to other liquid animal feed (e.g., drinking water).

[0505] In other embodiments, the animal feed is solid. In some such embodiments, the animal feed may comprise hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and sprouted grains and legumes. An antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen or a composition comprising same may be added to the solid animal feed before being given to the subject.

[0506] In some embodiments, an animal feed may comprise fats and fatty acids that further aid in reducing CH4 production in subjects. Based on a meta-analysis, fat supplementation reduced CH4 by 3.77% in cattle and 4.30% in sheep per 1% dietary fats. Fat decreases CH4 production (expressed as g / kg digestible dry matter (DM)) more from sheep than from cattle, which was attributed to the comparatively lower depression of DM digestion together with numerically larger depression of CH4 production (g / kg DM) by fat in sheep. Among fatty acids, C12:0, C18:3 and other polyunsaturated fatty acids (PUFA) are more potent than saturated fatty acids. The CFU-suppressing efficacy of fats generally persists, with persistent suppression being noted for 72 days and longer in cattle.

[0507] Fats supplemented up to 6% of the diet (DM) can also improve milk production while appreciably decreasing CH4 emissions (15%) in cattle, but higher concentrations decreased production efficiency due to a reduction of feed digestion and fermentation. Medium-chain fatty acids (MCFA) and PUFA can lower abundance and metabolic activities of rumen methanogens and change their species composition. PUFA can also directly inhibit protozoa and serve as H2 sink through biohydrogenation. Both MCFA and PUFA appear to damage the cell membrane, thereby abolishing the selective permeability of cell membrane, which is required for survival and growth of methanogens and other microbes. The inhibitory effect of fat on methanogenesis is more pronounced in cattle fed concentrate-based diets than in cattle fed forage-based diets. Because C12: and C14:0 is more inhibitory to M. ruminantium at pH 5 than at pH 7, the concentrate level-dependent anti-methanogenic efficacy of MCFA and PUFA is probably attributed to the lower pH associated with high-concentrate diets.

[0508] In some embodiments, the animal feed comprises fat and / or fatty acid. In some embodiments, the animal feed comprises fat and / or fatty acid that is at least about 1%, 2%, 3%, 4%, 5%, or 6% of the diet (e.g., diet based on dry matter).

[0509] Numerous animal feed and feed additives are known in the art. Any agent that reduces CH4 production in a subject (e.g., small molecule inhibitors, e.g., Table 4, probiotic bacterial strain, etc.; see below) described herein or those known in the art may be used as a feed additive. Certain exemplary feed additives include: berberine, nitrate, eucalyptus oil, alliin, diallyl disulfide (DADS), flavanone glycoside (e.g., neohesperidin, isonaringin, poncirin, hesperidin), 3-nitrooxypropanol, rac-4-Phenylbutane-l,2-diyl dinitrate, 2-(hydroxymethyl)-2- (nitrooxymethyl)-l,3-propanediol, N-ethyl-3-nitro-oxy-propionic sulfonyl amide, 5-nitrooxy- pentanenitrile, 5-nitrooxy-pentane, 3-nitro-oxy-propyl propionate, 1,3-bis-nitrooxypropane, 1 ,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, 3-nitro-oxy-propyl benzoate, 3-nitro-oxy- propyl hexanoate, 3-nitro-oxy-propyl 5-nitro-oxy-hexanoate, benzylnitrate, isosorbid- dinitrate, N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide, 3-nitrooxy propionic acid, methyl-3- nitrooxy propionate, ethyl-3-nitrooxy propionate, ethyl-4-nitrooxy butanoate, ethyl-3- nitrooxy butanoate, 5-nitrooxy pentanoic acid, ethyl-5 -nitrooxy pentanoate, 6-nitrooxy hexanoic acid, ethyl-6-nitrooxy hexanoate, ethyl-4-nitrooxy-cyclohexylcarboxylate, 8- nitrooxy octanoic acid, ethyl- 8-nitrooxy octanoate, 11 -nitrooxy undecanoic acid, ethyl-11- nitrooxy undecanoate, 5-nitrooxy-pentanoic amide, 5-nitrooxy-N-methyl-pentanoic amide, lauric acid, and haloform (e.g., bromoform, chloroform, iodoform).

[0510] AGENTS THAT REDUCE CH4PRODUCTION IN RUMINANTS

[0511] COMBINATION TREATMENT

[0512] Vaccines, antibodies, milk, animal feed, and agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, etc.) may be administered to a subject in any combination. By "in combination with," it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent (vaccines, antibodies, milk, animal feed, agents that reduce CH4 production in a subject) will be administered at a dose and / or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of combinations that may improve immune response against at least one methanogen, and / or reduce CH4 production by a subject.

[0513] The combinations can conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical compositions comprising a combination as defined above together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.

[0514] The individual compounds of such combinations can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

[0515] It will further be appreciated that (vaccines, antibodies, milk, animal feed, agents that reduce CH4 production in a subject) in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In some embodiments, the combinations, each or together may be administered according to the split dosing regimens described herein.

[0516] The term “conjoint” or “combination” administration, as used herein, refers to the administration of two or more agents that aid in reducing CH4 production in a subject. The different agents comprising the combination may be administered concomitant with, prior to, or following the administration of one or more agents.

[0517] In certain embodiments, combination administration can demonstrate synergisms between the two or more agents resulting in a greater CH4 reduction in subject than either agent alone.

[0518] Synergistic effects, also known as synergy, refer to the phenomenon where the combined effect of two or more factors or components is greater than the sum of their individual effects. In other words, when these factors or components interact, they create an amplified or enhanced effect that is greater than what would be expected based on their individual contributions. In certain cases, the agents may have different modes of action or mechanisms by which they exert their effects, for example targeting different methanogens or different methanogen enzymes. When these agents are combined, their actions can complement each other, targeting different aspects of a problem or working on multiple pathways simultaneously. This complementary action enhances their overall effectiveness, resulting in a better outcome than either agent could achieve alone.

[0519] In certain cases, synergism can significantly enhance the efficacy of the agents involved. For example, the agents may interact in a way that enhances their absorption, distribution, or bioavailability, increasing their effectiveness in treating a particular condition.

[0520] In certain cases, one agent may enhance the effects of the other without contributing much individually. This is known as potentiation. The presence of one agent can increase the uptake, binding affinity, or sensitivity of the other, making it more potent and effective. The combined effect is greater than what would be achieved by either agent on its own.

[0521] In certain cases, one or more agents may have inherent weaknesses or face resistance from target organisms or systems. By combining them with another agent, the synergistic interaction can bypass or counteract these obstacles, leading to a more effective outcome. For example, antibodies generated via vaccination of a first vaccine may face resistance to ruminal proteases, thus, combining with protease inhibitors or one or more additional vaccine towards certain ruminal proteases may reduce the resistance of the first vaccine.

[0522] In certain cases, combining two agents can amplify the positive effects or benefits they provide individually. For example, combining a vaccine for a methanogen with a vaccine towards a separate microorganism that is syntropic with the methanogen can amplify the positive effects or benefits of the vaccine by further reducing the fitness of the methanogen.

[0523] The level of synergism achieved when combining agents depends on numerous factors and is typically assessed through experimental studies or empirical observations specific to the agents and desired outcomes. Generally, combination of agents can lead to different degrees of synergisms ranging from No synergism to Supra-additive synergism.

[0524] No Synergism: In some cases, the combined effect of two or more agents may simply be additive or even less than additive. This means that the combined effect is equal to the sum of their individual effects or even lower. In such instances, no synergism is observed, and the agents may not interact in a way that amplifies their effects.

[0525] Mild to Moderate Synergism: A common outcome when combining agents is a mild to moderate level of synergism. This implies that the combined effect is greater than the sum 4 of their individual effects, but not dramatically so. The degree of synergism may vary depending on the specific agents and the conditions of their interaction.

[0526] Strong Synergism: In some cases, the combination of agents can lead to a strong synergistic effect. This means that the combined effect is significantly greater than the sum of their individual effects. Strong synergism often results in an amplified and more potent effect, exceeding what would be expected based on the additive effects of the individual agents.

[0527] Supra-additive Synergism: In rare instances, the combined effect of two or more agents can be supra-additive, meaning it surpasses even strong synergism. Supra-additive synergism results in an exceptionally powerful effect that far exceeds the sum of the individual effects. Such cases are usually considered highly beneficial, as they can provide remarkable outcomes in terms of efficacy, efficiency, or other desired parameters.

[0528] Any suitable degree of synergism (%synergism) can be demonstrated with a combination therapy comprising any two or more compositions or agents of the present disclosure (e.g., a vaccine and another agent), such as an improvement of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400% and / or not more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000%, for example 5-1000%, preferably 10-500%, more preferably 30-300%. In certain embodiments, the improvement is measured in the amount of CH4 reduced when administered a combination therapy as compared to either agent alone, for example %Synergism = CH4, combo * (CH4, vaccine)’1- In Certain embodiments, the %Synergism is measured in a herd of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 animals and / or not more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, or 2000 animals and the statistical significance, e.g., coefficient of variation of %synergism within the herd is at least 50, 60, 70, 75, 80, 85, 90, 95, 99, 99.5, or 100%.

[0529] In certain embodiments, the synergism resulting from the combinatorial therapy results in a prolonged efficacy of the treatment as compared to either alone. In certain embodiments, the combinatorial therapy is effective for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 20 months and / or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, or 24 months, for example 1-24 months. In certain embodiments, the length of efficacy of the treatment is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400% and / or not more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000% greater than either agent alone, for example 5-1000%, preferably 10-500%, more preferably 30-300%.

[0530] An exemplary combinatorial therapy includes vaccination with a first vaccine encoding one or more methanogen surface proteins in combination with at least one additional vaccine encoding one or more of different methanogen surface proteins. 4

[0531] Another exemplary combinatorial therapy includes vaccination with a vaccine encoding one or more methanogen surface proteins in combination with administration of a small molecule inhibitor of methanogenesis. Without wishing to be bound to theory, it is hypothesized that the small molecule inhibitor of methanogenesis may remove the plurality of ruminal methanogens, and antibodies generated from the vaccination prevent new methanogens for colonizing the methanogen-deficient rumen.

[0532] Accordingly, in certain aspects, a vaccine of the present disclosure is administered to a subject conjointly or in a combination with at least one inhibitor of CH4 production described herein or those known in the art. In some embodiments, the at least one inhibitor is selected from Table 4.

[0533] In some embodiments, the at least one inhibitor is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times. In some embodiments, the at least one inhibitor is administered to a subject daily, semiweekly, weekly, biweekly (every 2 weeks), monthly, semiannually, or annually. In some embodiments, the at least one inhibitor is administered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

[0534] 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,

[0535] 84, 85, 86, 87, 88, 89, or 90 days. In some embodiments, the at least one inhibitor is administered to a subject for a duration of at least, about, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks. In some embodiments, the at least one inhibitor is administered to a subject for at least 1 week but no more than 1 month. In some embodiments, the at least one inhibitor is administered to a subject orally, intravenously, intramuscularly, or subcutaneously. In preferred embodiments, the at least one inhibitor is administered to a subject orally. In some embodiments, the at least one inhibitor is administered to a subject as a feed additive.

[0536] In some embodiments, the at least one inhibitor is administered to a subject concomitant with, prior to, or following the vaccination with a vaccine of the present disclosure. In some embodiments, the at least one inhibitor is administered to a subject on the same day as the subject is vaccinated. In some embodiments, a subject is administered with the at least one inhibitor one or more times to reduce the CH4 production by the subject, and said subject is vaccinated as a maintenance regimen. In some embodiments, the at least one inhibitor comprises 3NOP. In some embodiments, a subject is administered at least about or no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

[0537] 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

[0538] 49, or 50 grams (g) of 3NOP per dose. In some embodiments, 3NOP is administered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

[0539] 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

[0540] 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,

[0541] 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days.

[0542] In some embodiments, at least about 0.5 g but no more than 25 g of 3NOP is administered to a subject in a given day. In preferred embodiments, about 2.5 g of 3NOP is administered to a subject in a given day. In some embodiments, about 2.5 g of 3NOP is administered to a subject per day for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0543] Table 4: Exemplary methanogenesis inhibitor

[0544]

[0545]

[0546] *Each of the references in Table 4 is incorporated herein by reference.

[0547] Other exemplary inhibitors of CH4 production are discussed below.

[0548] Probiotics that reduce CH4 production in animals

[0549] Probiotics are a class of beneficial active microorganisms or their cultures. Probiotics are useful in reducing CH4 emissions in animals (Table 8C). There are many types of probiotics, and different strains have different inhibitory effects on CH4 emissions. For example, the GA03 strain of Acetobacter is more effective at inhibiting CH4 production than other isolated strains. Most probiotics reduce CH4 production by influencing the activities of ruminal microorganisms, with no adverse effects on animals. In addition, probiotics enhance ruminal fermentation.

[0550] Lactic acid bacteria, which have been used as feed additives for a long time, not only reduce CH4 emissions per unit volatile fatty acid (VFA) output, but also improve the fermentation quality and fiber digestibility of silage. In addition, the denitrifying bacterium Bacillus 79R4 could prevent NO- 2-N poisoning and microbial ecosystems from impairing fermentation efficiency. Furthermore, Bacillus licheniformis reduces CH4 production and increases feed energy and protein utilization.

[0551] Table 5: Exemplary probiotic bacteria that reduce CH4 production

[0552] Prebiotics that reduce CH4 production

[0553] Prebiotics are substances that are not easily digested or absorbed by the host. They selectively stimulate the growth and activity of one or several ruminal microorganisms with a positive effect on ruminal fermentation. Prebiotics suppress ruminal CH4 production in subjects. Prebiotics mainly reduce rumen CH4 production by altering the bacterial community structure, influencing the permeability of the cell walls of methanogenic archaea, and stimulating other bacteria to compete with methanogens for H2 (Table 9).

[0554] The prebiotic chitosan can influence bacterial community structures by altering microbial population compositions, for example, by replacing fibrinolytic enzyme-producing microbes (Firmicutes and Fibrobacteres) with amylolytic enzyme-producing microbes (Bacteroides and Proteus); in turn, reducing CH4 production. Chitosan could influence the ruminal fermentation process by altering VFA distributions and increasing propionic acid concentrations, which reduces CH4 production in turn. However, the reduction in CH4 is associated with the degree of chitosan deacetylation, which could alter the permeability of the methanogen cell wall. In addition, various yeast products could reduce CH4 emissions by stimulating acetic acid-producing bacteria to compete with methanogens or metabolize H2.

[0555] Table 6: Exemplary prebiotic bacteria that reduce CH4 production

[0556] Other agents that reduce CH4 in animals

[0557] Among the CH4 mitigation options, inhibiting the growth or the metabolic activity of methanogens is the most effective approach. Another strategy is to modulate rumen microbiome so that fermentation is shifted toward decreased H2 production and increased production of reduced VFA (e.g., propionate). Provided herein are exemplary and non- exhaustive descriptions of anti-methanogenic compounds evaluated with a focus on their impact rumen methanogens.

[0558] Coenzyme M analogs

[0559] Methyl-CoM reductase (Mcr) mediates the final step of all the methanogenesis pathways and CoM (2-mercaptoethanesulfonic acid) is an essential cofactor serving as the methyl group carrier. Mcr reduces methyl-CoM to CH4. CoM is found in all known methanogens but not in other archaea or bacteria. Several halogenated sulfonated compounds, including 2-bromoethanesulfonate (BES), 2-chloroethanesulfonate (CES), and 3- bromopropanesulfonate (BPS), are structural analogs of CoM, and they can competitively and specifically inhibit Mcr activity, lowering CH4 production at relatively low concentrations. Different species of methanogens vary in sensitivity to these inhibitors. Of three species tested on BES, Mbb. Ruminantium was the most sensitive, while Methanosarcina mazei was the least sensitive, with Methanomicrobium mobile being intermediate. All three species appeared to be resistant to BPS up to 250 pmol / L in pure cultures. The different sensitivity to these CoM analogs has been attributed to varying ability to uptake these inhibitors into the cells. Methanogens able to synthesize their own CoM are less dependent on external CoM and are thus less sensitive. Mbb. Ruminantium is the only ruminal methanogen that requires CoM synthesized by other methanogens. 4

[0560] Halogenated aliphatic C1-C2 hydrocarbon

[0561] Halogenated aliphatic compounds with 1 or 2 carbons, such as chloroform, bromochloromethane (BCM), bromoform, bromodichloromethane, dibromochloromethane, carbon tetrachloride, trichloroacetamide, and trichloroethyladipate, can lower ruminal CH4 production. These halogenated compounds block the function of corrinoid enzymes and inhibit cobamide-dependent methyl group transfer in methanogenesis. These halogenated compounds also competitively inhibit CH4 production by serving as terminal electron (e“) acceptors. Drenching chloroform to cattle inhibited methanogenesis substantially for up to 32 days without affecting feed digestion or basic rumen function. The addition of BCM depressed CH4 production both in vitro and in vivo. In steers fed grain-based diets, BCM decreased CH4 production by 50 to 60% with no signs of toxicity or residues in meat. It was also reported that the abundance of total bacteria and protozoa was not changed, but methanogenesis and growth of methanogens were drastically inhibited by BCM in both batch cultures and continuous fermenters. While the commercial use of chloroform, a recognized carcinogen, is not practical, it provides validation for the class of BCM compounds in reducing CH4 production.

[0562] Some marine plants such as red seaweed, and algae, lichen, and fungi can contain high concentrations of organobromine compounds such as bromomethane and bromoform. A recent in vitro study showed that red seaweed Asparagopsis taxiformis lowered CH4 production by 99% at a dose of 2% of organic matter substrate. No adverse effect on feed digestion or fermentation was noted at <5% (of dry matter) inclusion. Thus, red seaweed, and probably other organobromine -rich plants, may offer a potentially practical natural approach to mitigate CH4 emission.

[0563] Nitrooxy compounds

[0564] 3-Nitrooxypropanol (3NOP) and ethyl-3NOP, two new synthetic compounds, have been shown to have specific anti-methanogenic properties. 3NOP appears to inactive Mcr by competitively binding to the Mcr active site and then oxidizing the Ni1+that is required for Mcr activity. Feeding of 3NOP at a dose rate of 2.5 g / day / cow mixed in diets decreased CH4 emission by 60% per kg of DM intake. In a study using beef cattle, 3NOP fed at 2.0 g / day / cow decreased CH4 yield by 59%, and the inhibition persisted for up to 112 days without much effect on feed intake, nutrient digestibility or total VFA concentrations. In one recent study, 3NOP fed at 40-80 mg / kg feed DM in dairy cows decreased CH4 production by about 30% persistently for up to 84 days. Similarly, 3NOP fed at 2.5 g / day / cow decreased CH4 yield by 37% in dairy cows. In sheep, 3NOP at 0.5 g / day also decreased CH4 production by 29% without adverse effect on digestion or rumen fermentation. However, when 3NOP was directly added to the rumen through rumen cannula at a daily dose of 0.50 or 2.5 g per cow (equivalent to 25 to 125 mg / kg feed dry matter), the degree of CH4 suppression declined to 7-10%. The later study suggests that 3NOP needs to be fed together with the diet to achieve higher efficacy. Thus, 3NOP could be used to lower CH4 emission from cows and sheep without adverse effects on nutrient utilization or animal performance. It has been demonstrated that 3NOP indeed decreased methanogen abundance while increasing the abundance of protozoa.

[0565] Table 7: Exemplary nitrogen-containing compounds that reduce CH4 production in animals

[0566] Pterin compounds 4

[0567] Pterin is a group of structural analogs of deazaflavin (F420), which is a coenzyme involved in two steps of the hydrogenotrophic methanogenesis pathway. Therefore, pterin compounds can competitively inhibit CH4 production. In one study, CH4 production by Mbb. Ruminantium, Ms. Mazei, and Mm. mobile was significantly decreased by lumazin (2,4- pteridinedione) in a dose-dependent manner from 0.06 to 0.24 mmol / L. As expected, pterin is much less efficacious in mixed rumen cultures than in pure methanogen cultures. It was suggested that lumazine could be degraded or transformed by some microbes in mixed cultures or adsorbed to solid particles where it becomes unavailable to methanogens. Some N-substituted derivatives of p-aminobenzoic acid, which are inhibitors of methanopterin synthesis in methanogens, decreased methanogenesis in ruminal cultures without inhibiting VFA production.

[0568] Hydroxymethylglutaryl-CoA (HMG-S-CoA) reductase inhibitors

[0569] All archaea contain long-chain isoprenoid alcohols as the major component of their cell membrane. Isoprenoid alcohols are unique to archaea. They are synthesized from mevalonate that is formed by reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG- S-CoA) catalyzed by HMG-S-CoA reductase. This enzyme is also used for the synthesis of the same precursor ultimately used in cholesterol synthesis in humans. As an inhibitor of HMG-S-CoA reductase, statins (e.g., lovastatin and mevastatin) can inhibit the growth of methanogens by inhibiting the synthesis of mevalonate. Lovastatin and mevastatin may also act as a potential inhibitor of F420-dependent NADP oxidoreductase as shown in the model structure of that enzyme. In the earliest reported study, mevastatin at 5.6 pmol / L inhibited the growth of all three strains of rumen Methanobrevibacter, but not rumen bacteria in vitro. In studies using a rumen simulation technique (Rusitec), lovastatin (150 mg / L) reduced CH4 production by approximately 42% without altering bacterial counts or nutrient fermentation.

[0570] The high cost of pure statins promoted a search for natural sources of statins as agents to mitigate CH4 production. Lovastatin is a secondary metabolite of idiophase of several fungal species (e.g., Penicillium spp., Aspergillus terreus, Monascus purpureus, and Pleurotus ostreatus), and it can reach a concentration up to 2.8% of the dry weight of P. ostreatus (oyster mushrooms) and 261 mg / kg fermented rice straw. Lovastatin extracted from fermented rice straw significantly reduced total CH4 production by rumen methanogens by nearly 28% after 48 h in vitro incubation. Extract from A. terreus-fermented rice straw containing lovastatin (97 mg / g dry mass) also significantly reduced CH4 production and abundance of methanogens, especially Methanobacteriales, and aerobic fungi, but increased 4 several fiber-degrading bacteria. Lovastatin also altered the morphology of M. smithii significantly, resulting in abnormal membrane formation and asymmetric cell divisions and increased HMG-S-CoA reductase gene expression. Fermented rice straw extract also modulated expression of several genes associated with methanogenesis, increasing expression of mtr, mta, and mcr while decreasing expression of hmd mAfno. Supernatant fractions containing statins produced by Mortierella wolfii also appeared promising to inhibit methanogenesis without reducing overall fermentation. In another study using sheep, fermented rice straw containing metabolites (possibly pravastatin and mevastatin) produced by Monascus spp. Decreased CH4 emission (by 30%), the abundance of methanogens, and ruminal acetate: propionate ratio compared with the unfermented rice straw.

[0571] Diallyl disulfide, the main ingredient of garlic oil, is known to inhibit HMG-S-CoA reductase. Garlic oil (300 mg / L) was more effective than lovastatin as an inhibitor of CH4 production (by up to 91% reduction); however, garlic oil also inhibited bacterial growth, which likely reduces the availability of methanogenesis substrates. Garlic oil lowered CH4 production in vitro and growth of methanogens, altered community structure of methanogens after 24 h incubation. Moreover, interestingly, the anti-methanogenic efficacy increased over time up to 18 days of incubation.

[0572] Plant secondary metabolites

[0573] Plants secondary metabolites (PSM), such as saponins, tannins, flavonoids, organosulphur compounds, and essential oils, have anti-microbial activities against several types of microorganisms. Numerous PSM extracts have been recognized as potential inhibitors of rumen methanogens and CH4 production. Some forage plants rich in tannins and saponins have also shown promise at mitigating CH4 emission from animals.

[0574] Table 8: Exemplary plant extracts that reduce CH4 production in animals

[0575] 1. Tannins: Tannins decrease CH4 production by directly inhibiting methanogens and indirectly decreasing H2 production as a result of decreased fiber digestion and protozoal population in the rumen. The inhibitory activity of tannins extracted from Lotus pedunculatus was demonstrated on pure cultures of methanogens. The inhibition of methanogen populations was also shown by tannins in the rumen of goats fed diets containing tannins. Studies on structure- activity relationships have shown that types and molecular weights of tannins are important in determining their potency in lowering CH4 production and abundance and diversity of rumen methanogens, with high molecular weight condensed tannins (CT) being more potent. Such structure-activity relationships have been demonstrated using members of Methanobacteriales including Methanobrevibacter.

[0576] 2. Flavonoids: Flavonoids have not been extensively evaluated with respect to rumen methanogenesis. It was reported that inclusion of flavone, myricetin, naringin, rutin, quercetin, or kaempferol decreased in-vitro CH4 production by 5 to 9 mL / g DM. Their potency ranked as follows: myricetin > kaempferol > flavone > quercetin > naringin > rutin > catechin. Catechin 4 decreased CH4 production both in vitro and in vivo. All the flavonoids, when fed at 0.2 g / kg DM, noticeably decreased relative abundances of hydrogenotrophic methanogens, and citrus Citrus aurantium) extract rich in mixed flavonoids and its pure flavonoid components, neohesperidin and naringin, appeared to result in the greatest inhibition. Methanosarcina spp. Were also inhibited by poncirin, neohesperidin, naringin and their mixture. Flavonoids directly inhibit methanogens and also likely acts as H2 sinks via cleavage of ring structures (e.g., catechin) and reductive dihydroxylation.

[0577] 3. Saponins: The effects of saponins on rumen fermentation, rumen microbial populations, and ruminant productivity have been examined extensively. Quillaja saponin at 1.2 g / L, but not at 0.6 g / L, lowered CH4 production in vitro and the abundance of methanogens (by 0.2-0.3 log) and altered their composition. Ivy fruit saponin decreased CH4 production by 40%, modified the structure of the methanogen community, and decreased its diversity. Saponins from Saponaria officinalis decreased CH4 and abundance of both methanogens and protozoa in vitro. It is hypothesized that saponins lower H2 production, thereby reduce CH4 production.

[0578] 4. Essential oils: The effects, mostly beneficial, of essential oils (EO) on rumen fermentation, microbial populations, and ruminant productivity have frequently been reviewed. Several EO compounds, either in pure form or in mixtures, are anti-methanogenic. The effects of EO on CH4 production and methanogens are variable depending on dose, types, and diet. Five EO (clove, eucalyptus, peppermint, origanum, and garlic oil) that have different chemical structures in vitro at three different doses (0.25, 0.50 and 1.0 g / L) were tested for their effect on CH4 production and archaeal abundance and diversity. Overall, all these EO suppressed CH4 production and abundance of archaea and protozoa in a dosedependent manner, but they differed in potency. Thyme oil or cinnamon oil fed to Holstein steers at 0.5 g / day decreased the relative abundance of total protozoa and methanogens. However, feeding beef cattle a blend of EO (CRINA®) did not affect CH4 production, methanogen abundance or its diversity. Overall, methanogens may be directly inhibited or indirectly inhibited by Eos via inhibition of protozoa and H2-producing bacteria in the rumen.

[0579] Alternative hydrogen sinks

[0580] Compounds with a redox potential higher than CO2 can thermodynamically outcompete CO2 for reducing equivalents produced during rumen fermentation. These compounds, thus, can be used as alternative e“ acceptors to redirect e“ flux away from methanogenesis. The commonly evaluated alternative e“ acceptors are discussed below. 4

[0581] 1. Nitrate and sulfate: Nitrate (NO31-) decreased CH4 production both in vitro and in vivo. Mechanistically, nitrate decreases CH4 production by outcompeting CO2 as an e“ acceptor, and its reduction intermediates, nitrite (NO21-) and nitrous oxide (N2O), also directly inhibit methanogens as well as some H2 producers. Sulfate also lowers CH4 production, but much less effectively than nitrate. Archaeal abundance declined in goats receiving nitrate. While nitrate is not toxic to methanogens, it is toxic to protozoa, fungi and to a lesser extent to select bacterial species, suggesting a more general toxicity of nitrate. Nitrate can replace a portion of the dietary nitrogen as it is reduced to ammonia.

[0582] 2. Nitrocompounds: A few organic nitrocompounds have been evaluated for their efficacy to decrease methanogens and CH4 production. These compounds can serve as e“ acceptors by some bacteria competing with methanogens for reducing equivalents. This is demonstrated by nitroethane that can be used as a terminal e“ acceptor by Dentitrobacterium detoxificans, thereby indirectly decreasing CH4 production. Nitrocompounds may also inhibit methanogenesis by directly inhibiting the activity of formate dehydrogenase / formate hydrogen lyase and hydrogenase, all of which are involved in the early step(s) of the hydrogenotrophic methanogenesis pathway, or inhibiting e“ transfer between ferredoxin and hydrogenase. Nitrocompounds generally are quite effective in lowering CH4 production, with 3-nitro-propionate, 2-nitropropanol, 2-nitroethanol and nitroethane being able to decrease CH4 production by 57 to 98% in vitro. Using sheep, it was shown that nitroethane decreased CH4 production by up to 45% and 69%, respectively, when orally administrated at 24 and 72 mg / kg body weight daily for 5 days. Although less effective than nitroethane, 2-nitropropanol also significantly lowered CH4 production (by 37%) in steers.

[0583] 3. Propionate and butyrate enhancers: Malate, acrylate, oxaloacetate, and fumarate are intermediates of carbohydrate fermentation. They can be converted to propionate or used in anabolism for the synthesis of amino acids or other molecules. They can accept reducing equivalents and thus stoichiometrically lower H2 available for CH4 production. When added at a concentration of 3.5 g / L, fumarate decreased CH4 production by 38% in continuous fermenters with forages as a substrate. Types of forages and their combinations appeared to affect the anti-methanogenic efficacy of fumarate, ranging from 6 to 27% inhibition at 10 mmol / L. Acrylate also depresses CH4 production in the rumen, but to a lesser extent than an equimolar level of fumarate. Malate was found to decrease CH4 production by beef cattle in a dose-dependent manner, with a 16% decrease being noted when fed at 7.5% of DM intake, which corresponds to a 9% reduction per unit of DM intake. Different studies reported different anti-methanogenic potencies of this type of e“acceptors. 4

[0584] Fumarate fed to goats at 10 g / day / goat was found to decrease the abundance of methanogens and CH4 production only by 11.9% while increasing concentrations of total VFA, acetate and propionate. Some of the intermediates of pyruvate conversion to butyrate can act as e“ acceptors, which could also decrease CH4 production.

[0585] 4. Unsaturated organic acids: Unsaturated fatty acids can act as H2 sinks during their biohydrogenation and thereby lower CH4 production. Propynoic acid (an unsaturated analog of propionic acid), 3-butenoic acid and 2-butynoic acid (both unsaturated analogs of butyric acid), and ethyl 2-butynoate each at 6 to 18 mmol / L have been evaluated as alternative e“ sinks to lower methanogenesis in vitro. Only propynoic acid and ethyl 2- butynoate markedly lowered CH4 production, by 65 to 76% and 24 to 79%, respectively. In another study, propynoic acid lowered CH4 production by 67% and 78% at 6 and 12 mmol / L, respectively and decreased methanogen abundance. Propynoic acid and ethyl 2-butynoate are directly toxic to methanogens, and species of methanogens vary in their sensitivity to these two inhibitors, with Mbb. Ruminantium being most sensitive, Ms. Mazei least sensitive, and Mm. mobile intermediate.

[0586] Inhibitors to hydrogen-producing bacteria

[0587] 1. Ionophores: Ionophores, such as monensin and lasalocid, are commonly used to improve rumen microbial metabolism. Being highly lipophilic ion carriers, they pass through the cell wall of Gram-positive bacteria and penetrate into the cell membrane. Therein, they serve as H+ / Na+and H+ / K+antiporters, dissipating ion gradients that are needed for ATP synthesis, nutrient transport, and other essential cellular activities and ultimately resulting in delayed cell division and even cell death. Ionophores preferentially inhibit Grampositive bacteria, including members of class Clostridia, including Ruminococcus species that produce acetate and H2. Ionophores can also inhibit some Gram-negative rumen bacteria, including bacteria that produce formate and H2. Therefore, ionophores may lower CH4 emission by decreasing H2 production. For examples, monensin fed at 24-35 mg / kg diet lowered CH4 production by up to 10% (g / kg DM intake), though no CH4 suppression was observed at 10-15 ppm. In a recent in vivo study, however, monensin at 60 mg / day / cow did not lower CH4 production by tropical cattle, though it decreased CH4 production by about 30% when fed at 250 mg / day / cow. As repeatedly noted, at such high supplementation level, DM intake was lowered, which explains most of the observed decrease in CH4 emission. Ionophores are not known to directly inhibit methanogens, but they can change the population dynamics of methanogen species. For example, monensin decreased the population of Methanomicrobium spp. While increasing that of Methanobrevibacter spp. Total methanogens were also decreased in cattle fed monensin. These can be explained by reduced availability of fband differences in affinity for H2 and growth kinetics among methanogen species.

[0588] 2. Bacteriocins: Bacteriocins are proteins or peptides produced by bacteria and inhibit select microbial species in the rumen and other habitats. There are only a few studies investigating the effect of bacteriocins on CH4 emission. Bovicin HC5, a bacteriocin produced by Streptococcus spp. From the rumen, was reported to suppress CH4 by 50% in vitro. Nisin, a bacteriocin produced by Lactobacillus lactis subsp. Lactis, has also been shown to decrease CH4 production in vitro by up to 40% depending upon its concentration. Similar to monensin, bacteriocins probably modulate rumen fermentation leading towards increased propionate, thereby decreasing CH4 production.

[0589] ADDITIONAL AGENTS THAT REDUCE CH4IN ANIMALS

[0590] BIOCHEMICAL PATHWAYS

[0591] In certain embodiments, the one or more deleterious atmospheric gases and / or precursors thereof are microbially derived through one or more biosynthetic pathway. The deleterious atmospheric gas can be any suitable deleterious atmosphere gas, such as CO2, CH4, nitrous oxide, or a combination thereof. The deleterious atmospheric gas precursor can be any suitable precursor, such as acetate, H2, CO2, methanol, monomethylamine, dimethylamine, trimethylamine, nitric oxide, or a combination thereof. In preferred embodiments, the deleterious atmosphere gas comprises CO2, H2, or CH4, more preferably CH4. In certain embodiments, wherein the resultant deleterious atmospheric gas comprises CH4, the one or more biosynthetic pathways include the acetoclastic, hydrogeno trophic, and methylotrophic pathways, which differ based on the starting substrates, i.e., precursor, (Fig. 1), more preferably the acetoclastic or hydrogenotrophic pathways, even more preferably the acetoclastic pathway.

[0592] The acetoclastic pathway comprises a series of enzymes that convert the precursor acetate through a series of enzymatic conversions to CH4. Starting from acetate, (1) acetate is converted to acetyl phosphate by acetate kinase (ack); (2) acetyl phosphate is converted to acetyl-CoA by phosphotransacetylase (pta); (3) the acetyl group from acetyla-CoA is transferred to a protein intermediate by acetyl-CoA decarbonylase ; (4) the acetyl group is then transferred to tetrhydrosarcinapterin to form 5-methyl-tetrahydrosarcinapterin by methyltetrahydrosarcinapterin methyltransferase; (5) 5-methyl-tetrahydrosarcinapterin is 4 converted to methyl-CoM by methyl-H4SPT:CoM methyltransferase (Mtr); and (6) methyl- CoM is reduced to CH4 by methyl-CoM reductase (Mcr) (Fig. 14 and Fig. 15).

[0593] The hydrogenotrophic pathway comprises a series of enzymes that convert the precursors H2 and CO2 to CH4. Starting from CO2 and H2, (1) a formylmethanofuran dehydrogenase (Fwd / Fmd) produces a formylmethanofuran, (2) which is further converted into 5-formyl-tetrahydromethanopterin by a formylmethanofuran iFUMPT formylatransfer (Ftr); (3) 5-formyl-tetrahydromethanopterin is further converted into 5,10- methenyltetrahydromethanop terin by methyl -H4M PT cyclohydrolase (Meh); (4) 5,10- methenyltetrahydromethanop terin is converted to Ne-methyltetrahydromethanop terin by F420- dependent methylene-FUMPT reductase (Mer); (5) Ne-methyltetrahydromethanopterin is converted to methyl-CoM by methyl-FUMPT: coenzyme M methyltransferase (Mtr); and (6) methyl-CoM is reduced to CH4 by methyl-CoM reductase (Mcr) (Fig. 14 and Fig. 15).

[0594] The methylotrophic pathway comprises a series of enzymes that convert one or more of dimethylamine, methanethiol, methanol, methylamine, methylthiopropanoate, tetramethylammonium, and / or trimethylamine into methyl-CoM, wherein methyl-CoM is reduced to CH4 by methyl-CoM reductase (Mcr) (Fig. 14 and Fig. 15).

[0595] In certain embodiments, provided herein are compositions, methods, and / or kits comprising one or more small molecules that reduce the activity of one or more enzymes in one or more CH4 biosynthetic pathways. The enzyme can be any suitable enzyme, such as 3- (methylthio)propanoate:coenzyme M methyltransferase, acetate kinase, acetyl-CoA decarbonylase, acetyl-CoA decarbonylase / synthase complex 0.202. acetyl-CoA decarbonylase / synthase complex , acetyl-CoA decarbonylase / synthase complex y5, acetyl- CoA synthase, carbon monoxide dehydrogenase, carbonic anhydrase, Co-methyltransferase, coenzyme M reductase, cyclohydrolase, dehydrogenase, dimethylamine- [corrinoid protein] Co-methyltransferase, F420-dependent methylene-FUMPT reductase, F420-dependent methylene-FUSPT dehydrogenase, formylmethanofuran dehydrogenase, formylmethanofuran iFUMPT formyltransferase, formylmethanofuramFUSPT formyltransferase, formyltransferase, FF-forming methylene-FUMPT dehydrogenase, methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, methenyl-FUMPT cyclohydrolase, methyl-coenzyme M reductase, methyl-FUSPTiCoM methyltransferase, methylated [methylamine-specific corrinoid protein]: coenzyme M methyltransferase, methylcobamide:CoM methyltransferase, methylthiol: coenzyme M methyltransferase, methyltransferase, MtaC protein: coenzyme M methyltransferase, phosphotransacetylase, tetrahydromethanopterin S -methyltransferase, tetramethylammonium methyltransferase, trimethylamine-corrinoid protein Co-methyltransferase, or a combination thereof. In a preferred embodiment, the enzyme comprises methyl-CoM reductase (Mcr) (Fig. 16).

[0596] COMPOSITIONS FOR REDUCING PRODUCTION OF DELETERIOUS ATMOSPHERIC GASES AND / OR PRECURSORS THEREOF

[0597] In certain embodiments provided herein are compositions. In certain embodiments, provided herein are compositions comprising one or more small molecules. In preferred embodiments, provided herein are compositions comprising one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors thereof. The small molecule can be any suitable small molecule for reducing the production of one or more greenhouse gases and / or precursors thereof, for example a small molecule that interferes with the uptake and / or conversion of acetate, H2, CO2, methanol, monomethylamine, dimethylamine, trimethylamine, nitric oxide, or a combination thereof, and / or a small molecule that interfere with the production of CO2, H2 nitrous oxide, or a combination thereof. In preferred embodiments, the small molecule interferes with the uptake and / or conversion of acetate, H2 and / or CO2 and / or the production of CO2 or CH4, more preferably with the production of CH4.

[0598] Small molecules that affect production of deleterious atmospheric gases and / or precursors thereof

[0599] In certain embodiments, provided herein is a composition for reducing emissions of deleterious atmospheric gasses and / or precursors thereof comprise: one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and / or precursors thereof. The one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and / or precursors can be any suitable molecule.

[0600] In certain embodiments, the one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and / or precursors comprises a compound with the formula the formula R1-[CH2]n-ONO2 wherein n is an integer from 1 to 15;

[0601] R1is selected from the group consisting of H, Ci-Cealkyl, phenyl, — OH, — NH2, — CN,

[0602] — COOH, — O(C=O)R3, — NHC(=O)R3, SO2NHR3, or — ONO2, — SH and R3is Ci-

[0603] Cealkyl, phenyl, pyridyl; with the proviso that when n is >3 the hydrocarbon chain may be interrupted by — O— or — NH— .

[0604] In some embodiments, the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors comprises 3-nitrooxypropanol, 9-nitrooxynonanol, 5-nitrooxy pentanoic acid, 6-nitrooxy hexanoic acid, bis(2- hydroxyethyl)amine dinitrate, 1 ,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, or any combination thereof. Preferably, the one or more small molecules is 3-nitrooxypropanol (3NOP).

[0605] In some embodiments, the composition comprises about 1 to about 25% by weight of the small molecule, about 5 to about 20% by weight of the small molecule, or about 5 to about 15% by weight of the small molecule.

[0606] Solid carriers

[0607] In certain embodiments, the composition further comprises one or more solid carriers. As used herein, the term “solid carrier” includes additives commonly used in the preparation of powderous formulations such as thickeners, for example gums or cellulose derivatives such as xanthan gum, karaya gum and / or ethylcellulose. The one or more solid carriers can be any agriculturally suitable carrier, such as attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, a natural or synthetic derivative thereof, or a combination thereof.

[0608] In certain embodiments, the one or more solid carriers comprises any carrier suitable for ingestion, such as a saccharide comprising cellulose, xantham gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, or a combination thereof.

[0609] In some embodiments, the carrier comprises attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, a natural or synthetic derivative thereof, or a combination thereof.

[0610] In other embodiments, the carrier comprises attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, silica, propylene glycol, hemp protein, biochar, montmorillonite, activated charcoal, lignin, wood flour, hemp protein, pea protein, soy protein, gelatin, casein, chitosan, talc, calcium phosphate, arginine, lysine, calcium carbonate, 4 carbon black, glutamine, betaine, bismuth phosphate, bismuth citrate, iron phosphate, or any combination thereof.

[0611] In some embodiments, the carrier comprises the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, or a combination thereof.

[0612] In some embodiments, the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, carrageenan, cellulose acetate, hydroxypropyl cellulose, cellulose acteate phthalate, maltrodextran, dextran, inulin, corn starch, amylopectin, sodium starch glycolate, pentaerthritol, cyclodextrin, or a combination thereof.

[0613] In certain preferred embodiments, the solid carrier comprises silica and ethylcellulose, more particularly about 10% to about 50% by weight of the silica and about 50 to about 90% by weight of the ethylcelluose.

[0614] In other preferred embodiments, the solid carrier comprises silica and activated charcoal, particularly about 10% to about 90% by weight of the silica and about 10% to about 90% by weight of the activated charcoal.

[0615] In certain embodiments, the binder further comprises arginine, lysine, or both arginine and lysine. While not being bound by theory, it is believed that arginine and lysine are capable of forming hydrogen bonds with the small molecule, such as 3NOP, thereby altering the release rate.

[0616] In other preferred embodiments, the carrier comprises activated charcoal and ethylcellulose, particularly about 10% to about 50% by weight of the activated charcoal and about 40 to about 90% by weight of the ethylcellulose.

[0617] In some embodiments, the carrier further comprises about 1 to about 10% by weight of sodium lignosulfate. While not being bound by theory, it is believed that sodium lignosulfate improves coating adhesion to the tablet resulting in a reduction in release rate of the small molecule.

[0618] In other embodiments, the carrier comprises arginine and polycaprolactone, such as about 10 to about 60% by weight of the arginine and about 30 to about 90% by weight of the polycaprolactone. 4

[0619] In other preferred embodiments, the carrier comprises 25% silica, 66% polycaprolactone, such as about 10 to about 60% by weight of the silica and about 30 to about 90% by weight of the polycaprolactone.

[0620] In certain embodiments, the composition comprises a granular shape. The composition may comprise any suitable shape, such as a spherical-, square-, rectangular-, capsular-, cylindrical-, conical-, ovular-, triangular-, diamond-, disk-like shape, or a combination thereof. In certain embodiments, the shape of the particle affects the rate of dissolution of the particle.

[0621] The granular particle can comprise any suitable texture, for example hard or soft. In certain embodiments, the texture of the particle affects the rate of dissolution of the particle. In certain embodiments, the composition comprises a combination of differently textured pellets each of which release the small molecule at different rates.

[0622] In certain embodiments, the granular particles comprise a uniform size distribution, for example about ± 20%, ± 15%, ±10%, ± 5%, ± 2%, or ± 1% size distribution in the median particle size. In certain embodiments, the granular particles comprise a non-uniform size distribution, for example greater than about ± 20%. In certain embodiments, the granular particles comprise a plurality of differently sized populations of granular particles each of which comprise a uniform size distribution.

[0623] In certain embodiments, the one or more solid carrier dissolves and thereby releases the one or more small molecules that reduce the production of greenhouse gases and / or precursors thereof. In a preferred embodiment, the one or more solid carriers will dissolve in water.

[0624] Extended and delayed release

[0625] It may be necessary to vary the rate of dissolution of the composition. For example, one may want to produce an extended-release formulation, wherein the composition releases the one or more small molecules over a period of time to maintain a suitable environmental concentration of the one or more small molecules. This can be beneficial to reduce the frequency of applications, for example to reduce labor costs and / or applications in rural and / or hard to reach environments. In certain embodiments, complete dissolution of the composition and full release of the one or more small molecules occurs over at least about 1 , 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, and / or nor more than about 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, or 147 days, for example about 1 to about 147 days, preferably 4 about 7 to about 63 days, more preferably about 7 to about 42 days even more preferably about 14 to about 42 days yet even more preferably about 14 to about 28 days.

[0626] In certain embodiments, the extended-release formula may comprise any suitable extended-release formula. In certain embodiments, the extended-release formula comprises one or more additives that reduce the rate of dissolution of the composition, one or more additives that reduce the rate of dissolution of the small molecule from the composition, one or more additives that comprise a membrane that dissolves over time, wherein the rate of dissolution of the membrane controls the rate of release of the one or more small molecules, a suitable alternative, or a combination thereof.

[0627] In certain embodiments, the population of granular particles comprises a plurality of populations of granular particles wherein each population comprises a different formulation and / or shape. In certain embodiments, the population of granular particles comprises a first population and a second population. In certain embodiments, the population of granular particles further comprises at least 1, 2, 3, 4, 5, 5, 6, 8, or 9 and / or no more than 4, 5, 6, 7, 8, 9, or 10 additional populations, for example a total of 3-10 additional populations, preferably 3-7 additional populations, more preferable 3-5 additional populations. In a preferred embodiment, each of the additional populations comprises a different formulation than the others.

[0628] In certain embodiments, the rate of dissolution of the granular particles is modulated by the size of the granular particle. In certain embodiments, smaller granular particles dissolve faster than larger granular particles, such that each successive larger population in the plurality of populations of differently size particles provides a delayed release compared to the smaller populations of particles. In certain embodiments, an increased proportion of larger to smaller granular particles in a population of granular particles results in slower rates of dissolution of the population of granular particles.

[0629] In certain embodiments, the first population of particles comprises an immediate release formulation. In certain embodiments, the second population comprises a delayed release formulation, wherein the second population dissolves and / or releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and / or precursors after the first population. In certain embodiments, each additional population comprises a delayed release formulation, wherein each population dissolves and / or releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and / or precursors at a different time than each of the other populations. 4

[0630] In some embodiments, an immediate release formulation releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gases with about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days. In some embodiments, a delayed release formulation releases the one or small molecules after about 1 week or more, for example up to about 21 weeks.

[0631] In some embodiments, the first population of particles provides has a half-life for the one or more small molecules (such as about 1 to 12 hrs), the second population has a longer half-life (such as about 24 or more hrs), each additional population has a longer half-life than the previous population such that an effective amount of the one or more small molecules is maintained for weeks or months. Thus, the present compositions advantageously do not, in such embodiments, require repeated, frequent applications.

[0632] In certain embodiments, the extended release happens within the first 10% of the halflife and then the 1st delayed happens with 1-2 half lives, then the next with 1-2 of the delayed release.

[0633] In some embodiments, the particles have a size ranging from about 1 mm to about 20 mm, about 1 to about 15 mm, about 1 to about 10 mm, about 5 to about 20 mm, about 5 to about 15 mm, or about 5 to about 10 mm.

[0634] Coatings

[0635] In certain embodiments, the composition may comprise a coating, for examples particles or tablet having a coating. The coating can comprise any suitable coating, such as a wax, a fat, or a synthetic polymer. In certain embodiments, the wax comprises organic compounds consisting of long alkyl chains, natural waxes (plant, animal) which are typically esters of fatty acids and long chain alcohols as well as synthetic waxes, which are long-chain hydrocarbons lacking functional groups. In certain embodiments, the fat comprises a wide group of compounds which are soluble in organic solvents and largely insoluble in water such as hydrogenated fats (or saturated fats) which are generally triesters of glycerol and fatty acids. Suitable fats can have natural or synthetic origin. In certain embodiment, the fat comprises glycerine monostearate, carnauba wax, candelilla wax, sugarcane wax, palmitic acid, stearic acid hydrogenated cottonseed oil, hydrogenated palm oil and hydrogenated rapeseed oil, or combinations thereof. Any suitable synthetic polymer can be used, such as poly-L-glutamic acid (PGA) and polylactic acid (PLA). In preferred embodiments, the synthetic polymer is at least partially water soluble.

[0636] The coating may be single layer or multiple layers, preferably two layers. In some embodiments, the coating is selected from cellulose acetate phlalate, ethyl cellulose, hydroxypropyl cellulose, polycaprolactone, alginate, chitosan, polyethylene glycol, cellulose acetate, triacetin, propylene glycol, n-methyl-2-pyrollidone, and any combination thereof.

[0637] In certain preferred embodiments, the coating comprises two or more poly electrolytes, such as polystyrene sulfonate, polyethyleneimine, sodium lignosulfate, polyglutamic acid and poly-L-lysine, poly-L-arginine, polyallylamine hydrochloride, polyacrylic acid, or any combination thereof.

[0638] In some preferred embodiments, the poly electrolytes comprise poly allylamine hydrochloride and sodium lignosulfate.

[0639] In some preferred embodiments, the poly electrolytes comprise poly allylamine hydrochloride and polystyrene suylfonate.

[0640] In other preferred embodiments, the polyelectrolytes comprise sodium lignosulfate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine, and sodium lignosulfate.

[0641] In still other preferred embodiments, the polyelectrolytes comprise polystyrene sulfonate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine.

[0642] The polyelectrolytes may, in certain embodiments, be chemically cross-linked with a cross-linking agent.

[0643] In certain embodiments, the composition comprises one or more coatings applied with minimal to no bubbles. Additionally or alternatively, the composition comprises one or more coatings that comprise a foam or a plurality of air bubbles. In certain cases, the foamed coating can temporarily alter the buoyancy of the composition. One such example includes a composition comprising a foamed coating that floats when initially applied, then, after a period of time, the air pockets in the foamed coating fill with water resulting in the composition sinking to the bottom.

[0644] Additives with a density greater than water

[0645] In certain embodiments, the composition further comprises one or more additives with a density greater than water. For example, the additive may have a density greater than 1.1, preferably about 1.1 mg / mL to about 3 mg / mL, about 1.5 to about 3 mg / mL, about 1.5 to about 2.5 mg / mL, or about 1.5 to about 2 mg / mL. Suitable additives include silica, attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, or a combination thereof. In certain embodiments, the one or more additives with a density greater than water result in the composition sinking below the surface. In certain embodiments, the one or more additives with a density greater than water result in the composition partially or completely sinking to the bottom. In preferred embodiments, the composition completely sinks to the bottom. In certain embodiments, the composition comprising the additive with a density greater than water has a density of at least 1.1, preferably about 1.1 mg / mL to about 3 mg / mL, about 1.5 to about 3 mg / mL, about 1.5 to about 2.5 mg / mL, or about 1.5 to about 2 mg / mL.

[0646] Agriculturally beneficial additives

[0647] In certain embodiments, the composition further comprises one or more agriculturally beneficial additives. The agriculturally beneficial additive can be any suitable additive depending on the application, such a vitamin, a nutrient, an antibiotic, a fungicide, or a combination thereof.

[0648] In certain embodiments, the additive includes one or more suitable components that reduce methanogenesis by methanogens, such as, seaweed (e.g., Asparagopsis taxiformis), kelp, 3-nitrooxypropanol, anthraquinones, ionophores (e.g., monensin and / or lasalocid), polyphenols (e.g., saponins, tannins), organosulfurs (e.g., garlic extract), flavonoids (e.g., quercetin, rutin, kaempferol, naringin, and anthocyanidins; bioflavonoids from green citrus fruits, rose hips and black currants), carboxylic acid, terpenes (e.g., D-limonene, pinene and citrus extracts), or a combination thereof.

[0649] METHODS FOR REDUCING PRODUCTION OF DELETERIOUS ATMOSPHERIC GASES AND / OR PRECURSORS THEREOF

[0650] In certain embodiments provided herein are methods. In certain embodiments, provided herein are methods for using one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors thereof. In certain embodiments, provided herein are methods for applying one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors thereof to any suitable environment. The suitable environment can comprise any suitable environment. In some embodiments, the suitable environment comprises an environment in which a ruminant (unvaccinated or vaccinated) lives or occupies, e.g., a habitat for a ruminant. In some embodiments, the suitable environment comprises the rumen.

[0651] In certain embodiments, the method for reducing emissions of deleterious atmospheric gasses and / or precursors thereof comprises applying a composition comprising one or more small molecules that reduce the production of the deleterious atmospheric gasses and / or precursors thereof. The composition can comprise any suitable composition. In a 4 preferred embodiment, the composition comprises any one of the compositions as described in the Compositions for reducing production of deleterious atmospheric gases and / or precursors thereof section above. In a more preferred embodiments, the composition comprises 3NOP. In certain embodiments, the composition is applied to a water source, such as a trough or a pond, from which a ruminant ingests the composition or a portion thereof from the water source into the rumen. For example as illustrated in Figs. 17-18.

[0652] In certain cases, the composition needs to be reapplied periodically to maintain a suitable concentration of the one or more small molecules. In certain embodiments, the method further comprises reapplying after a period of time a composition comprising one or more small molecules that reduce the production of the deleterious atmospheric gasses and / or precursors thereof. In certain embodiments, the method further comprises, reapplying again after a period of time a composition comprising one or more small molecules that reduce the production of the deleterious atmospheric gasses and / or precursors thereof. Any suitable number of reapplications may be performed as needed to maintain an effective amount of the one or more small molecules. In some embodiments, the composition is reapplied after about 7 to about 28 days, about 7 to 46 days, about 7 to 92 days or about 7 to 147 days.

[0653] In certain embodiments, the composition is delivered to one or more water sources.

[0654] In certain cases, the concentration of the one or more small molecules can be measured to ensure the presence of a suitable concentration of the one or more small molecules. Any suitable method may be used to measure the concentration, such as a strip test, liquid chromatography, or thin layer chromatography. The method can be performed with or without human intervention.

[0655] Kits for reducing production of deleterious atmospheric gases and / or precursors thereof

[0656] In certain embodiments, provided herein are kits. In certain embodiments, the kit comprises any one of the compositions as described in the Compositions for reducing production of deleterious atmospheric gases and / or precursors thereof section. In certain embodiments, the kit further comprises a suitable container for shipping.

[0657] METHODS OF USING VACCINES, ANIMAL FEED, ANTIBODIES, MILK

[0658] Provided herein are methods of using the vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, etc.), or any combination thereof. In certain aspects, provided herein are methods of inducing an immune response against at least one methanogen in a subject, the method comprising administering to the subject the vaccines or pharmaceutical compositions of the present disclosure.

[0659] In some embodiments, the immune response comprises a B cell response (e.g., to produce the antibodies). The antibodies produced in response to the vaccine are transferred to the saliva of the subjects, which are swallowed by the subjects to enter the rumen. Once in the rumen, the antibodies come in contact with at least one methanogen to bind / neutralize said methanogen.

[0660] As used herein, the term “neutralization of a methanogen” encompasses any reduction in one or more activities that are normally carried out by the methanogen in the absence of the antibodies that bind the methanogen.

[0661] In some embodiments, the activity of a methanogen includes but is not limited to, the activity that aids in producing CH4 gas. For example, binding of the antibodies to the methanogen may reduce the ability of the methanogen to carry out biochemical reactions that are necessary to produce CH4, e.g., reduce the ability to convert H2 and CO2 or acetate into CH4 and ATP. In some embodiments, the reduced ability to produce CH4 may lower the fitness of methanogen in the rumen.

[0662] In some embodiments, the activity of a methanogen includes but is not limited to, the activity that aids in forming a granular colony with other bacteria. In some embodiments, such activity may be disrupted physically - e.g., antibodies binding to the methanogen would prevent physical association and / or film formation of the granular colony of bacteria. In some embodiments, a reduction in the activity of forming a granular colony may lead to the reduced ability of a methanogen to remain in the rumen. In some embodiments, such reduced ability may result in the reduction of the total number of methanogens inside the rumen.

[0663] In certain aspects, provided herein are methods of reducing the activity, number, and / or type of methanogens in the gut of a subject, the method comprising administering to the subject the vaccines or pharmaceutical compositions of the present disclosure.

[0664] In certain aspects, provided herein are methods of reducing the amount of CH4 produced by a subject, the method comprising administering to the subject the vaccines or pharmaceutical compositions of the present disclosure.

[0665] In preferred embodiments, any one of the methods produces an antibody against at least one methanogen. In some embodiments, the antibody is an IgG or an IgA. In preferred embodiments, the antibody is an IgA. The IgA isoform, at least in cattle, may be more stable p in the rumen. For example, IgA levels in cattle saliva were reduced by only 40% after 8h exposure to rumen contents while IgG levels were reduced by 80%.

[0666] In some embodiments, the antibody is produced in an amount sufficient to: (a) carry the antibody to the gut; (b) reduce the number and / or type of methanogens in the gut; and / or (c) reduce the amount of CH4 produced by the subject.

[0667] In some embodiments, the method reduces the CH4 emission from the subject by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared with a control.

[0668] Additionally or alternatively, the method reduces the H2 emission from the subject from at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared with a control.

[0669] Additionally or alternatively, the method increases the feed conversion efficiency of the subject by about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

[0670] Additionally or alternatively, the method increases the concentration of one or more volatile fatty acids (e.g., propionate, butyrate, acetate) in the rumen of the subject by about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

[0671] Additionally or alternatively, the method increases the average daily gain (ADG) of the subject by about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

[0672] Additionally or alternatively, the method increases the dry matter intake (DMI) of the subject by about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control. Additionally or alternatively, the method increases the milk production of the subject by about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

[0673] In some embodiments, the control is an accepted reference, or the amount of CH4 production in a subject that has not been vaccinated.

[0674] Notably, CH4 emission or production of CH4 by a subject can occur at any part of its intestinal track, which includes, e.g., a rumen and a lower bowel (lower intestinal track). The rumen accounts for 90% of all CH4 production. The rumen has no adaptive immune response. 4

[0675] Thus, to be effective in reducing the level of CH4 production, the rumen or a methanogen therein must be exposed to neutralizing antibodies that bind and inactivate the methanogen. By contrast, the lower bowel, which accounts for 10% of all CH4 production, has adaptive immune response such that any immune response due to a vaccine can be amplified in the lower bowel. Accordingly, in preferred embodiments, the result of immune response from a vaccine is exposed to the lower bowel of a subject, which then further amplifies the effect of the vaccine. In other words, in preferred embodiments, a vaccine of the present disclosure or the immune response it elicits is exposed to a methanogen in a lower bowel of the subject. Thus, the present disclosure encompasses a method of reducing (i) CH4 production and / or (ii) activity, number, and / or type of methanogens in the lower intestinal track of a subject, the method comprising administering to the subject a vaccine comprising at least one methanogen cell surface protein or a fragment thereof.

[0676] As described herein, in some embodiments, the vaccine or the pharmaceutical composition is administered to the subject via a route selected from intramuscular administration, intradermal administration, subcutaneous administration, and nasal administration.

[0677] In some embodiments, the subject is administered with at least one dose of the vaccine or pharmaceutical composition.

[0678] In some embodiments, the subject is administered with at least one or two repeat doses of the vaccine or pharmaceutical composition (e.g., booster dose).

[0679] In some embodiments, the subject is administered with the repeat dose of the vaccine or pharmaceutical composition after at least about 2 weeks, 1 month, 6 months, or 12 months from the time the subject is administered with the preceding dose of the vaccine.

[0680] In some embodiments, the subject is administered with the repeat dose of the vaccine or pharmaceutical composition no more than about 3 months, 6 months, 12 months, or 24 months from the time the subject is administered with the preceding dose of the vaccine.

[0681] In some embodiments, the subject receives the repeat dose of the vaccine after at least about 2 weeks and no more than about 18 months from the time the subject is administered with the preceding dose of the vaccine.

[0682] In some embodiments, the subject receives a repeat dose of the vaccine after at least about 4 weeks and no more than about 12 months from the time the subject is administered with the preceding dose of the vaccine. 4

[0683] In certain embodiments, the methods of the present disclosure further comprises administering to the subject at least one agent (e.g., at least one additional agent) that reduces the level of CH4 produced by the subject.

[0684] The vaccine composition may be administered before, concurrently with, or after, any agent, milk, antibody, animal feed, or any composition of the present disclosure.

[0685] In some embodiments, the at least one agent is selected from 3-Nitrooxypropanol (3N0P), ethyl-3NOP, 2-bromoethanesulfonate (BES), 2-chloroethanesulfonate (CES), 3- bromopropanesulfonate (BPS), bromochloromethane (BCM), bromoform, bromodichloromethane, dibromochloromethane, carbon tetrachloride, trichloroacetamide, trichloroethyladipate, lumazin (2,4-pteridinedione), p- aminobenzoic acid, lovastatin, mevastatin, pravastatin, diallyl disulfide, garlic oil, saponins, tannins, flavonoids, nitrate, nitroethane, -nitro-propionate, 2-nitropropanol, 2-nitroethanol, malate, acrylate, oxaloacetate, fumarate, propynoic acid, 3-butenoic acid, 2-butynoic acid, ethyl 2-butynoate, monensin, lasalocid, bovicin HC5, nisin, and any combination thereof.

[0686] In preferred embodiments, the agent is 3N0P or ethyl-3NOP.

[0687] In certain aspects, provided herein are methods of reducing CH4 production in a subject, the method comprising orally administering to and / or feeding the subject the antibody, the milk and / or the derivatives thereof, and / or the animal feed of the present disclosure.

[0688] In certain aspects, the methods of the present disclosure relate to a subject. In some embodiments, the subject is selected from a cow, a bull, a bison, a yak, a buffalo, an antelope, a goat, a sheep, a deer, a giraffe, a caribou, a gazelle, a macropod, a llama, a camel, and an alpaca.

[0689] In some embodiments, the subject is an offspring (e.g., calf) of the vaccinated female subject that received the milk comprising an antibody that binds at least one methanogen.

[0690] In some embodiments, the subject is an adult subject. In other embodiments, the subject is a young subject (e.g., a calf). In some embodiments, a young subject includes a subject from birth to weaning. In some embodiments, a young subject includes a subject from birth up to two years of age, such from birth up to 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Accordingly, in some embodiments, a young subject may be at least, about, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months in age. In preferred embodiments, a young subject is administered with a vaccine, antibodies, milk, animal feed, agent (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other composition of the present p disclosure prior to establishment of methanogens. In some such embodiments, a lower dose of vaccine or other agents may be required.

[0691] In some embodiments, a subject is a subject born from a vaccinated parent(s). In some embodiments, a subject is a subject born from a vaccinated mother such that the subject received a high level of methanogen-neutralizing antibodies in the colostrum and milk fed to the subject at birth. Such a subject or a subject who received an early treatment may have low initial methanogen establishment, thereby enhancing a long term performance of the vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces CH4 production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure.

[0692] In some embodiments, a subject is vaccinated with each change in hands and / or environment, such as from one owner to another, one ranch to another, or one geographical area to another. Typical ranch operations will vaccinate a newly arrived animal to manage disease, and vaccinating with a vaccine of the present disclosure (e.g., a vaccine that targets at least one methanogen cell surface protein) will fall into this standard agronomic practice.

[0693] In some embodiments, a subject is vaccinated with a vaccine of the present disclosure (e.g., a vaccine that targets at least one methanogen cell surface protein) at a time when the subject is subject to at least one other vaccination. For example, a subject (e.g., a domesticated ruminant, a dairy cow, a beef cow) is subject to vaccination against infectious bovine rhinotracheitis (IBR), bovine virus diarrhea (BVD), parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), clostridia, E. Coli mastitis, leptospirosis, mannheimia hemolytica, brucella, vibriosis, Campylobacter, trichomonas, trichomoniasis, rotavirus, foot and mouth disease virus (FMDV), coronavirus, and / or respiratory disease. An exemplary vaccination regimen for a ruminant throughout the life cycle is shown in Table 9.

[0694] Additionally or alternatively, CH4 reducing vaccine compositions may be combined with one or more additional antigens configured to target infectious bovine rhinotracheitis (IBR), bovine virus diarrhea (BVD), parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), clostridia, E. Coli mastitis, leptospirosis, mannheimia hemolytica, brucella, vibriosis, Campylobacter, trichomonas, trichomoniasis, rotavirus, foot any mouth disease virus (FMDV) coronavirus, and / or respiratory disease to form a multivalent vaccine composition, which is then administered to the subject as disclosed herein.

[0695] Table 9: Exemplary administration schedule of USDA-approved vaccinations for a ruminant

[0696] Vaccinating a large number of animals (e.g., in a ranch, e.g., herding ruminants) is a challenging process. Thus, in preferred embodiments, the administration of a vaccine of the present disclosure is incorporated into a pre-existing vaccination program that an animal is already subject to. Such a method reduces the cost, time, and efforts in administering a vaccine of the present disclosure to an animal.

[0697] METHODS FOR DETECTION OF CH4PRODUCTION

[0698] Various methods for detecting the level of CH4 produced by animals are known in the art and can be applied to the methods of the present disclosure. 4

[0699] In some embodiments, portable respiration hoods for tethered and non-tethered animals (Garnsworthy et al. (2012) J. Dairy Sci. 95:3166-3180; Garnsworthy et al. (2019) Animals 9:837; Zimmerman and Zimmerman W02011130538; each of which is incorporated herein by reference) directly measure the gas concentration of incoming and exhaust air from individual animals.

[0700] In some embodiments, tracer-ratio gas releases from the animal (Johnson et al. (1994) Environ. Sci. Technol., 28, 359-362, which is incorporated herein by reference), such as Sulfur hexafluoride (SF6) (Grainger et al. (2007) J. Dairy Sci., 90:2755-2766; Vechi et al. (2022) Agriculture, Ecosystems and Environment 330:107885; each of which is incorporated herein by reference), assumes that the tracer gas and the emitted CH4 have similar transport paths, so that a tracer measurement can establish the CH4 emission rate.

[0701] In some embodiments, micrometeorological techniques are typically considered a herd-scale measurement, where the emission rate is calculated from the measurement of enhanced gas concentrations downwind of an animal herd (Harper et al. (2011) Anim. Feed Sci. Tech., 166-167, 227-239, which is incorporated herein by reference), and these include the mass balance technique (Laubach et al. (2008) Aust. J. Exp. Agr., 48:132-137; Lockyer and Jarvis (1995) Environ. Pollut. 90:383-390; each of which is incorporated by reference), eddy covariance (Dengel et al. (2011) Glob. Change Biol., 17:3524-3533; Felber et al. (2015) Biogeosciences, 12:3925-3940; each of which is incorporated herein by reference), and inverse dispersion techniques (Flesch et al., (2005) Atmos. Environ., 39 :4863^-874; Todd et al. (2014) J. Environ. Qual., 43 : 1125-1130 ; Bai et al. (2021) Atmos. Meas. Tech., 14:3469- 3479; each of which is incorporated herein by reference). The main advantage of micrometeorological techniques is that they do not interfere with the animals or the environment.

[0702] There are also devices that measure the level of CH4 (see e.g., Rey et al. (2019) Animals 9:563, Mapfumo et al. (2018) Pastoralism: Research, Policy and Practice 8:15; each of which is incorporated herein by reference). For example, the laser CH4 detector (LMD) is a handheld open path laser measuring device (e.g., LaserMethaneMini (Tokyo Gas Engineering Co., Ltd. Anritsu Devices Co., Ltd., Tokyo, Japan)). The principle of the LMD measuring technology is described (Chagunda et al. (2013) Animal, 7:394-400; Garnsworthy et al. (2012) J. Dairy Sci. 95:3166-3180; and Chagunda et al. (2009) Comput. Electron. Agric. 68:157-160; each of which is incorporated herein by reference). Briefly, this device is based on infrared absorption spectroscopy using a semiconductor laser for CH4 detection. The device must be pointed towards the nostrils of the cow from a fixed distance. Then, the LMD measures the density of the air column between the device and the animal’s nostrils. The reflected laser beam is detected by the device, and its signal is processed and converted to the cumulative CH4 concentration along the laser path in ppm-m. The LMD is connected to a tablet (Samsung Galaxy Tab A6, New Jersey, USA) running GasViewer app (Tokyo Gas Engineering Solutions, Tokyo, Japan) via Bluetooth connection for exporting and storing the data in real time at 0.5 s intervals. The effect of atmospheric ambient CH4 concentration from the measurements is discounted using the offset function of the LMD.

[0703] The non-dispersive infrared analyzer CH4 analyzer (NDIR) (Guardian NG Edinburg Instruments Ltd., Livinstong, UK) is one of the so-called sniffer methods that measure CH4 concentration (ppm) in breath or exhaled air. These methods have been previously used (e.g., by Garnsworthy et al. (2012) J. Dairy Sci. 95:3166-3180) to assess the CH4 production of dairy cows at commercial farms. Briefly, a gas sampling tube from the front of a cow’s head to a gas analyzer to continuously measure CH4 concentration in the cow’s breath is used. Then, air is drawn through the instrument by an integral pump between the gas inlet port and analyzer. The device can have a range of 0 to 10,000 ppm, and air can be sampled continuously at a rate of 1 L / min through an 8 mm polyamide tube, using approximately 2 m of tube from the analyzer to cow’s nostrils. CH4 concentration can be recorded at 1 s intervals and stored in a datalogger (Data Recorder SRD-99; Simex Sp. Z 0.0, Gdansk, Poland). Baseline or ambient CH4 concentration can be calculated as mean CH4 concentration before starting the measurements and subtracted from the measured data. Each day before starting measurements, the NDIR analyzer should be verified using standard mixtures of CH4 in N2 (0.0%, 0.25%, 0.50%, 0.75% and 1.0%; MESA International Technologies INC, Santa Ana, CA, USA).

[0704] Certain methods and devices are described further below and in Table 10.

[0705] Table 10: Features of exemplary methods for measuring CH4 output by individual animals

[0706] As indicated above and shown in Table 10, exemplary methods include respiration chambers, the sulfur hexafluoride (SF6) tracer technique, breath sampling during milking or feeding, the GreenFeed system, and the laser CH4 detector. Each method measures different 4 components of CH4 output. Only respiration chambers measure total emissions from the animal via the oral, nasal and anal routes; all other methods ignore emissions via the anus and only measure CH4 emitted in breath. Breath measurements are justified because 99% of CH4 is emitted from the mouth and nostrils, and only 1% via the anus. The SF6 technique samples breath over 24 h, whereas other techniques use spot samples of breath over periods of min. throughout the day, so diurnal variation has to be considered. The majority of CH4 (87%) is released by eructation, which provides a clear signal for sample processing.

[0707] RESPIRATION CHAMBER

[0708] Respiration chambers for open- or closed-circuit indirect calorimetry are considered the ‘Gold Standard’, and were used extensively in nutrition studies when establishing the Metabolizable Energy system. A single animal...

Claims

CLAIMSWhat is claimed is:

1. A vaccine composition comprising cells and / or cell parts of at least one hydrogenotroph or at least one methanogen, optionally wherein the at least one hydrogenotroph or at least one methanogen is selected from the hydrogenotrophs or methanogens in Table A and / or Table B.

2. The vaccine composition of claim 1, wherein the vaccine comprises at least one methanogen.

3. The vaccine composition of claim 1 or 2, wherein the at least one methanogen is of a genus Methanobrevibacter.

4. The vaccine composition of any one of claims 1-3, wherein the at least one methanogen comprises Methanobrevibacter ruminantium, optionally Methanobrevibacter ruminantium Ml.

5. The vaccine composition of any one of claims 1-4, wherein the at least one methanogen comprises Methanobrevibacter gottschalkii, optionally Methanobrevibacter gottschalkii DSM11977.

6. The vaccine composition of any one of claims 1-5, wherein the vaccine composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 methanogens, preferably at least 8 methanogens.

7. The vaccine composition of any one of claims 1-6, wherein the vaccine composition comprises two methanogens.

8. The vaccine composition of claim 7, wherein the ratio of the two methanogens is at least about 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9: 1 by cell number.

9. The vaccine composition of claim 7 or 8, wherein the two methanogens are Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii.

10. The vaccine composition of claim 9, wherein the ratio of Methanobrevibacter ruminantium to Methanobrevibacter gottschalkii is at least about 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1 by cell number.

11. The vaccine composition of any one of claims 1-10, wherein at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells and / or cell parts are cells (i.e., whole cells).

12. The vaccine composition of any one of claims 1-11, wherein the vaccine composition comprises at least about 106, 107, 108, 109, or 1010cells per mL; and / or no more than about 107, 108, 109, 1010, 1011, or 1012cells per mL.

13. The vaccine composition of any of claims 1-12, wherein the vaccine composition comprises:(a) at least about 106but no more than about 1012cells per mL;(b) at least about 108but no more than about 1012cells per mL;(c) at least about 108but no more than about 1011cells per mL; or(d) at least about 108but no more than about 1010cells per mL.

14. The vaccine composition of claim 12 or 13, wherein the cells per mL is equivalent to events per mL.

15. The vaccine composition of any one of claims 1-14, wherein the vaccine composition comprises:(a) at least about 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, or 4 mg of total proteins; and / or(b) no more than about 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 4, 6, 8, or 10 mg of total proteins.

16. The vaccine composition of any one of claims 1-15, wherein the vaccine composition comprises:(a) at least about 0.2 but no more than about 10 mg of total protein; or(b) at least about 0.6 but no more than about 5 mg of total protein.

17. The vaccine composition of any one of claims 1-16, wherein the cells and / or cell parts are killed, fixed, and / or irradiated.

18. The vaccine composition of claim 17, wherein the cells and / or cell parts are:(a) killed by heat, steam, freezing, or sonicated;(b) fixed by formaldehyde or formalin; and / or(c) irradiated by UV or gamma irradiation.

19. The vaccine composition of any one of claims 1-18, wherein the cells and / or cell parts are lyophilized.

20. The vaccine composition of any one of claims 1-19, wherein the vaccine composition is a pharmaceutical composition.

21. The vaccine composition of claim 20, wherein the pharmaceutical composition comprises at least one carrier, at least one excipient, at least one cryoprotectant, and / or at least one inactive ingredient.

22. The vaccine composition of any one of claims 1-21, wherein the vaccine composition comprises at least one adjuvant.

23. The vaccine composition of claim 22, wherein the at least one adjuvant comprises:(a) inorganic salts, preferably aluminum and calcium salts;(b) an oil emulsion;(c) saponins;(d) lipid or liposomes(e) an immune-stimulating complex;(f) a carrier protein (e.g., keyhole limpet hemocyanin (KLH), optionally conjugated to cells and / or cell parts);(g) a bacterial product or derivatives thereof (e.g., bacterial toxins, lipopolysaccharide, etc.);(h) a cytokine; or(i) any combination of two or more selected from (a)-(h).

24. The vaccine composition of claim 22 or 23, wherein the at least one adjuvant comprises Complete Freund’s adjuvant, Incomplete Freund’s adjuvant, Montanide ISA70, Montanide ISA61, Saponin, chitosan thermogel, lipid (e.g., monophosphoryl lipid A), a lipid nanoparticle / cationic liposome adjuvant, Emulsigen-D, Emulsigen, Emulsigen-P, Polygen, ENABL 06, Montainde ISA 201, Montanide Gel 02, or any combination of two or more thereof.

25. The vaccine composition of claim 24, wherein the at least one adjuvant comprises Complete Freund’s adjuvant or Incomplete Freund’s adjuvant.

26. The vaccine composition of any one of claims 1-25, wherein the vaccine composition induces immune response against at least one cell surface protein or a fragment thereof of the at least one methanogen.

27. The vaccine composition of claim 26, wherein the at least one cell surface protein or a fragment thereof is selected from an adhesin-like protein, adhesin-like protein with cysteine protease domain, tetrahydromethanopterin S -methyltransferase subunit, a fragment thereof, and / or any combination thereof.

28. A method of treating a disease in a subject, the method comprising administering to the subject a vaccine composition of any one of claims 1-27.

29. The method of claim 28, wherein the disease is a periodontal disease, Inflammatory Bowel Disease (IBD), gingivitis, bloat, and / or liver abscess.

30. The method of claim 28, wherein the disease is associated with elevated, increased, or severe lactic acidosis.

31. A method of inducing an immune response against at least one methanogen in a subject, the method comprising administering to the subject the vaccine of any one of claims 1-27.

32. The method of claim 31, wherein the immune response comprises a B cell response and / or a T cell response, preferably a B cell response.

33. A method of reducing (i) lactate in a digestive tract and / or (ii) increasing pH in a digestive tract in a subject, the method comprising administering to the subject the vaccine of any one of claims 1-27.

34. A method of reducing the activity, number, and / or type of methanogens in a digestive tract of a subject, the method comprising administering to the subject the vaccine composition of any one of claims 1-27.

35. The method of claim 33 or 34, wherein the digestive track comprises rumen, reticulum, omasum, abomasum, stomach, small intestine, large intestine, and / or rectum, preferably rumen.

36. A method of reducing the amount of methane (CH4) and / or hydrogen (H2) emitted by a subject, preferably eructated and / or exhaled, the method comprising administering to the subject the vaccine composition of any one of claims 1-27.

37. The method of claim 36, wherein the amount of methane (CH4) and / or hydrogen (H2) is reduced by about 5-100%, preferably by about 10-100%, compared to a control, optionally wherein the amount of methane (CH4) and / or hydrogen (H2) is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

38. The method of claim 36 or 37, wherein the amount of methane (CH4) and / or hydrogen (H2) is reduced by about 20-100%, preferably by about 30-100%, compared to a control.

39. The method of any one of claims 36-38, wherein the amount of methane (CH4) is reduced by (a) about 1 kg - about 50 kg within 8 weeks from the time of first vaccination, or (b) about 5 kg - about 300 kg within a year from the time of first vaccination, compared to a control.

40. The method of any one of claims 36-39, wherein the amount of methane (CH4) normalized to an amount of CO2 emitted by the subject (i.e., CH4 / CO2) is reduced by about 5-100%, preferably by about 10-100%, compared to a control, optionally wherein the amount of methane (CH4) normalized to the amount of CO2 is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

41. The method of claim 40, wherein the amount of methane (CH4) normalized to the amount of CO2 is reduced by about 20-100%, preferably by about 30-100%, compared to a control.

42. The method of any one of claims 36-41, wherein the amount of hydrogen (H2) is reduced by (a) about 10 g - about 500 g within 8 weeks from the time of first vaccination, or (b) about 50 g - about 3 kg within a year from the time of first vaccination, compared to a control.

43. A method of increasing the amount of carbon dioxide (CO2) emitted by a subject, preferably eructated and / or exhaled, the method comprising administering to the subject the vaccine composition of any one of claims 1-27.

44. The method of claim 43, wherein the amount of carbon dioxide (CO2) is increased by about 1-100%, preferably by about 1-20%, compared to a control, optionally wherein the amount of CO2 is increased by at least about !%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

45. The method of claim 43 or 44, wherein the amount of carbon dioxide (CO2) is increased by about 3-10%, preferably by about 3-20%, compared to a control.

46. The method of any one of claims 43-45, wherein the amount of carbon dioxide (CO2) is increased by (a) about 29.8 kg - about 1,490 kg within 8 weeks from the time of first vaccination, or (b) about 149 kg - about 8,940 kg within a year from the time of first vaccination, compared to a control.

47. The method of any one of claims 37-42 and 44-46, wherein the control is:(a) an accepted reference;(b) the amount of methane, CCh-normalized methane, hydrogen, or carbon dioxide emitted by an unvaccinated subject; or(c) the amount of methane, CCh-normalized methane, hydrogen, or carbon dioxide emitted by the vaccinated subject prior to vaccination.

48. The method of any one of claims 28-47, wherein the subject produces an antibody against at least one methanogen.

49. The method of claim 48, wherein the antibody is an IgG, IgM, or an IgA, preferably an IgA or IgM.

50. The method of claim 48 or 49, wherein the antibody is produced in an amount sufficient to:(a) be detectable in the digestive tract;(b) reduce the number and / or alter the relative abundances of methanogens in the digestive tract;(c) reduce the amount of methane, the CC -normalized methane, and / or hydrogen emitted by the subject; and / or(d) increase the amount of CO2 emitted by the subject.

51. The method of any one of claims 28-50, wherein the vaccine composition is administered to the subject via a route selected from intramuscular administration, intradermal administration, subcutaneous administration, and nasal administration.

52. The method of any one of claims 28-51, wherein the vaccine composition is administered to the subject via intramuscular administration or subcutaneous administration, preferably subcutaneous administration.

53. The method of any one of claims 28-52, wherein the subject is administered with at least one repeat dose of the vaccine composition of any one of claims 1-27.

54. The method of claim 53, wherein the subject is administered with at least two repeat doses of the vaccine composition.

55. The method of claim 53 or 54, wherein the subject is administered with the vaccine composition at least 3 times per year.

56. The method of any one of claims 53-55, wherein the at least one repeat dose comprises the same dose or a different dose (e.g., number or amount of cells and / or cell parts) compared to the preceding dose of the vaccine composition.

57. The method of any one of claims 53-56, wherein the at least one repeat dose comprises the same adjuvant or a different adjuvant compared to the preceding dose of the vaccine composition.

58. The method of any one of claims 53-57, wherein the subject is administered with the repeat dose of the vaccine composition after at least about 2 weeks, about 3 weeks, about 1 month, about 6 months, or about 12 months from the time the subject is administered with the preceding dose of the vaccine composition.

59. The method of any one of claims 53-58, wherein the subject is administered with the repeat dose of the vaccine composition no later than about 1 month, about 2 months, about 3 months, 6 months, 12 months, 18 months, or 24 months from the time the subject is administered with the preceding dose of the vaccine composition.

60. The method of any one of claims 53-59, wherein the subject receives the repeat dose of the vaccine after at least about 2 weeks and no more than about 18 months from the time the subject is administered with the preceding dose of the vaccine.

61. The method of any one of claims 53-60, wherein the subject is administered with at least two repeat doses of the vaccine composition, and the subject receives:(a) the first repeat dose (“boost 1”) after about 2 weeks to about 4 weeks from the time the subject is administered with the initial vaccine dose (“prime”); and / or(b) the second repeat dose (“boost 2”) after about 3 months to about 6 months from the time the subject is administered with the initial vaccine dose (“prime”).

62. The method of any one of claims 28-61, wherein the subject is administered with a dosage of between about 103cells per vaccine dose per kg of animal body weight and 109cells per vaccine dose per kg of animal body weight of the vaccine composition each time of vaccination.

63. The method of any one of claims 28-62, further comprising administering to the subject (a) at least one agent that reduces the level of methane and / or hydrogen produced by the subject; and / or (b) at least one agent that increases production efficiency.

64. The method of claim 63, wherein the at least one agent is administered to a subject concomitant with, prior to, or after the vaccination.

65. The method of claim 63 or 64, wherein the at least one agent is administered to a subject after the vaccination.

66. The method of any one of claims 63-65, wherein the at least one agent is administered to a subject daily, semiweekly, weekly, biweekly (every two weeks), or monthly.

67. The method of any one of claims 63-66, wherein the at least one agent is administered to a subject for a duration of at least 1 week but no more than 1 month.

68. The method of any one of claims 63-67, wherein the at least one agent comprises:(a) an agent selected from the agents listed in Tables 4-8;(b) 3-Nitrooxypropanol (3NOP), ethyl-3NOP, 2-bromoethanesulfonate (BES), 2- chloroethanesulfonate (CES), 3-bromopropanesulfonate (BPS), bromochloromethane (BCM), bromoform, bromodichloromethane, dibromochloromethane, carbon tetrachloride, trichloroacetamide, trichloroethyladipate, lumazin (2,4-pteridinedione), p- aminobenzoic acid, lovastatin, mevastatin, pravastatin, diallyl disulfide, garlic oil, saponins, tannins, flavonoids, nitrate, nitroethane, -nitro-propionate, 2-nitropropanol, 2-nitroethanol, malate, acrylate, oxaloacetate, fumarate, propionic acid, 3-butenoic acid, 2-butynoic acid, ethyl 2- butynoate, monensin, lasalocid, bovicin HC5, nisin, or any combination thereof; or(c) monensin (Rumensin®), Optigrid® 45 (ractopamine hydrochloride), Amprolium (Corid), Bacitracin (Albac, BMD), Bambermycin (GainPro), Decoquinate (Deccox), Fenbendazole (Safe-Guard), Laidlomycin (Cattlyst), Lasalocid (Bovetec), Melengestrol Acetate (MGA), Methoprene (Altosid), Morantel (Rumatel), Poloxalene (Bloat Guard), Ractopamine (Optaflexx, Actogain), Tetraclovinphos (Rabon), or any combination thereof.

69. The method of any one of claims 63-68, wherein the at least one agent comprises 3N0P or ethyl-3NOP.

70. The method of claim 69, wherein the subject is administered with at least about 0.5 g but no more than 25 g of 3NOP per day.

71. The method of claim 69 or 70, wherein the subject is administered with at least about 1 g but no more than 5 g of 3NOP per day.

72. The method of any one of claims 69-71, wherein the subject is administered with about 2.5 g of 3NOP per day.

73. The method of any one of claims 69-72, wherein the subject is administered with 3NOP for a duration of at least 1 week but no more than 1 month.

74. The method of any one of claims 63-73, wherein the at least one agent comprises Monensin (Rumensin®).

75. The method of any one of claims 63-74, wherein the at least one agent is formulated in animal feed.

76. The method of any one of claims 63-75, wherein the at least one agent is a composition comprising one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors thereof and one or more agriculturally suitable carriers.

77. The method of claim 76, wherein the one or more agriculturally suitable carriers comprises a solid carrier.

78. The method of claim 77, wherein the one or more solid carriers comprises attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, a natural or synthetic derivative thereof, or a combination thereof.

79. The method of claim 77, wherein the one or more solid carriers comprises attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, silica, propylene glycol, hemp protein, biochar, montmorillonite, activated charcoal, lignin, wood flour, hemp protein, pea protein, soy protein, gelatin, casein, chitosan, talc, calcium phosphate, arginine, lysine, calcium carbonate, carbon black, glutamine, betaine, bismuth phosphate, bismuth citrate, iron phosphate, or any combination thereof.

80. The method of any one of claims 77-79, wherein the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, or a combination thereof.

81. The method of any one of claims 77-79, wherein the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, carrageenan, cellulose acetate, hydroxypropyl cellulose, cellulose acteate phthalate, maltrodextran, dextran, inulin, corn starch, amylopectin, sodium starch glycolate, pentaerthritol, cyclodextrin, or a combination thereof.

82. The method of any one of claims 77-81, wherein the solid carrier comprises silica and ethylcellulose.

83. The method of claim 82, wherein the carrier comprises about 10% to about 50% by weight of the silica and about 50 to about 90% by weight of the ethylcelluose.

84. The method of any one of claims 76-83, wherein the carrier comprises silica and activated charcoal.

85. The method of any one of claims 76-84, wherein the carrier comprises about 10% to about 90% by weight of the silica and about 10% to about 90% by weight of the activated charcoal.

86. The method of any one of claims 85, wherein the carrier further comprises arginine, lysine, or both arginine and lysine.

87. The method of any one of claims 76-86, wherein the carrier comprises activated charcoal and ethylcellulose.

88. The method of claim 87, wherein the carrier comprises about 10% to about 50% by weight of the activated charcoal and about 40% to about 90% by weight of the ethylcellulose.

89. The method of claim 87 or 88, wherein the carrier further comprises about 1 to about 10% by weight of sodium lignosulfate or about 1 to about 10% by weight of hydroxyethyl cellulose.

90. The method of any one of claims 76-89, wherein the carrier comprises arginine and polycaprolactone.

91. The method of claim 90, wherein the carrier comprises about 10 to about 60% by weight of the arginine and about 30 to about 90% by weight of the polycaprolactone.

92. The method of any one of claims 76-91, wherein the carrier comprises silica and polycaprolactone.

93. The method of claim 92, wherein the carrier comprises about 10 to about 60% by weight of the silica and about 30 to about 90% by weight of the polycaprolactone.

94. The method of any one of claims 77-93, wherein the one or more solid carriers is inert.

95. The method of any one of claims 77-94, wherein the one or more solid carriers is water soluble.

96. The method of any one of claims 77-95, further comprising one or more additives with a density greater than water and / or one or more additives that reduces the rate of dissolution of the composition in water.

97. The method of claim 96, wherein the composition has a density of at least 1.1, preferably about 1.1 mg / mL to about 3 mg / mL, about 1.5 to about 3 mg / mL, about 1.5 to about 2.5 mg / mL, or about 1.5 to about 2 mg / mL.

98. The method of claim 96 or 97, wherein the one or more additives with a density greater than water comprises silica, attapulgite, kaolinite, fuller’s earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, or any combination thereof.

99. The method of any one of claims 96-98, wherein the one or more additives that reduces the rate of dissolution of the composition further reduces a rate of release of the one or more small molecules into water.

100. The method of claim 99, wherein the composition dissolves over at least about 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, and / or nor more than about 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, or 147 days, about 1 to about 147 days, more preferably 7-63 days, more preferably about 7-42 days, even more preferably 14-42 days yet even more preferably 14-28 days.

101. The method of any one of claims 76-100, wherein about 40 to about 80% of the small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors thereof is released in water after 15 days.

102. The method of any one of claims 76-101, wherein the composition comprises particles having a uniform size distribution.

103. The method of any one of claims 76-102, wherein the composition comprises particles having a non-uniform size distribution.

104. The method of claim 102 or 103, wherein the particles comprise a spherical-, square-, rectangular-, capsular-, cylindrical-, conical-, ovular-, triangular-, diamond-, or disk-like shape.

105. The method of any one of claims 76-104, wherein the particles have a size ranging from about 1 mm to about 20 mm, about 1 to about 15 mm, about 1 to about 10 mm, about 5 to about 20 mm, about 5 to about 15 mm, or about 5 to about 10 mm.

106. The method of any one of claims 76-105, wherein the particles further comprise a coating.

107. The method of claim 106, wherein the coating comprises at least two layers.

108. The method of claim 106 or 107, wherein the coating is selected from cellulose acetate phlalate, ethyl cellulose, hydroxypropyl cellulose, polycaprolactone, alginate, chitosan, polyethylene glycol, cellulose acetate, triacetin, propylene glycol, n- methyl-2-pyrollidone, and any combination thereof.

109. The method of claim 106 or 107, wherein the coating comprises two or more polyelectrolytes.

110. The method of claim 109, wherein the polyelectrolytes comprise polystyrene sulfonate, polyethyleneimine, sodium lignosulfate, polyglutamic acid and poly-L-lysine, poly-L-arginine, polyallylamine hydrochloride, polyacrylic acid, or any combination thereof.

111. The method of claim 109 or 110, wherein the poly electrolytes comprise polyallylamine hydrochloride and sodium lignosulfate.

112. The method of claim 109 or 110, wherein the polyelectrolytes comprise polyallylamine hydrochloride and polystyrene sulfonate.

113. The method of claim 109 or 110, wherein the poly electrolytes comprise sodium lignosulfate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine, and sodium lignosulfate.

114. The method of claim 109 or 110, wherein the polyelectrolytes comprise polystyrene sulfonate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine.

115. The method of any one of claims 109-114, wherein the two or more polyelectrolytes are crosslinked.

116. The method of any one of claims 76-115, wherein the one or more small molecules comprise a molecule that interferes with the uptake and / or conversion of acetate,H2, CO2, methanol, monomethylamine, dimethylamine, trimethylamine, nitric oxide, or a combination thereof.

117. The method of any one of claims 76-116, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors interact with an enzyme selected from the group consisting of 3- (methylthio)propanoate:coenzyme M methyltransferase, acetate kinase, acetyl-CoA decarbonylase, acetyl-CoA decarbonylase / synthase complex 0.

282. acetyl-CoA decarbonylase / synthase complex , acetyl-CoA decarbonylase / synthase complex y5, acetyl- CoA synthase, carbon monoxide dehydrogenase, carbonic anhydrase, Comethyltransferase, coenzyme M reductase, cyclohydrolase, dehydrogenase, dimethylamine- -[corrinoid protein] Co-methyltransferase, F420-dependent methylene- H4MPT reductase, F420-dependent methylene-FUSPT dehydrogenase, formylmethanofuran dehydrogenase, formylmethanofura FUMPT formyltransferase, formylmethanofuran iFUS PT formyltransferase, formyltransferase, FF-forming methylene-FUMPT dehydrogenase, methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, methenyl-FUMPT cyclohydrolase, methyl-coenzyme M reductase, methyl- FUSPTiCoM methyltransferase, methylated [methylamine- specific corrinoid protein] :coenzyme M methyltransferase, methylcobamide:CoM methyltransferase, methylthiokcoenzyme M methyltransferase, methyltransferase, MtaC protein: coenzyme M methyltransferase, phosphotransacetylase, tetrahydromethanopterin S -methyltransferase, tetramethylammonium methyltransferase, trimethylamine— corrinoid protein Co-methyltransferase, and any combination thereof.

118. The method of claim 117, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors interact with methyl-coenzyme M reductase (MCR).

119. The method of any one of claims 76-118, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors comprise a compound having the formula R1-[CH2]n-ONO2 wherein n is an integer from 1 to 15;R1is selected from the group consisting of H, Ci-Cealkyl, phenyl, — OH, — NH2, — CN,— COOH, — O(C=O)R3, — NHC(=O)R3, SO2NHR3, or — ONO2, — SH and R3is Ci-Cealkyl, phenyl, pyridyl; with the proviso that when n is >3 the hydrocarbon chain may be interrupted by — O— or — NH— .

120. The method of claim 119, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors is selected from 3-nitrooxypropanol, 9-nitrooxynonanol, 5-nitrooxy pentanoic acid, 6-nitrooxy hexanoic acid, bis(2-hydroxyethyl)amine dinitrate, 1 ,4-bis-nitrooxybutane, 1,5-bis- nitrooxypentane, and any combination thereof.

121. The method of claim 119, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors comprises 3-nitrooxypropanol (3NOP).

122. The method of any one of claims 76-121, wherein the composition comprises about 1 to about 25% by weight of the small molecule, about 5 to about 20% by weight of the small molecule, or about 5 to about 15% by weight of the small molecule.

123. The method of any one of claims 76-122, wherein the composition comprises a plurality of populations of particles, wherein each population or particles comprises a different formulation, a different shape, and / or a different size distribution.

124. The method of claim 123, wherein the plurality of populations of particles comprises a first population and a second population.

125. The method of claim 124, wherein the population of granular particles further comprises at least 1, 2, 3, 4, 5, 5, 6, 8, or 9 and / or no more than 4, 5, 6, 7, 8, 9, or 10 additional populations, for example 3-10 additional populations, preferably 3-7 additional populations, more preferably 3-5 additional populations.

126. The method of claim 124 or 125, wherein the first population comprises an immediate release formulation.

127. The method of any one of claims 124-126, wherein the second population comprises a delayed release formulation.

128. The method of any one of claims 125-127, wherein each additional population comprises a delayed release formulation, wherein each population dissolves and / or releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and / or precursors at a different time than each of the other populations.

129. An antibody produced by the method of any one of claims 28-128, or a fragment thereof.

130. The antibody of claim 129, wherein the antibody is a monoclonal antibody.

131. The antibody of claim 129 or 130, wherein the antibody is an IgM, an IgG or an IgA, preferably an IgA or an IgM.

132. The antibody of any one of claims 129-131, wherein the antibody is lyophilized.

133. The antibody of any one of claims 129-132, wherein the antibody is in a pharmaceutical composition comprising at least one excipient and / or carrier.

134. Milk and / or a derivative thereof produced by the subject of any one of claims 28-128.

135. The milk and / or a derivative thereof of claim 134, wherein the milk and / or derivatives thereof comprises an antibody that binds at least one methanogen.

136. The milk and / or a derivative thereof of claim 134 or 135, wherein the milk and / or derivatives thereof is pasteurized and / or homogenized.

137. The milk and / or a derivative thereof of any one of claims 134-136, wherein the milk and / or derivatives thereof is lyophilized or evaporated to form dry milk powder (e.g., boiling at low pressure at low temperature).

138. The milk and / or a derivative thereof of any one of claims 134-137, further comprising at least one agent that reduces methane and / or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Tables 4-8.

139. An animal feed comprising:(a) the antibody of any one of claims 129-133;(b) at least one agent that reduces methane and / or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Tables 4-8;(c) the milk of any one of claims 134-138; or(d) any combination of two or more of (a)-(c).

140. The animal feed of claim 139, wherein the animal feed is liquid (e.g., drinking water, milk) or solid (e.g., fodder).

141. The animal feed of claim 139 or 140, wherein the animal feed comprises fat and / or fatty acid, optionally wherein the animal feed comprises fat and / or fatty acid that is at least about 1%, 2%, 3%, 4%, 5%, or 6% of the diet.

142. A method of reducing methane and / or hydrogen production in a subject, the method comprising orally administering to and / or feeding the subject the antibody of any one of claims 129-133, the milk and / or a derivative thereof of any one of claims 134-138, the animal feed of any one of claims 139-141, or any combination of two or more thereof.

143. The method of claim 142, further comprising administering at least one agent that reduces methane and / or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Tables 4-8.

144. The method of claim 142 or 143, further comprising administering the subject with the at least one vaccine composition of any one of claims 1-27, optionally according to the method of any one of claims 28-128.

145. The method of any one of claims 28-128 and 142-144, wherein the subject is a mammal, a human, or a ruminant.

146. The method of claim 145, wherein the ruminant is selected from a cow, a bull, a bison, a yak, a buffalo, an antelope, a goat, a sheep, a deer, a giraffe, a caribou, a gazelle, a macropod, a llama, a camel, and an alpaca.

147. The method of any one of claims 28-128 and 142-146, wherein the subject is cattle.

148. The method of claim 147, wherein the cattle is selected from a pregnant cow, heifer, bull, and steer.

149. The method of any one of claims 28-128 and 142-148, wherein the subject is an adult.

150. The method of claim 149, wherein the subject is adult cattle selected from:(a) a cow that is about 6-8 months to about 2-7 years old;(a) a beef adult that is about 1.5 years to about 2.5 years old; and(b) a dairy adult that is about 4 years to 7 years old.

151. The method of any one of claims 28-128 and 142-148, wherein the subject is a young subject (e.g., before weaning or below 2 years of age).

152. The method of claim 151, wherein the subject is young cattle selected from:(a) a newborn calf and a pre- weaned calf that is about 0 month to about 3 months old; and(b) a weaned cow that is about 3 months to 8 months old.

153. The method of any one of claims 28-128 and 142-152, wherein the subject is a pregnant female subject.

154. The method of any one of claims 28-128 and 142-153, wherein the subject is an offspring (e.g., calf) of the vaccinated female subject that received the milk comprising an antibody that binds at least one methanogen.

155. The method of any one of claims 28-128 and 142-154, wherein the vaccine is administered to a subject as a part of a pre-existing vaccination program to which the subject is subject (e.g., Table 9).

156. The method of any one of claims 28-128 and 142-155, wherein the vaccine is administered to a subject when the subject is subject to or receives at least one other vaccine, wherein the at least one other vaccine is against infectious bovine rhinotracheitis (IBR), bovine virus diarrhea (BVD), parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), clostridia, E. Coli mastitis, leptospirosis, mannheimia hemolytica, brucella, vibriosis, Campylobacter, trichomonas, trichomoniasis, rotavirus, coronavirus, and / or respiratory disease.

157. The method of any one of claims 28-128 and 142-156, wherein the vaccine is administered to a subject when the subject changes in hands and / or a changes in environment.

158. The method of any one of claims 28-128 and 142-157, wherein the vaccine reduces methane and / or hydrogen production in the lower intestinal track (lower bowel) or the rumen of the subject.

159. The method of claim 158, wherein the method results in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% reduction in the level of methane and / or produced by the subject, optionally wherein the reduction in the level of methane and / or hydrogen is compared to an untreated subject.

160. A kit comprising the vaccine composition of any one of claims 1-27.

161. The kit of claim 160, wherein the vaccine composition comprises no more than one methanogen.

162. The kit of claim 160, wherein the vaccine composition comprises at least two methanogens.

163. The kit of claim 160, wherein the kit comprises at least two vaccine compositions comprising same or different methanogens.

164. The kit of claim 163, wherein the at least two vaccine compositions comprise different methanogens, and the different methanogens are in separate containers.

165. The kit of any one of claims 160-164, wherein the kit comprises about or at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 500, 1000, 1500, 2000, 2500, or 3000 doses of the vaccine composition.

166. The kit of claim 165, wherein(a) all doses are in a single container;(b) each dose is in an individual container; or(c) about or at least about 2, 3, 4, 5, 10, 25, 50, or 100 doses are pooled in a single container.

167. The kit of any one of claims 160-166, further comprising at least one adjuvant.

168. The kit of claim 167, wherein the vaccine composition and the at least one adjuvant are in separate containers.

169. The kit of claim 167 or 168, wherein the kit comprises at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 500, 1000, 1500, 2000, 2500, or 3000 doses; and(a) all doses are in a single container, and the at least one adjuvant for all doses are in a separate single container;(b) each dose is in an individual container, and the at least one adjuvant for all doses are in a separate single container;(c) each dose is in an individual container, and the at least one adjuvant for each dose is in a separate individual container; or(d) about or at least about 2, 3, 4, 5, 10, 25, 50, or 100 doses are pooled in a single container, and the pooled amount of at least one adjuvant for the pooled doses are in a separate single container.

170. The kit of any one of claims 167-169, wherein the kit comprises at least two adjuvants that are different.

171. A system comprising a growth chamber (e.g., bioreactor), wherein the growth chamber comprises a growth medium under an anaerobic atmosphere comprising less than 80% H2, optionally wherein the system further comprises at least one hydrogenotroph.

172. The system of claim 171, wherein the anaerobic atmosphere comprises less than 50% H2.

173. The system of claim 171 or 172, wherein the anaerobic atmosphere comprises at least 0.5% but no more than 20% H2, preferably at least 0.5% but no more than 4% H2.

174. The system of any one of claims 171-173, wherein the anaerobic atmosphere comprises at least 1% but no more than 4% H2, preferably at least 2% but no more than 4% H2.

175. The system of any one of claims 171-174, wherein the anaerobic atmosphere comprises CO2.

176. The system of claim 175, wherein the anaerobic atmosphere comprises at least about 1 part of CO2 for every 4 parts of H2.

177. The system of any one of claims 171-176, wherein the anaerobic atmosphere comprises inert gas.

178. The system of claim 177, wherein the inert gas is N2.

179. The system of any one of claims 171-178, wherein the anaerobic atmosphere consists essentially of H2, CO2, and inert gas, preferably wherein the inert gas is N2.

180. The system of any one of claims 171-179, wherein the at least one hydrogenotroph is under the pressure of less than about 180 kPa, about 150 kPa, about 125 kPa, about 120 kPa, about 110 kPa, or about 105 kPa.

181. The system of any one of claims 171-180, wherein the growth chamber is at least about 0.1 L, 0.5 L, 1 L, 5 L, 10 L, 50 L, 100 L, 250 L, 500 L, or 1000 L in volume.

182. The system of any one of claims 171-181, wherein a volume of the growth medium is at least about 0.1 L, 0.5 L, 1 L, 5 L, 10 L, 50 L, 100 L, 250 L, 500 L, or 1000 L.

183. The system of any one of claims 171-182, wherein the growth medium comprises(a) at least one component listed in Table D or Table E;(b) at least 10, 20, 30, or 40 components listed in Table D;(c) at least 10, 20, 30, 40, or 50 components listed in Table E;(d) all components listed in Table D or Table E;(e) medium according to Table D or Table E (e.g., having an exemplary concentration of at least one component, or having a preferred range of concentrations (or a value or a sub -range of concentrations therein) of at least one component);(f) BY medium; or(g) SD0001 medium.

184. The system of any one of claims 171-183, wherein the growth medium does not comprise a non-hydrogenotrophic additive (e.g., acetate, methanol, ethanol).

185. The system of any one of claims 171-184, wherein the system comprises at least one auxiliary instrument.

186. The system of claim 185, wherein the at least one auxiliary instrument is selected from gas supply, gas inlet, gas outlet, gas mixer upstream of the growth chamber, temperature control, gas flow control, H2 sensor, CO2 sensor, CH4 sensor, spectrophotometer, turbimeter, fluorometer, pH sensor, oxygen sensor, liquid flow control, pressure sensor, foam sensor, H2S sensor, scale, flow meter, camera, redox sensor, contamination sensor, motor, magnetic stirrer, shaker, agitator, impeller, sparger, sampling port (e.g., cells, gas, media), methane conversion system, and any one or more of the instruments listed in Table C.

187. The system of any one of claims 171-186, wherein the system further comprises at least one hydrogenotroph.

188. The system of claim 187, wherein the at least one hydrogenotroph comprises:(e) at least one hydrogenotroph selected from the hydrogenotrophs in Table A and / or Table B;(f) at least one methanogen;(g) at least one methanogen, wherein the at least one methanogen comprises a methanogen of a genus Methanobrevibacter, and / or(h) at least one methanogen, wherein the at least one methanogen comprises Methanobrevibacter ruminantium and / or Methanobrevibacter gottschalkii.

189. A method of growing at least one hydrogenotroph, the method comprising incubating the at least one hydrogenotroph in the system of any one of claims 171-188.

190. A method of growing at least one hydrogenotroph, the method comprising the steps of:(a) purging a growth chamber (e.g., bioreactor) with an anaerobic gaseous mixture comprising less than 80% H2, optionally less than 50% H2;(b) incubating the at least one hydrogenotroph in growth medium in the growth chamber purged in step (a).

191. The method of claim 190, wherein the anaerobic gaseous mixture comprises at least 0.5% but no more than 20% H2, preferably at least 0.5% but no more than 4% H2.

192. The method of claim 190 or 191, wherein the anaerobic gaseous mixture comprises at least 1% but no more than 4% H2, preferably at least 2% but no more than 4% H2.

193. The method of any one of claims 190-192, wherein the anaerobic gaseous mixture comprises CO2.

194. The method of claim 193, wherein the anaerobic gaseous mixture comprises at least about 1 part of CO2 for every 4 parts of H2.

195. The method of any one of claims 190-194, wherein the anaerobic gaseous mixture comprises inert gas.

196. The method of claim 195, wherein the inert gas is N2.

197. The method of any one of claims 190-196, wherein the anaerobic gaseous mixture consists essentially of H2, CO2, and inert gas, preferably wherein the inert gas is N2.

198. The method of any one of claims 190-197, wherein the gases of the anaerobic gaseous mixture are pre-mixed before entering the growth chamber.

199. The method of any one of claims 190-197, wherein the gases of the anaerobic gaseous mixture are mixed in a gas mixer upstream of the growth chamber.

200. The method of any one of claims 190-197, wherein the gases of the anaerobic gaseous mixture enter the growth chamber individually, and the gases are mixed in the growth chamber.

201. The method of any one of claims 190-200, wherein the anaerobic gaseous mixture is sparged into the growth media.

202. The method of any one of claims 190-201, wherein the anaerobic gaseous mixture is supplied continuously during the culturing of the at least one hydrogenotroph.

203. The method of any one of claims 190-202, wherein the gases of the anaerobic gaseous mixture is supplied from a single gas tank.

204. The method of any one of claims 190-202, wherein the gases of the anaerobic gaseous mixture is supplied from at least two gas cylinders, each of which comprises a different gas or gas mixture.

205. The method of any one of claims 190-204, wherein the at least one hydrogenotroph is under the pressure of less than about 180 kPa, about 150 kPa, about 125 kPa, about 120 kPa, about 110 kPa, or about 105 kPa.

206. The method of any one of claims 190-205, wherein the pressure of the supplied gas at the gas inlet to the growth chamber is at least about 102 kPa but no more than about 125 kPa.

207. The method of any one of claims 190-206, wherein the growth chamber is at least about 0.1 L, 0.5 L, 1 L, 5 L, 10 L, 50 L, 100 L, 250 L, 500 L, or 1000 L in volume.

208. The method of any one of claims 190-207, wherein a volume of the growth medium is at least about 0.1 L, 0.5 L, 1 L, 5 L, 10 L, 50 L, 100 L, 250 L, 500 L, or 1000 L.

209. The method of any one of claims 190-208, wherein the growth medium comprises(a) at least one component listed in Table D or Table E;(b) at least 10, 20, 30, or 40 components listed in Table D;(c) at least 10, 20, 30, 40, or 50 components listed in Table E; or(d) all components listed in Table D or Table E;(e) medium according to Table D or Table E (e.g., having an exemplary concentration of at least one component, or having a preferred range of concentrations (or a value or a sub -range of concentrations therein) of at least one component);(f) BY medium; or(g) SD0001 medium.

210. The method of any one of claims 190-209, wherein the growth medium does not comprise a non-hydrogenotrophic additive (e.g., acetate, methanol, ethanol).

211. The method of any one of claims 190-210, wherein the growth chamber further comprises at least one auxiliary instrument.

212. The method of claim 211, wherein the at least one auxiliary instrument is selected from gas supply, gas inlet, gas outlet, gas mixer upstream of the growth chamber, temperature control, gas flow control, H2 sensor, CO2 sensor, CH4 sensor, spectrophotometer, turbimeter, fluorometer, pH sensor, oxygen sensor, liquid flow control, pressure sensor, foam sensor, H2S sensor, scale, flow meter, camera, redox sensor, contamination sensor, motor, magnetic stirrer, shaker, agitator, impeller, sparger, sampling port (e.g., cells, gas, media), methane conversion system, and any one of the instruments listed in Table C.

213. The method of claim 211 or 212, wherein the at least one auxiliary instrument comprises a methane conversion system, wherein the gases from the growth medium are circulated back into the growth chamber.

214. The method of any one of claims 190-213, wherein the at least one hydrogenotroph comprises at least one hydrogenotroph selected from the hydrogenotrophs in Table A and / or Table B.

215. The method of claim 214, wherein the at least one hydrogenotroph comprises:(d) at least one methanogen;(e) at least one methanogen, wherein the at least one methanogen is of a genus Methanobrevibacter; and / or(f) at least one methanogen, wherein the at least one methanogen comprises Methanobrevibacter ruminantium and / or Methanobrevibacter gottschalkii.

216. A culture medium comprising(a) at least one component listed in Table D or Table E;(b) at least 10, 20, 30, or 40 components listed in Table D;(c) at least 10, 20, 30, 40, or 50 components listed in Table E;(d) all components listed in Table D or Table E; or(e) medium according to Table D or Table E (e.g., having a preferred range of concentrations (or a value or a sub-range therein) of at least one component).

217. A method of reducing CH4 emissions in a ruminant comprising administering to the ruminant a vaccine composition comprising a cell and / or cell part of at least one methanogen, wherein the CH4 emissions are reduced by at least about 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20% as compared to an untreated control ruminant.

218. A method of reducing H2 emissions in a ruminant comprising administering to the ruminant a vaccine composition comprising a cell and / or cell part of at least one methanogen, wherein the H2 emissions are reduced by at least 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% as compared to an untreated control ruminant.

219. A method of increasing the productivity of a ruminant comprising administering to the ruminant a vaccine composition comprising a cell and / or cell part of at least one methanogen, wherein the productivity is increased by at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5,14, 14.5, or 15% as compared to an untreated control ruminant.

220. The method of any one of claims 217-219, wherein the vaccine composition comprises the vaccine composition of any one of claims 1-27.

221. The method of any one of claims 217-220, wherein the ruminant is cattle.

222. An animal injected subcutaneously with the vaccine composition of any one of claims 1-27, wherein the vaccine composition comprises about 107, 108, 109, IO10, 1011, 1012, 1013, or 1014methanogen cells, for example about 107- about 1014methanogen cells.

223. A method of producing a low carbon animal product, the method comprising:(a) administering to an animal (e.g., a mammal, a ruminant) a vaccine composition of any one of claims 1-27;(b) determining an amount of emissions of at least one greenhouse gas (e.g., CO2, CH4, N2O, and / or H2) from the animal of (a) following administration until animal product harvesting;(c) determining a first carbon intensity as a ratio of a first amount of emissions from the vaccinated animal in (b) and the amount of harvested animal product;(d) determining a second carbon intensity as a ratio of a second amount of carbon emissions from an untreated animal and the same amount of harvested animal product; and(e) determining the difference between the first carbon intensity and the second carbon intensity.

224. The method of claim 223, wherein determining the amount of emissions comprises measuring the emissions using a GreenFeed system.

225. The method of claim 223, wherein the animal product is selected from the group consisting of meat, milk, and wool.

226. The method of claim 223, further comprising administering to the animal at least one agent that reduces methane production.

227. The method of claim 223, further comprising certifying the animal product as a low carbon intensity product based on the determined difference between the first carbon intensity and the second carbon intensity.

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