Compositions and methods for reducing enteric methane emission in ruminants

Abstract

The present disclosure teaches methods of utilizing isolated microorganisms, microbial ensembles, and compositions for improving feed efficiency and reducing enteric methane emissions in cattle. The disclosure also provides a method of quantifying enteric methane emissions in cattle at commercial scale.

Description

COMPOSITIONS AND METHODS FOR REDUCING ENTERIC METHANE EMISSION IN RUMINANTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 499,452, filed on May 1, 2023 and U.S. Provisional Application No. 63 / 510,911, filed on June 29, 2023, each of which is herein incorporated by reference in their entirety.FIELD

[0002] The present disclosure relates to isolated and biologically pure microorganisms that have applications, inter alia, in improving feed efficiency and reducing enteric methane emission in cattle. The present disclosure also provides a quantification method of measuring enteric methane emissions in cattle.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The sequence listing associated with this application is provided in XML format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The contents of the electronic sequence listing (ASBI_030_01WO_SeqList_ST26.xml; Size: 17,722,130 bytes; and Date of Creation: April 30, 2024) are herein incorporated by reference in its entirety.BACKGROUND

[0004] In light of global climate change, population growth, and food production demand, an environmentally sustainable and resilient agricultural system requires the development and implementation of climate-smart solutions to promote sustainable agriculture. Based on a 2020 EPA survey, 33% of livestock agriculture greenhouse gas emitted was from dairy cattle with 53% from enteric methane emissions (EME) generated in the rumen. Thus, one of the necessary components of climate-smart agriculture is developing effective and sustainable methods that reduce EME or EME intensity in the dairy industry.

[0005] Existing EME quantification methods rely on expensive equipment, including respiration chambers and wind tunnels. This equipment is generally difficult to transport and can only measure a limited number of cows at a time. Furthermore, measurement requires animals to be removed from their familiar environment, potentially influencing animalbehavior, resulting in measurements that do not represent true on-farm EME and EME intensity. On a larger scale, previous studies have used micrometeorology techniques to predict EME levels on-farm. This method measures methane emissions over an entire farm, which includes manure methane emissions and herd-level EME. Since these methane emissions are predicted over a large and variable area (e.g., weather and wind speed) and not an animalspecific measurement, it is not possible to assess the effects of an EME intervention on-farm.

[0006] Therefore, there remains a need in the art for improved methods of an accurate and high-throughput method of measuring EME in the dairy industry to effectively identify approaches to reduce EME in the environment.SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides a method for reducing enteric methane emission (EME) and / or EME intensity by a ruminant, the method comprising: administering a microbial supplement to the ruminant, wherein the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; (b) a. Pichia sp. comprising an ITS nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067; and a carrier suitable for ruminant administration.

[0008] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

[0009] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO:

[0010] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (b) a. Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

[0011] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1; and (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067.

[0012] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1; and (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

[0013] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1; and (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

[0014] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1; and (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

[0015] In some embodiments, the microbial supplement is a feed supplement. In some embodiments, the microbial supplement is administered orally to the ruminant.

[0016] In some embodiments, the carrier is calcium bicarbonate. In some embodiments, the carrier is zeolite.

[0017] In some embodiments, the bacteria and / or fungi are encapsulated. In some embodiments, the bacteria is a spore.

[0018] In some embodiments, the microbial supplement is encapsulated. In some embodiments, a Clostridium sp., a Pichia sp., a Ruminococcus sp., a Butyrivibrio sp. is encapsulated. In some embodiments, the Clostridium sp., the Pichia sp., the Ruminococcus sp. and / or the Butyrivibrio sp. is a spore.

[0019] In some embodiments, the ruminant is a cow, a steer, a heifer, or a calf. In some embodiments, the ruminant is a dairy cow.

[0020] In some embodiments, the abundance of methanogenic microbial populations is reduced in the ruminant administered the microbial supplement compared to a ruminant not administered the microbial supplement.

[0021] In some embodiments, wherein the EME is reduced in the ruminant administered the microbial supplement compared to a ruminant not administered the microbial supplement.

[0022] In some embodiments, wherein the EME intensity is reduced in the ruminant administered the microbial supplement compared to a ruminant not administered the microbial supplement.

[0023] In some embodiments, the present disclosure provides a method of quantifying enteric methane emissions in a ruminant, the method comprising: (a) collecting a sample from the ruminant; (b) extracting DNA from the sample; (c) performing next generation sequencing on the DNA to determine a microbiome of the ruminant; and (d) quantifying enteric methane emissions in the ruminant using a biomarker from step (c) that is determined using a machine learning model.

[0024] In some embodiments, the sample is an oral swab sample, a rumen sample, or a saliva sample.

[0025] In some embodiments, the machine learning model associates the microbiome with EME.

[0026] In some embodiments, the machine learning model uses random forest regression. In some embodiments, the machine learning model uses variational autoencoders or dynamical variational autoencoders.

[0027] In some embodiments, the machine learning model associates microbial composition, microbial diversity, specific taxonomic groups, and / or core members of the microbiome with EME.

[0028] In some embodiments, the machine learning model associates microbial composition, microbial diversity, specific taxonomic groups, and / or core members of the microbiome and additional animal characteristics with EME.

[0029] In some embodiments, the additional animal characteristics comprise milk production, dry matter intake, intake, energy corrected milk production, feed efficiency, fat percentage, protein percentage.

[0030] In some embodiments, the machine learning model associates microbial composition, microbial diversity, specific taxonomic groups, and / or core members of the microbiome with EME, and wherein the microbial composition, microbial diversity, specific taxonomic groups, and / or core members are represented as a ratio.

[0031] In some embodiments, the next generation sequencing is 16S rRNA gene amplicon sequencing. In some embodiments, the next generation sequencing is mcrA gene amplicon sequencing.

[0032] In some embodiments, the ruminant is a cow, a steer, a heifer, or a calf. In some embodiments, the ruminant is a dairy cow.

[0033] In some embodiments, the present disclosure provides a machine learning model to predict EME by a cow, wherein the machine learning model associates oral microbiome of the cow with EME.

[0034] In some embodiments, the present disclosure also provides a method of quantifying enteric methane emissions in a ruminant, the method comprising: (a) collecting a sample from the ruminant; (b) extracting DNA from the sample; (c) performing qPCR on the DNA to determine the copy number of a particular marker gene; and (d) quantifying enteric methane emissions in the ruminant based on copy number.

[0035] In some embodiments, the sample is an oral swab sample, a rumen sample or a saliva sample.

[0036] In some embodiments, the marker gene is selected from genes specific to methanogens. In some embodiments, the marker gene encodes a subunit of methyl coenzyme M or methyltransferase. In some embodiments, the marker gene is mcrA, mcrB, mcrG, mtaB, or mtbA gene.

[0037] In some embodiments, the ruminant is a cow, a steer, a heifer, or a calf. In some embodiments, the ruminant is a dairy cow.

[0038] In some embodiments, the present disclosure provides a microbial supplement for reducing EME and / or EME intensity by a ruminant, wherein the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; (b) Pichia sp. comprising an ITS nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067; and a carrier suitable for ruminant administration.

[0039] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

[0040] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

[0041] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (b) a. Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.DESCRIPTION OF THE DRAWINGS

[0042] Fig. 1 shows a general workflow of one embodiment of the method for determining the absolute abundance of one or more active microorganism strains.

[0043] Fig. 2 shows a general workflow of one embodiment of a method for determining the co-occurrence of one or more, or two or more, active microorganism strains in a sample with one or more metadata (environmental) parameters, followed by leveraging cluster analysis and community detection methods on the network of determined relationships.

[0044] Fig. 3 depicts a schematic diagram that illustrates an example process flow for use with an exemplary microbe interaction analysis and selection system, including the determination of maximal information coefficient (MIC) scores discussed throughout the present disclosure.

[0045] Fig. 4A shows schematic of how rumen microorganisms can improve rumen microbial interactions and lead to reduced EME and improved milk yield.

[0046] Fig. 4B shows a general workflow of one embodiment of a method for a microbial feed supplement (MFS) development.

[0047] Figs. 5A — 5B show the synchronized interaction between cellulolytic and amylolytic populations in dairy rumen microbiome (Fig. 5A) and the improvement in energy-corrected milk yield upon the supplementation of the MFS (Fig. 5B).

[0048] Fig. 6 shows EME signal as detected by GreenFeed systems in a controlled academic study at the first location in Brookings, SD.

[0049] Fig. 7 shows the effect of microbial feed supplement (MFS) on milk yield in dairy cows in comparison to control without the MFS administration.

[0050] Fig. 8 shows the effect of microbial feed supplement (MFS) on methane yield in dairy cows in comparison to control without the MFS administration.

[0051] Fig. 9 shows the effect of microbial feed supplement (MFS) on methane intensity in dairy cows in comparison to control without the MFS administration.

[0052] Fig. 10 shows EME signal as detected by GreenFeed systems in a controlled academic study at the second location in Tulare, CA.

[0053] Fig. 11 shows the effect of microbial feed supplement (MFS) on milk yield in dairy cows in comparison to control without the MFS administration.

[0054] Fig. 12 shows the effect of microbial feed supplement (MFS) on energy corrected milk (ECM) in dairy cows in comparison to control without the MFS administration.

[0055] Fig. 13 shows the effect of microbial feed supplement (MFS) on feed efficiency in dairy cows in comparison to control without the MFS administration.

[0056] Fig. 14 shows the effect of microbial feed supplement (MFS) on methane intensity in dairy cows in comparison to control without the MFS administration.

[0057] Fig. 15 shows the effect of microbial feed supplement (MFS) on methane production in dairy cows in comparison to control without the MFS administration.

[0058] Fig. 16 shows mcrA copy number correlations to GreenFeed measurements over time of day.

[0059] Fig. 17 shows mcrA copy number correlations to GreenFeed measurements. R = rumen, S = saliva, F = Fecal

[0060] Figs. 18A-18B show Lasso (Fig. 18A) and Random Forest (Fig. 18B) approaches by machine learning models to predict EME.

[0061] Fig. 19 shows predicted and observed EME rate by DVAE model approach.BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THEDEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENTPROCEDURES

[0062] The microorganisms described in this Application were deposited with the American Type Culture Collection (ATCC®), located at 10801 University Blvd., Manassas, VA 20110, USA; the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Culture Collection (NRRL®), located at 1815 N. University St., Peoria, IL 61604, USA; and / or the National Collection of Type Cultures (NCTC), Culture Collections of UK Health Security Agency located at 61 Colindale Avenue, London NW9 5EQ, United Kingdom; and / or the Bigelow National Center for Marine Algae and Microbiota, located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA.

[0063] The deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and / or type strain rules and procedures. The deposit accession numbers for the Budapest Treaty deposits and type strains are provided in Table 2. The depository, accession numbers, and corresponding dates of deposit for the microorganisms described in this application are separately provided in Table 7.

[0064] The Tables below show the closest predicted hits for taxonomy of the microbes as predicted by BLAST. The International Code of Nomenclature of Prokaryotes only recognizes physical cultures of microorganisms as type material. Because of this, uncultured microorganisms that have been identified through sequencing and in silico methods do not have formal names and taxonomic assignment. There is no formal process for naming microorganisms identified using cultivation-independent approaches. Furthermore, the sequencing of novel microorganisms currently outpaces the cultivation and isolation of novel microorganisms, thus creating a large gap and poor classification of several branches of microbial taxonomy. See Murray AE et al., Nat Microbiol 5, 987-994 (2020); Konstantinidis et al., The ISME J 11, 2399-2406 (2017). Taxonomic comparisons performed in BLAST utilizing the NCBI databases only leverages taxonomic names recognized by international entities, that is, only cultivated species. Thus, the genus and / or species names listed in the Tables below are matches to each in silico detected organism’s closest cultivated and named relative. Depending on the percent similarity, the closest relatives may not be in the same species, genus, family, etc. due to the novelty and difficulty of cultivation of the microorganism presented.

[0065] The strains designated in the Tables below have been deposited in the labs of Native Microbials, Inc. since at least before the date of filing of the present application and before the date of deposit with the noted depository institutions.Table 1. Microbes of the Present DisclosureTable 2. Microbial Deposits Corresponding to the Microbes of Table 11Deposit number applicable to Budapest treaty and / or type strain rules and procedures2Deposit number applicable to Budapest treaty and / or type strain rules and procedures3Deposit number applicable to Budapest treaty and / or type strain rides and procedures4Deposit number applicable to Budapest treaty and / or type strain rules and proceduresTable 3. Microbes of the Present DisclosureTable 4. Microbes of the Present DisclosureTable 5. Microbes of the Present DisclosureTable 6. Microbes of the Present DisclosureTable 7. Budapest Treaty Deposits of the Disclosure5Deposit number applicable to Budapest treaty and / or type strain rules and procedures6Deposit number applicable to Budapest treaty and / or type strain rules and procedures7Deposit number applicable to Budapest treaty and / or type strain rules and procedures8Deposit number applicable to Budapest treaty and / or type strain rules and proceduresDETAILED DESCRIPTIONDefinitions

[0066] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0067] The term “a” or “an” may refer to one or more of that entity, i.e. can refer to plural referents. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0068] Reference throughout this specification to “one embodiment”, “an embodiment”, “one aspect”, or “an aspect” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0069] As used herein, in particular embodiments, the terms “about” or “approximately” when used in conjunction with numerical values and / or ranges generally refer to those numerical values and / or ranges near to a recited numerical value and / or range. In some instances, the terms “about” and “approximately” may mean the value plus or minus a range of 10%. The terms “about” and “approximately” may be used interchangeably.

[0070] As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, Bacteria and Archaea, eukaryotic fungi and protists, as well as viruses. In some embodiments, the disclosure refers to the “microbes” of Table 1 and Tables 3-6, or the “microbes” incorporated by reference. This characterization can refer to not only the predicted taxonomic microbial identifiers of the Table, but also the identified strains of the microbes listed in the Table.

[0071] The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest (e.g.increased milk production in a ruminant, increased feed efficiency in beef cattle, or decreased methane emissions in a ruminant). The community may comprise two or more species, or strains of a species, of microbes. In some instances, the microbes coexist within the community symbiotically.

[0072] The term “bioensemble,” “microbial ensemble,” or “synthetic ensemble” refers to a composition comprising one or more active microbes identified by methods, systems, and / or apparatuses of the present disclosure and that do not naturally exist in a naturally occurring environment and / or at ratios or amounts that do not exist in nature. A bioensemble is a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest (e.g. increased feed efficiency in feedlot cattle, increased milk production in a ruminant, or decreased methane emissions in a ruminant). The bioensemble may comprise two or more species, or strains of a species, of microbes. In some instances, the microbes coexist within the community symbiotically.

[0073] The term “microbial community” means a group of microbes comprising two or more species or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest (e.g. increased milk production in a ruminant or feed efficiency in beef cattle).

[0074] As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, animal tissue).

[0075] Microbes of the present disclosure may include spores and / or vegetative cells. In some embodiments, microbes of the present disclosure include microbes in a viable but non- culturable (VBNC) state, or quiescent state. See Liao and Zhao (US Publication US2015267163A1). In some embodiments, microbes of the present disclosure include microbes in a biofilm. See Merritt et al. (U.S. Patent 7,427,408).

[0076] Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with an acceptable carrier.

[0077] As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures, however spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be nonconductive to the survival or growth of vegetative cells.

[0078] As used herein, “microbial composition” refers to a composition comprising one or more microbes of the present disclosure, wherein a microbial composition, in some embodiments, is administered to animals of the present disclosure.

[0079] As used herein, “carrier”, “acceptable carrier”, or “pharmaceutical carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solution, saline solutions, and aqueous dextrose and glycerol solutions are preferably employed as carriers, in some embodiments as injectable solutions. In some embodiments, gelling agents are used as carriers. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. The choice of carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. See Hardee and Baggo (1998. Development and Formulation of Veterinary Dosage Forms. 2ndEd. CRC Press. 504 pg.); E.W. Martin (1970. Remington’s Pharmaceutical Sciences. 17thEd. Mack Pub. Co.); and Blaser et al. (US Publication US20110280840 Al).

[0080] In some aspects, carriers may be granular in structure, such as sand or sand particles. In further aspects, the carriers may be dry, as opposed to a moist or wet carrier. In some aspects, carriers can be nutritive substances and / or prebiotic substances selected from fructooligosaccharides, inulins, isomalto-oligosaccharides, lactitol, lactosucruse, lactulose, pyrodextrines, soy oligosaccharides, transgalacto-oligosaccharides, xylo-oligosaccharides, trace minerals, and vitamins. In some aspects, carriers can be in solid or liquid form. In some aspects, carriers can be zeolites, calcium carbonate, magnesium carbonate, silicon dioxide, ground corn, trehalose, chitosan, shellac, albumin, starch, skim-milk powder, sweet-whey powder, maltodextrin, lactose, and inulin. In some aspects, a carrier is water or physiological saline.

[0081] In certain aspects of the disclosure, the isolated microbes exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970)(discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff’d in part, rev ’d in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B 12 produced by microbes), incorporated herein by reference.

[0082] As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.

[0083] As used herein, “microbiome” refers to the collection of microorganisms that inhabit the digestive tract or gastrointestinal tract of an animal (including the rumen if said animal is a ruminant) and the microorgansims’ physical environment (i.e. the microbiome has a biotic and physical component). The microbiome is fluid and may be modulated by numerous naturally occurring and artificial conditions (e.g., change in diet, disease, antimicrobial agents, influx of additional microorganisms, etc.). The modulation of the microbiome of a rumen that can be achieved via administration of the compositions of the disclosure, can take the form of: (a) increasing or decreasing a particular Family, Genus, Species, or functional grouping of microbe (i.e. alteration of the biotic component of the rumen microbiome) and / or (b) increasing or decreasing volatile fatty acids in the rumen, increasing or decreasing rumen pH, increasing or decreasing any other physical parameter important for rumen health (i.e. alteration of the abiotic component of the rumen microbiome).

[0084] As used herein, “probiotic” refers to a substantially pure microbe (z.e., a single isolate) or a mixture of desired microbes, and may also include any additional components that can be administered to a ruminent for restoring microbiota. Probiotics or microbial inoculant compositions of the disclosure may be administered with an agent to allow the microbes to survive the environment of the gastrointestinal tract, ie., to resist low pH and to grow in the gastrointestinal environment. In some embodiments, the present compositions (e.g., microbial compositions) are probiotics in some aspects.

[0085] As used herein, “prebiotic” refers to an agent that increases the number and / or activity of one or more desired microbes. Non-limiting examples of prebiotics that may be useful in the methods of the present disclosure include fructooligosaccharides (e.g., oligofructose, inulin, inulin-type fructans), galactooligosaccharides, amino acids, alcohols, and mixtures thereof. See Ramirez -Farias et al. (2008. Br. J. Nutr. 4: 1-10) and Pool-Zobel and Sauer (2007. J. Nutr. 137:2580-2584 and supplemental).

[0086] The term “growth medium” as used herein, is any medium which is suitable to support growth of a microbe. By way of example, the media may be natural or artificial including gastrin supplemental agar, LB media, blood serum, and tissue culture gels. It should be appreciated that the media may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients.

[0087] The term “relative abundance” as used herein, is the number or percentage of a microbe present in the gastrointestinal tract or other organ system, relative to the number or percentage of total microbes present in said tract or organ system. The relative abundance may also be determined for particular types of microbes such as bacteria, fungi, viruses, and / or protozoa, relative to the total number or percentage of bacteria, fungi, viruses, and / or protozoa present. In one embodiment, relative abundance is determined by PCR. In another embodiment, relative abundance is determined by colony forming unit assays (cfu) or plaque forming unit assays (pfu) performed on samples from the gastrointestinal tract or other organ system of interest.

[0088] The medium may be amended or enriched with additional compounds or components, for example, a component which may assist in the interaction and / or selection of specific groups of microorganisms. For example, antibiotics (such as penicillin) or sterilants (for example, quaternary ammonium salts and oxidizing agents) could be present and / or the physical conditions (such as salinity, nutrients (for example organic and inorganic minerals (such as phosphorus, nitrogenous salts, ammonia, potassium and micronutrients such as cobalt and magnesium), pH, and / or temperature), methionine, prebiotics, ionophores, and beta glucans could be amended.

[0089] As used herein, the term “ruminant” includes mammals that are capable of acquiring nutrients from plant-based food by fermenting it in a specialized stomach (rumen) prior to digestion, principally through microbial actions. Ruminants included cattle, goats, sheep, giraffes, yaks, elk, deer, antelope, and others.

[0090] As used herein, the term “bovid” includes any member of family Bovidae, which include hoofed mammals such as antelope, sheep, goats, buffalo, bison, and cattle, among others.

[0091] As used herein, the term “steer” includes any member, species, variant, or hybrid of Bos indicus, Bos taurus indicus, or Bos taurus taurus. The term “steer” further includes reference to cow (mature female), steer (castrated male), heifer (immature female not having born offspring), bull (mature uncastrated male), and calf (immature males or females).

[0092] As used herein, the terms “beef cattle” and “feedlot cattle” are used synonymously to refer to cattle that are grown and utilized for the production of beef. Said cattle of the present disclosure include varieties such as the following: Africander, Angus, Aubrac, Barzona, Bazadaise, Beef Shorthorn, Beefalo, Beefmaster, Belgian Blue, Belmont Red, Belted Galloway, Black Angus, Blonde d’ Aquitaine, Bonsmara, Boran, Bradford, Brahman, Brahmousin, Brangus, British White, Buelingo, Canchim, Caracu, Charolais, Chianina, Composite, Corriente, Devon, Dexter, Drakensberger, Droughtmaster, English Longhorn, Galloway, Gelbvieh, Gloucester, Hays Converter, Hereford, Highland, Holstein, Hybridmaster, Limousin, Lincoln Red, Lowline, Luing, Maine-Anjou, Rouge des Pres, Marchigiana, Miniature Hereford, Mirandesa, Mongolian, Murray Grey, Nelore, Nguni, Parthenais, Piemontese, Pinzgauer, Red Angus, Red Poll, Retinta, Romagnola, Salers, Sanganer, Santa Cruz, Santa Gertrudis, Senepol, Shetland, Simbrah, Simmental, South Devon, Speckle Park, Square Meaters, Sussex, Tarentaise, Texas Longhorn, Tuli, Wagyu, Watusi, Welsh Black, Whitebred Shorthorn, and Zebu; or hybrids and / or crosses thereof.

[0093] As used herein, “dairy cattle” or “dairy cows” are used synonymously to refer to cows that are grown and utilized for the production of milk.

[0094] As used herein, “performance” should be taken to be an improvement in one or more desirable traits in a ruminant.

[0095] As used herein, “energy-corrected milk” or “ECM” represents the amount of energy in milk based upon milk volume, milk fat, and milk protein. ECM adjusts the milk components to 3.5% fat and 3.2% protein, thus equalizing animal performance and allowing for comparison of production at the individual animal and herd levels over time. An equation used to calculateECM, as related to the present disclosure, is: ECM = (0.327 x milk pounds) + (12.95 x fat pounds) + (7.2 x protein pounds)

[0096] As used herein, “improved” should be taken broadly to encompass improvement of a characteristic of interest, as compared to a control group, or as compared to a known average quantity associated with the characteristic in question. For example, “improved” milk production, feed efficiency, or methane emssions associated with application of a beneficial microbe, microbial consortia, or microbial ensemble of the disclosure can be demonstrated by comparing the milk, feed efficiency, or methane emissions of a treated ruminant compared to an untreated ruminant. In the present disclosure, “improved” does not necessarily demand that the data be statistically significant (i.e. p < 0.05); rather, any quantifiable difference demonstrating that one value (e.g. the average treatment value) is different from another (e.g. the average control value) can rise to the level of “improved.”

[0097] As used herein, “inhibiting and suppressing” and like terms should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.

[0098] The term “marker” or “unique marker” as used herein is an indicator of unique microorganism type, microorganism strain or activity of a microorganism strain. A marker can be measured in biological samples and includes without limitation, a nucleic acid-based marker such as a ribosomal RNA gene, a peptide- or protein-based marker, and / or a metabolite or other small molecule marker.

[0099] The term “metabolite” as used herein is an intermediate or product of metabolism. A metabolite in one embodiment is a small molecule. Metabolites have various functions, including in fuel, structural, signaling, stimulatory and inhibitory effects on enzymes, as a cofactor to an enzyme, in defense, and in interactions with other organisms (such as pigments, odorants and pheromones). A primary metabolite is directly involved in normal growth, development and reproduction. A secondary metabolite is not directly involved in these processes but usually has an important ecological function. Examples of metabolites include but are not limited to antibiotics and pigments such as resins and terpenes, etc. Some antibiotics use primary metabolites as precursors, such as actinomycin which is created from the primary metabolite, tryptophan. Metabolites, as used herein, include small, hydrophilic carbohydrates; large, hydrophobic lipids and complex natural compounds.

[0100] As used herein, the term “genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism, or group of organisms.

[0101] As used herein, the term “allele(s)” means any of one or more alternative forms of a gene, all of which alleles relate to at least one trait or characteristic. In a diploid cell, the twoalleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. Since the present disclosure, in embodiments, relates to QTLs, i.e. genomic regions that may comprise one or more genes or regulatory sequences, it is in some instances more accurate to refer to “haplotype” (i.e. an allele of a chromosomal segment) instead of “allele”, however, in those instances, the term “allele” should be understood to comprise the term “haplotype”. Alleles are considered identical when they express a similar phenotype. Differences in sequence are possible but not important as long as they do not influence phenotype.

[0102] As used herein, the term “locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.

[0103] As used herein, the term “genetically linked” refers to two or more traits that are coinherited at a high rate during breeding such that they are difficult to separate through crossing.

[0104] A “recombination” or “recombination event” as used herein refers to a chromosomal crossing over or independent assortment. The term “recombinant” refers to an organism having a new genetic makeup arising as a result of a recombination event.

[0105] As used herein, the term “molecular marker” or “genetic marker” refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Markers further include polynucleotide sequences encoding 16S or 18S rRNA, and internal transcribed spacer (ITS) sequences, which are sequences found between small-subunit and large-subunit rRNA genes that have proven to be especially useful in elucidating relationships or distinctions among when compared against one another. Mapping of molecular markers in the vicinity of an allele is a procedure which can be performed by the average person skilled in molecular-biological techniques.

[0106] The primary structure of major rRNA subunit 16S comprise a particular combination of conserved, variable, and hypervariable regions that evolve at different rates and enable the resolution of both very ancient lineages such as domains, and more modern lineages such as genera. The secondary structure of the 16S subunit include approximately 50 helices which result in base pairing of about 67% of the residues. These highly conserved secondary structural features are of great functional importance and can be used to ensure positional homology in multiple sequence alignments and phylogenetic analysis. Over the previous few decades, the16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone for the current systematic classification of bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

[0107] A sequence identity of 94.5% or lower for two 16S rRNA genes is strong evidence for distinct genera, 86.5% or lower is strong evidence for distinct families, 82% or lower is strong evidence for distinct orders, 78.5% is strong evidence for distinct classes, and 75% or lower is strong evidence for distinct phyla. The comparative analysis of 16S rRNA gene sequences enables the establishment of taxonomic thresholds that are useful not only for the classification of cultured microorganisms but also for the classification of the many environmental sequences. Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

[0108] As used herein, the term “trait” refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, quantity of milk fat produced relates to the amount of triglycerides, triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, cholesterol, glycolipids, and fatty acids present in milk; efficiency of feed utilization, particularly with com-intensive diets; amounts of feces produced; susceptibility to gut pathogens; decrease in motality rates; decreased methane emissions; among others. In some embodiments, desirable traits include, but are not limited to: predominance of short chain fatty acids, medium chain fatty acids, and long chain fatty acids; quantity of carbohydrates such as lactose, glucose, galactose, and other oligosaccharides; quantity of proteins such as caseins and whey; quantity of vitamins, minerals, milk yield / volume; reductions in methane (CF ) emissions or manure; improved efficiency of nitrogen utilization; improved dry matter intake; improved feed efficiency and digestibility; increased degradation of cellulose, lignin, and hemicellulose; increased rumen concentrations of fatty acids such as acetic acid, propionic acid, and butyric acid. In some embodiments, desirable traits include, but are not limited to: an increase in weight; an increase in average daily weight gain; an increase of musculature; an increase of fatty acid concentration in the gastrointestinal tract; an improved efficiency in feed utilization and digestibility; an increase in polysaccharide and lignin degradation; an increase in fat, starch, and / or protein digestion; an increase in fatty acid concentration in the rumen; pH balance in the rumen, an increase in vitamin availability; an increase in mineral availability; an increase in amino acid availability; a reduction in methane and / or nitrous oxide emissions; a reduction in manure production; an improved dry matter intake; an improved efficiency of phosphorous utilization; an increased resistance to colonization of pathogenic microbes that colonize cattle; reduced mortality; increased production of antimicrobials; increased clearance of pathogenic microbes; increased resistance to colonization of pathogenic microbes thatcolonize cattle; increased resistance to colonization of pathogenic microbes that infect humans; reduced incidence of acidosis or bloat; increased meat marbling, increased or decreased red coloring of meat, increased or decreased texture / coarseness of meat; increased amount of USDA Prime, USDA Choice, and USDA Select quality meat per animal, increased in the number of animals producing USDA Prime, USDA Choice, and USDA Select quality meat; increase or reduced concentration or presence of volatile compounds in the meat; reduced prevalence of acidosis or bloat; reduced body temperature; and any combination thereof; wherein said increase or reduction is determined by comparing against an animal not having been administered said composition.

[0109] A trait may be inherited in a dominant or recessive manner, or in a partial or incompletedominant manner. A trait may be monogenic (i.e. determined by a single locus) or polygenic (i.e. determined by more than one locus) or may also result from the interaction of one or more genes with the environment.

[0110] In the context of this disclosure, traits may also result from the interaction of one or more ruminant genes and one or more microorganism genes.[OHl] As used herein, the term “homozygous” means a genetic condition existing when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism. Conversely, as used herein, the term “heterozygous” means a genetic condition existing when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.

[0112] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., a ruminant), or group of organisms which results from the interaction between that individual’s genetic makeup (z.e., genotype) and the environment.

[0113] As used herein, the term “chimeric” or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid, or a protein sequence, that links at least two heterologous polynucleotides, or two heterologous polypeptides, into a single macromolecule, or that re-arranges one or more elements of at least one natural nucleic acid or protein sequence. For example, the term “recombinant” can refer to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0114] As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturallyoccurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.

[0115] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence” are used interchangeably.

[0116] As used herein, the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and / or the regulatory sequences required fortheir expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0117] As used herein, the term “homologous” or “homologue” or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar or strain. For purposes of this disclosure homologous sequences are compared. “Homologous sequences” or “homologues” or “orthologs” are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and / or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined usingsoftware programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (FM. Ausubel etal., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0118] As used herein, the term “nucleotide change” refers to, e.g., nucleotide substitution, deletion, and / or insertion, as is well understood in the art. For example, mutations contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.

[0119] As used herein, the term “protein modification” refers to, e.g., amino acid substitution, amino acid modification, deletion, and / or insertion, as is well understood in the art.

[0120] As used herein, the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule. A fragment of a polynucleotide of the disclosure may encode a biologically active portion of a genetic regulatory element. A biologically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the disclosure that comprises the genetic regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full length polypeptide. The length of the portion to be used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide useful as an epitope may be as short as 4 amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.

[0121] Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91 : 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et a / .(1997) Nature Biotech. 15:436-438; Moore et a / .(1997) J. Mol. Biol. 272:336-347; Zhang eta / .(1997) PNAS 94:4504-4509; Crameri etal. (1998) Nature 391 :288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458. For PCR amplifications of the polynucleotides disclosed herein, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

[0122] The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A / T vs. G / C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

[0123] The terms “stringency” or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ion,typically about 0.01 to 1.0 M Na+ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or “conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C and a wash in 2*SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, IM NaCl, 1% SDS at 37° C, and a wash in 0.1 *SSC at 60° C. Hybridization procedures are well known in the art and are described by e.g. Ausubel et al., 1998 and Sambrook etal., 2001. In some embodiments, stringent conditions are hybridization in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecyl sulfate at 45°C, such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by a wash in 5*SSC, containing 0.1% (w / v) sodium dodecyl sulfate, at 55°C to 65°C.

[0124] As used herein, “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.

[0125] As used herein, a “constitutive promoter” is a promoter which is active under most conditions and / or during most development stages. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or scorable markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory transcription system; production of compounds that requires ubiquitous activity in the organism; and production of compoundsthat are required during all stages of development. Non-limiting exemplary constitutive promoters include, CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.

[0126] As used herein, a “non-constitutive promoter” is a promoter which is active under certain conditions, in certain types of cells, and / or during certain development stages. For example, tissue specific, tissue preferred, cell type specific, cell type preferred, inducible promoters, and promoters under development control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues.

[0127] As used herein, “inducible” or “repressible” promoter is a promoter which is under chemical or environmental factors control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.

[0128] As used herein, a “tissue specific” promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, in the art sometimes it is preferable to use promoters from homologous or closely related species to achieve efficient and reliable expression of transgenes in particular tissues. This is one of the main reasons for the large amount of tissue-specific promoters isolated from particular tissues found in both scientific and patent literature.

[0129] As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the disclosure can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3 ' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.

[0130] As used herein, the phrases “recombinant construct”, “expression construct”, “chimeric construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the disclosure. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others. Vectors can be plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating. As used herein, the term “expression” refers to the production of a functional end-product e.g., an mRNA or a protein (precursor or mature).

[0131] In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term “heterologous trait” refers to a phenotype imparted to a transformed host cell or transgenic organism by an exogenous DNA segment, heterologous polynucleotide or heterologous nucleic acid. Various changes in phenotype are of interest to the present disclosure, including but not limited to modifying the fatty acid composition in milk, altering the carbohydrate content of milk, increasing yield of an economically important trait (e.g., milk, milk fat, milk proteins, weight, etc.), reducing methane emissions, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in organisms using the methods and compositions of the present disclosure.

[0132] As used herein, the term “MIC” means maximal information coefficient. MIC is a type of nonparamentric network analysis that identifies a score (MIC score) between active microbial strains of the present disclosure and at least one measured metadata (e.g., milk fat).Further, U.S. Application No. 15 / 217,575, filed on July 22, 2016 (issued as U.S. Patent No. 9,540,676 on January 10, 2017) is hereby incorporated by reference in its entirety.

[0133] The maximal information coefficient (MIC) is then calculated between strains and metadata (3021a), and between strains (3021b); as seen in Fig. 3. Results are pooled to create a list of all relationships and their corresponding MIC scores (3022). If the relationship scores below a given threshold (3023), the relationship is deemed / identified as irrelevant (3023b). If the relationship is above a given threshold (3023), the relationship deemed / identified as relevant (3023a), and is further subject to network analysis (3024). The following code fragment shows an exemplary methodology for such analysis, according to one embodiment:Read total list of relationships file as links threshold = 0.8 for i in range(len(links)): if links >= threshold multiplierfi] = 1 else multiplierfi] = 0 end if links temp = multiplier*links final links = links_temp[links_temp != 0] savetxt(output_file,final_links) output_file.close()

[0134] Based on the output of the network analysis, active strains are selected (3025) for preparing products (e.g., ensembles, aggregates, and / or other synthetic groupings) containing the selected strains. The output of the network analysis can also be used to inform the selection of strains for further product composition testing.

[0135] The use of thresholds is discussed above for analyses and determinations. Thresholds can be, depending on the implementation and application: (1) empirically determined (e.g., based on distribution levels, setting a cutoff at a number that removes a specified or significant portion of low level reads); (2) any non-zero value; (3) percentage / percentile based; (4) onlystrains whose normalized second marker (i.e., activity) reads is greater than normalized first marker (cell count) reads; (5) log2 fold change between activity and quantity or cell count; (6) normalized second marker (activity) reads is greater than mean second marker (activity) reads for entire sample (and / or sample set); and / or any magnitude threshold described above in addition to a statistical threshold (i.e., significance testing). The following example provides thresholding detail for distributions of RNA-based second marker measurements with respect to DNA-based first marker measurements, according to one embodiment.

[0136] In some embodiments, the compositions of the present disclosure comprise one or more bacteria and / or one or more fungi that have a MIC score of at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.65, at least about 0.7, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.9, or at least about 0.95.

[0137] In some embodiments, the compositions of the present disclosure comprise one or more bacteria and / or one or more fungi that have a MIC score of at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.80, at least 0.85, at least 0.9, or at least 0.95.

[0138] As used herein “shelf-stable” refers to a functional attribute and new utility acquired by the microbes formulated according to the disclosure, which enable said microbes to exist in a useful / active state outside of their natural environment in the rumen (i.e. a markedly different characteristic). Thus, shelf-stable is a functional attribute created by the formulations / compositions of the disclosure and denoting that the microbe formulated into a shelf-stable composition can exist outside the rumen and under ambient conditions for a period of time that can be determined depending upon the particular formulation utilized, but in general means that the microbes can be formulated to exist in a composition that is stable under ambient conditions for at least a few days and generally at least one week. Accordingly, a “shelf-stable ruminant supplement” is a composition comprising one or more microbes of the disclosure, said microbes formulated in a composition, such that the composition is stable under ambient conditions for at least one week, meaning that the microbes comprised in the composition (e.g. whole cell, spore, or lysed cell) are able to impart one or more beneficial phenotypic properties to a ruminant when administered (e.g. increased milk yield, improved milk compositional characteristics, improved rumen health, and / or modulation of the rumen microbiome).Feedlot Catle v.s. Dairy Cows

[0139] One of ordinary skill in the art would be aware that the diet of a dairy cow would be distinct from that of a steer on a beef feedlot. The steer on the beef feedlot would be fed a high- energy high-grain diet in order to quickly increase the rate of weight gain and to increase the maximum weight prior to rendering. The cow on the dairy farm would be fed a different diet that is optimized for the production of milk with little consideration for rapid weight gain or highest maximum weight. The two diets would result in the rumen of the animals in the two environments to yield drastically different microbiota. Thus, the microorganisms in the rumen of the dairy cow and that of the feedlot steer are expected to be different from one another. However, in some instances, dairy cows can be fed a ration that contains more grain than normal. In these cases, the dairy cow rumen may resemble that of a steer on a high-grain diet.Isolated Microbes

[0140] In some aspects, the present disclosure provides isolated microbes, including novel strains of microbes, presented in Table 1 and Tables 3-6.

[0141] In other aspects, the present disclosure provides isolated whole microbial cultures of the microbes identified in Table 1 and Tables 3-6. These cultures may comprise microbes at various concentrations.

[0142] In some aspects, the disclosure provides for utilizing one or more microbes selected from Table 1 and Tables 3-6 to increase a phenotypic trait of interest (e.g., reducing methane emissions) in a ruminant.

[0143] Furthermore, the disclosure relates to microbes having characteristics substantially similar to that of a microbe identified in Table 1 and Tables 3-6.

[0144] The isolated microbial species, and novel strains of said species, identified in the present disclosure, are able to impart beneficial properties or traits to ruminants. For instance, the isolated microbes described in Table 1 and Tables 3-6, or consortia of said microbes, are able to reduce enteric methane emissions in a ruminant. The reduction can be quantitatively measured, for example, by measuring the effect that said microbial application has upon methane emissions.

[0145] In some embodiments, the isolated microbial strains are microbes of the present disclosure that have been genetically modified. In some embodiments, the genetically modified or recombinant microbes comprise polynucleotide sequences which do not naturally occur in said microbes. In some embodiments, the microbes may comprise heterologouspolynucleotides. In further embodiments, the heterologous polynucleotides may be operably linked to one or more polynucleotides native to the microbes.

[0146] In some embodiments, the heterologous polynucleotides may be reporter genes or selectable markers. In some embodiments, reporter genes may be selected from any of the family of fluorescence proteins (e.g., GFP, RFP, YFP, and the like), P-galactosidase, luciferase. In some embodiments, selectable markers may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, aminoglycoside adenyltransferase, dihydrofolate reductase, acetolactase synthase, bromoxynil nitrilase, P-glucuronidase, dihydrogolate reductase, and chloramphenicol acetyltransferase. In some embodiments, the heterologous polynucleotide may be operably linked to one or more promoter.

[0147] In some embodiments, the disclosure provides isolated microbial strains of Clostridium sensu stricto. In some embodiments, the isolated microbial strain of Clostridium sensu stricto comprises the 16S nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the isolated microbial strain of Clostridium sensu stricto comprising the 16S nucleic acid sequence of SEQ ID NO: 28 comprises the deposit accession number described in Table 2.

[0148] In some embodiments, the disclosure provides isolated microbial strains of Pichia sp. In some embodiments, the isolated microbial strain of Pichia sp. comprises the ITS nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the isolated microbial strain of Pichia sp. comprising the ITS nucleic acid sequence of SEQ ID NO: 32 comprises the deposit accession number described in Table 2.

[0149] In some embodiments, the disclosure provides isolated microbial strains of Butyrivibrio sp. In some embodiments, the isolated microbial strain of Butyrivibrio sp. comprises the 16S nucleic acid sequence of SEQ ID NO: 2067. In some embodiments, the isolated microbial strain of Butyrivibrio sp. comprising the 16S nucleic acid sequence of SEQ ID NO: 2067 comprises the deposit accession number described in Table 2.

[0150] In some embodiments, the disclosure provides isolated microbial strains of Ruminococcus sp. In some embodiments, the isolated microbial strain of Ruminococcus bovis comprises the 16S nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the isolated microbial strain of Ruminococcus bovis comprising the 16S nucleic acid sequence of SEQ ID NO: 1 comprises the deposit accession number described in Table 2.

[0151] In some embodiments, the isolated microbial strain of Ruminococcus bovis comprises the 16S nucleic acid sequence of SEQ ID NO: 2108. In some embodiments, the isolated Ruminococcus bovis comprises a 16S rRNA sequence of SEQ ID NO: 2108 that differs by two nucleotides to the original Ruminococcus bovis of SEQ ID NO: 1. In some embodiments, theisolated microbial strain of Ruminococcus bovis comprises the deposit accession number PTA- 125917, NRRL B-67764, TSD-225, or NCTC 14479. In some embodiments, the isolated strain of Ruminococcus bovis comprises one or more mutations selected from the group consisting of: (a) a G^T substitution at position 297 of SEQ ID NO: 2109; (b) a CC^TA substitution at positions 301-302 of SEQ ID NO: 2111; (c) a T^G substitution at position 307 of SEQ ID NO: 2111; (d) a -A deletion at position 300 of SEQ ID NO: 2113; (e) a CCA— >TTC substitution at positions 116-118 of SEQ ID NO: 2115; (f) a +T insertion between positions 105-106 of SEQ ID NO: 2117; (g) a C^T substitution at position 298 of SEQ ID NO: 2119; (h) a C^A substitution at position 298 of SEQ ID NO: 2121; and (i) a +AC insertion between positions 43-44 of SEQ ID NO: 2123. In some embodiments, the isolated strain of Ruminococcus bovis comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 2110, 2112, 2114, 2116, 2118, 2120, 2122, or 2124.Microbial Compositions

[0152] In some aspects, the disclosure provides microbial compositions comprising a combination of at least any two microbes selected from amongst the microbes identified in Table 1 and Tables 3-6.

[0153] In certain embodiments, the compositions of the present disclosure comprise two microbes, or three microbes, or four microbes, or five microbes, or six microbes, or seven microbes, or eight microbes, or nine microbes, or ten or more microbes. Said microbes of the consortia are different microbial species, or different strains of a microbial species.

[0154] In particular aspects, the disclosure provides microbial consortia, comprising species as grouped in Tables 8-14. In one example, the letters A through I in Tables 8-14 represent a non-limiting selection of microbes of the present disclosure, defined as:A = Strain designation Ascusb_3138 (SEQ ID NO: 28) identified in Table 1;B = Strain designation Ascusf_15 (SEQ ID NO: 32) identified in Table 1;C = Strain designation Ascusb_5 (SEQ ID NO: 1 or 2108) identified in Table 1;D = Strain designation Ascusb_826 (SEQ ID NO: 2067) identified in Table 1;E = Strain designation Ascusbbf_4D (SEQ ID NO: 10654) identified in Table 6;F = Strain designation Ascusbbf_154B (SEQ ID NO: 10589) identified in Table 6; G = Strain designation Ascusbbf_876E (SEQ ID NO: 10402) identified in Table 6;H = Strain designation MP1D12T(SEQ ID NO: 10940) identified in Table 5; andI = Strain designation Ascusf_249 (SEQ ID NO: 50) identified in Table 1.

[0155] In another example, the letters A through I in Tables 8-14 represent a non-limiting selection of microbes of the present disclosure, selected from the group consisting of strain designation Ascusb_17 (SEQ ID NO: 4943), Ascusb_409 (SEQ ID NO: 4944), Ascusb_17848 (SEQ ID NO: 4945), Ascusb_18 (SEQ ID NO: 4104), Ascusb_126 (SEQ ID NO: 4828), Ascusb_51 (SEQ ID NO: 4364), Ascusb_64 (SEQ ID NO: 4620), Ascusb_299 (SEQ ID NO: 4921), Ascusb_ 341 (SEQ ID NO: 4814), Ascusb_ 1785 (SEQ ID NO: 4116), Ascusbbf_154 (SEQ ID NO: 5020); Ascusbbf_4 (SEQ ID NO: 5031); and Ascusbbf_876 (SEQ ID NO: 4962) identified in Tables 4-6.Table 8. Eight and Nine Strain ConsortiaTable 9. Seven Strain ConsortiaTable 10. Six Strain ConsortiaTable 11. Five Strain ConsortiaTable 12. Four Strain ConsortiaTable 13. Three Strain ConsortiaTable 14. Two Strain Consortia

[0156] In some embodiments, the microbial consortia may be selected from any member group from Tables 8-14.

[0157] The present disclosure provides microbial supplements comprise: (i) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28; (ii) Pichia sp. comprising an ITS nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32; (iii) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 2108; and / or (iv) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 2067. In some embodiments of the methods, the microbial supplement further comprises a carrier suitable for ruminant administration.

[0158] The present disclosure provides microbial supplements comprise: microbial supplements comprise: (i) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (ii) a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; (iii) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1 or 2108; and / or (iv) a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067. In some embodiments of the methods, the microbial supplement further comprises a carrier suitable for ruminant administration.

[0159] The present disclosure provides microbial supplements comprise: (i) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28; (ii) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 or 2108; and (iii) a. Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2067. In some embodiments of the methods, the microbial supplement further comprises a carrier suitable for ruminant administration.

[0160] The present disclosure provides microbial supplements comprise: microbial supplements comprise: (i) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (ii) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1 or 2108; and (iii) a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067. In some embodiments of the methods, the microbial supplement further comprises a carrier suitable for ruminant administration.Isolated Microbes — Microbial Culture Techniques

[0161] The microbes of the present disclosure were obtained, among other places, at various locales in the United States from the gastrointestinal tract of cows.

[0162] The microbes of Table 1 and Tables 3-6 were matched to their nearest taxonomic groups by utilizing classification tools of the Ribosomal Database Project (RDP) for 16s rRNA sequences and the User-friendly Nordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences. Examples of matching microbes to their nearest taxa may be found in Lan et al. (2012. PROS one. 7(3):e32491), Schloss and Westcott (2011. Appl. Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005. New Phytologist. 166(3): 1063-1068).

[0163] The isolation, identification, and culturing of the microbes of the present disclosure can be effected using standard microbiological techniques. Examples of such techniques may be found in Gerhardt, P. (ed.) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.) Manual of Clinical Microbiology, Third Edition. American Society for Microbiology, Washington, D.C. (1980), each of which is incorporated by reference.

[0164] Isolation can be effected by streaking the specimen on a solid medium (e.g., nutrient agar plates) to obtain a single colony, which is characterized by the phenotypic traits described hereinabove (e.g., Gram positive / negative, capable of forming spores aerobically / anaerobically, cellular morphology, carbon source metabolism, acid / base production, enzyme secretion, metabolic secretions, etc.) and to reduce the likelihood of working with a culture which has become contaminated.

[0165] For example, for microbes of the disclosure, biologically pure isolates can be obtained through repeated subculture of biological samples, each subculture followed by streaking onto solid media to obtain individual colonies or colony forming units. Methods of preparing, thawing, and growing lyophilized bacteria are commonly known, for example, Ghema, R. L. and C. A. Reddy. 2007. Culture Preservation, p 1019-1033. In C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. Snyder, eds. American Society for Microbiology, Washington, D.C., 1033 pages; herein incorporated by reference. Thus freeze dried liquid formulations and cultures stored long term at -70° C in solutions containing glycerol are contemplated for use in providing formulations of the present disclosure.

[0166] The microbes of the disclosure can be propagated in a liquid medium under aerobic conditions, or alternatively anaerobic conditions. Medium for growing the bacterial strains of the present disclosure includes a carbon source, a nitrogen source, and inorganic salts, as well as specially required substances such as vitamins, amino acids, nucleic acids and the like. Examples of suitable carbon sources which can be used for growing the microbes include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, maltose, and the like; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid and the like; alcohols such as ethanol and glycerol and the like; oil or fat such as soybean oil, rice bran oil, olive oil, com oil, sesame oil. The amount of the carbon source added varies according to the kind of carbon source and is typically between 1 to 100 gram(s) per liter of medium. Preferably, glucose, starch, and / or peptone is contained in the medium as a major carbon source, at a concentration of 0.1-5% (W / V). Examples ofsuitable nitrogen sources which can be used for growing the bacterial strains of the present disclosure include, but are not limited to, amino acids, yeast extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia or combinations thereof. The amount of nitrogen source varies according to the type of nitrogen source, typically between 0.1 to 30 gram per liter of medium. The inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, sodium chloride, calcium carbonate, sodium carbonate can be used alone or in combination. The amount of inorganic acid varies according to the kind of the inorganic salt, typically between 0.001 to 10 gram per liter of medium. Examples of specially required substances include, but are not limited to, vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, dried yeast and combinations thereof. Cultivation can be effected at a temperature, which allows the growth of the microbial strains, essentially, between 20°C and 46°C. In some aspects, a temperature range is 30°C-39°C. For optimal growth, in some embodiments, the medium can be adjusted to pH 6.0-7.4. It will be appreciated that commercially available media may also be used to culture the microbial strains, such as Nutrient Broth or Nutrient Agar available from Difco, Detroit, MI. It will be appreciated that cultivation time may differ depending on the type of culture medium used and the concentration of sugar as a major carbon source.

[0167] In some aspects, cultivation lasts between 24-96 hours. Microbial cells thus obtained are isolated using methods, which are well known in the art. Examples include, but are not limited to, membrane filtration and centrifugal separation. The pH may be adjusted using sodium hydroxide and the like and the culture may be dried using a freeze dryer, until the water content becomes equal to 4% or less. Microbial co-cultures may be obtained by propagating each strain as described hereinabove. In some aspects, microbial multi-strain cultures may be obtained by propagating two or more of the strains described hereinabove. It will be appreciated that the microbial strains may be cultured together when compatible culture conditions can be employed.

[0168] In some embodiments, the microorganisms of the present disclosure are subjected to a serial preservation challenge to improve microbial viability. In some embodiments, the microorganisms are subjected to a serial preservation challenge to improve stability. In some embodiments, the microorganisms of the present disclosure are subjected to at least onepreservation challenge. In some embodiments, the microorganisms of the present disclosure are subjected to at least two, three, four, five, or more preservation challenges.

[0169] In some embodiments, the serial preservation method comprises: (a) subjecting a population of target microbial cells to a first preservation challenge to provide a first population of challenged microbial cells; (b) harvesting viable challenged microbial cells from the first population of challenged microbial cells to provide a first population of viable challenged microbial cells; (c) subjecting the first population of viable challenged microbial cells to a second preservation challenge to provide a second population of challenged microbial cells; (d) harvesting viable challenged microbial cells from the second population of challenged microbial cells to provide a second population of viable challenged microbial cells; (e) subjecting the second population of viable challenged microbial cells to a third preservation challenge to provide a third population of challenged microbial cells; (f) harvesting viable challenged microbial cells from the third population of challenged microbial cells to provide a third population of viable challenged microbial cells; (g) preserving the third population of viable challenged microbial cells to provide a population of preserved viability-enhanced microbial cells; and (h) preparing a product using the population of preserved viability- enhanced microbial cells.

[0170] In some embodiments, each of the preservation challenges are the same type of preservation challenge. For example, in some embodiments, the microorganisms of the present disclosure are subjected to two, three, four, five, or more preservation challenges before final preservation for storage and / or incorporation into a product, wherein each of the preservation challenges are of the same type. Types of preservation challenges include, but are not limited to, freeze drying / lyophilization, vitrification / glass formation, evaporation, foam formation, vaporization, cryopreservation, spray drying, adsorptive drying, extrusion, and fluid bed drying. Methods of microbial preservation are further described in PCT Application No. PCT / US2020 / 020311, herein incorporated by reference in its entirety.

[0171] In some embodiments, the preservation challenges are different types of preservation challenges. For example, in some embodiments, the microorganisms of the present disclosure are subjected to a first and a second preservation challenge, wherein the first and the second preservation challenges are different challenges types. For example, in some embodiments, the first preservation challenge is a cryopreservation challenge and the second preservation challenge is a freeze-drying preservation challenge.

[0172] In some embodiments, any one of the microorganisms listed in Table 1 and Tables 3- 6 may be subjected to serial preservation challenge. In some embodiments, serial preservationresults in one or more mutations in the genome of a microorganism. In some embodiments, serial preservation results in one or more mutations in the genome of any one of the microorganisms listed in Table 1 and Tables 3-6.

[0173] In some embodiments, serial preservation of the microorganisms of the present disclosure results in an increase in microbial viability of at least 5%. In other words, the viability of the population of microbes present at the conclusion of the serial preservation challenges is increased by at least 5% compared to the viability of the population of microbes that were present prior to any preservation challenges. In some embodiments, serial preservation of a microbial culture results in an increase in microbial viability between about 5% and about 30%, about 5% and about 25%, about 5% and about 20%, about 5% and about 15%, about 5% and about 10%, about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, or about 25% and about 30%. In some embodiments, serial preservation of a microbial culture results in an increase in microbial viability between about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, about 25% and about 30%, about 10% and about 25%, about 10% and about 20%, or about 10% and about 15%. In some embodiments, serial preservation of a microbial culture results in an increase in microbial viability of at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30% or more.

[0174] In some embodiments, serial preservation of the microorganisms of the present disclosure results in an increase in microbial stability of at least 5%. In other words, the stability of the population of microbes present at the conclusion of the serial preservation challenges is increased by at least 5% compared to the stability of the population of microbes that were present prior to any preservation challenges. In some embodiments, serial preservation of a microbial culture results in an increase in stability between about 5% and about 30%, about 5% and about 25%, about 5% and about 20%, about 5% and about 15%, about 5% and about 10%, about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, or about 25% and about 30%. In some embodiments, serial preservation of a microbial culture results in an increase in stability between about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, about 25% and about 30%, about 10% and about 25%, about 10% and about 20%, or about 10% and about 15%. In some embodiments, serial preservation of a microbial culture results in an increase in stability of at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30% or more.Isolated Microbes — Microbial Strains

[0175] Microbes can be distinguished into a genus based on polyphasic taxonomy, which incorporates all available phenotypic and genotypic data into a consensus classification (Vandamme et al. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996, 60:407-438). One accepted genotypic method for defining species is based on overall genomic relatedness, such that strains which share approximately 70% or more relatedness using DNA-DNA hybridization, with 5°C or less A7m(the difference in the melting temperature between homologous and heterologous hybrids), under standard conditions, are considered to be members of the same species. Thus, populations that share greater than the aforementioned 70% threshold can be considered to be variants of the same species. Another accepted genotypic method for defining species is to isolate marker genes of the present disclosure, sequence these genes, and align these sequenced genes from multiple isolates or variants. The microbes are interpreted as belonging to the same species if one or more of the sequenced genes share at least 97% sequence identity.

[0176] The 16S or 18S rRNA sequences, or ITS sequences are often used for making distinctions between species and strains, in that if one of the aforementioned sequences share less than a specified percent sequence identity from a reference sequence, then the two organisms from which the sequences were obtained are said to be of different species or strains.

[0177] Thus, one could consider microbes to be of the same species, if they share at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity across the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

[0178] Further, one could define microbial strains of a species, as those that share at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence sequence identity across the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

[0179] In one embodiment, microbial strains of the present disclosure include those that comprise polynucleotide sequences that share at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.1%, at least about 95.2%, at least about 95.3%, atleast about 95.4%, at least about 95.5%, at least about 95.6%, at least about 95.7%, at least about 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, at least about 96.7%, at least about 96.8%, at least about 96.9%, at least about 97%, at least about 97.1%, at least about 97.2%, at least about 97.3%, at least about 97.4%, at least about 97.5%, at least about 97.6%, at least about 97.7%, at least about 97.8%, at least about 97.9%, at least about 98%, at least about 98.1%, at least about 98.2%, at least about 98.3%, at least about 98.4%, at least about 98.5%, at least about 98.6%, at least about 98.7%, at least about 98.8%, at least about 98.9%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100% sequence identity with any one of SEQ ID NOs: 1-10940.

[0180] In some embodiments, the purified population of bacteria comprises a 16S nucleic acid sequence and / or purified population of fungi comprises an ITS nucleic acid sequence that shares at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.1%, at least about 95.2%, at least about 95.3%, at least about 95.4%, at least about 95.5%, at least about 95.6%, at least about 95.7%, at least about 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, at least about 96.7%, at least about 96.8%, at least about 96.9%, at least about 97%, at least about 97.1%, at least about 97.2%, at least about 97.3%, at least about 97.4%, at least about 97.5%, at least about 97.6%, at least about 97.7%, at least about 97.8%, at least about 97.9%, at least about 98%, at least about 98.1%, at least about 98.2%, at least about 98.3%, at least about 98.4%, at least about 98.5%, at least about 98.6%, at least about 98.7%, at least about 98.8%, at least about 98.9%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100% sequence identity with any one of SEQ ID NOs: 1-10940.

[0181] In some embodiments, the present disclosure provides a method for reducing enteric methane emission (EME) and / or EME intensity by a ruminant, the method comprising administering a microbial supplement to the ruminant, wherein the microbial supplement comprises: a purified population of bacteria comprising a 16S nucleic acid sequence and / or purified population of fungi comprising an ITS nucleic acid sequence that shares at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.1%, at least about 95.2%, at least about 95.3%, at least about 95.4%, at least about 95.5%, at least about 95.6%, at least about 95.7%, at least about 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, at least about 96.7%, at least about 96.8%, at least about 96.9%, at least about 97%, at least about 97.1%, at least about 97.2%, at least about 97.3%, at least about 97.4%, at least about 97.5%, at least about 97.6%, at least about 97.7%, at least about 97.8%, at least about 97.9%, at least about 98%, at least about 98.1%, at least about 98.2%, at least about 98.3%, at least about 98.4%, at least about 98.5%, at least about 98.6%, at least about 98.7%, at least about 98.8%, at least about 98.9%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100% sequence identity with any one of SEQ ID NOs: 1-10940.

[0182] In some embodiments, the purified population of bacteria comprises a 16S nucleic acid sequence and / or purified population of fungi comprises an ITS nucleic acid sequence that shares at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.1%, at least about 95.2%, at least about 95.3%, at least about 95.4%, at least about 95.5%, at least about 95.6%, at least about 95.7%, at least about 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, at least about 96.7%, at least about 96.8%, at least about 96.9%, at least about 97%, at least about 97.1%, at least about 97.2%, at least about 97.3%, at least about 97.4%, at least about 97.5%, at least about 97.6%, at least about 97.7%, at least about 97.8%, at least about 97.9%, at least about 98%, at least about 98.1%, at least about 98.2%, at least about 98.3%, at least about 98.4%, at least about 98.5%, at least about 98.6%, at least about 98.7%, at least about 98.8%, at least about 98.9%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or at least about 100% sequence identity with SEQ ID NOs: 1, 28, 32, 2067, and / or 2108.

[0183] In some embodiments, the purified population of bacteria comprises a 16S nucleic acid sequence and / or purified population of fungi comprises an ITS nucleic acid sequence that shares at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least about 100% sequence identity with SEQ ID NOs: 10589, 10654, 10402, and / or 10940.

[0184] In some embodiments, the microbial composition comprises a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10940, wherein the 16S nucleic acid sequence is at least one nucleotide different from a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10402. In some embodiments, the microbial composition comprises a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10940, wherein the 16S nucleic acid sequence is at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleotides different from a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10402. In some embodiments, the microbial composition comprises a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10940, wherein the 16S nucleic acid sequence is one nucleotide different from a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10402. In some embodiments, the microbial composition comprises a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10940, wherein the 16S nucleic acid sequence is two nucleotides different from a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10402. In some embodiments, the microbial composition comprises a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10940, wherein the 16S nucleic acid sequence is three nucleotides different from a purified population of bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10402.

[0185] In some embodiments, the present disclosure provides a method for reducing EME and / or EME intensity by a ruminant, the method comprising: administering a microbial supplement to the ruminant, wherein the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; (b) Pichia sp. comprising an ITS nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1 or 2108; and / or (d) a Butyrivibrio sp.comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067; and a carrier suitable for ruminant administration.

[0186] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1 or 2108; and / or (d) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

[0187] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1 or 2108; and / or (d) a. Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

[0188] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (b) aPichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; (c) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1 or 2108; and / or (d) aButyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

[0189] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1 or 2108; and (c) aButyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067.

[0190] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1 or 2108; and (c) aButyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

[0191] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1 or 2108; and (c) a Butyr ivibr io sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

[0192] In some embodiments, the microbial supplement comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; (b) a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1 or 2108; and (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

[0193] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5031; (b) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5020; and / or (c) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0194] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 5031; (b) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 5020; and / or (c) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0195] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5031; (b) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5020; and / or (c) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NOs: 10402, 10939, or 10940; and (d) a carrier suitable for ruminant administration.

[0196] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant.In some embodiments, the microbial composition comprises: (a) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 5031; (b) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 5020; and / or (c) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NOs: 10402, 10939, or 10940; and (d) a carrier suitable for ruminant administration.

[0197] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10654; (b) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10589; and / or (c) a bacteria comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0198] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10654; (b) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10589; and / or (c) a bacteria comprising a 16S nucleic acid sequence of SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0199] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5031; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5020; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0200] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 5031; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 5020; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0201] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5031; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 5020; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NOs: 10402, 10939, or 10940; and (d) a carrier suitable for ruminant administration.

[0202] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 5031; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 5020; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence of SEQ ID NOs: 10402, 10939, or 10940; and (d) a carrier suitable for ruminant administration.

[0203] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10654; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10589; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0204] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 10654; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 10589; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 10939 or 10940; and (d) a carrier suitable for ruminant administration.

[0205] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10654; (b) aSuccinivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 10589; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NOs: 10402, 10939, or 10940; and (d) a carrier suitable for ruminant administration.

[0206] In some embodiments, the present disclosure provides a method of reducing EME and / or EME intensity in a ruminant by administering a microbial composition to the ruminant. In some embodiments, the microbial composition comprises: (a) Prevotella sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 10654; (b) a Succinivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 10589; and / or (c) a Chordicoccus sp. comprising a 16S nucleic acid sequence of SEQ ID NOs: 10402, 10939, or 10940; and (d) a carrier suitable for ruminant administration.

[0207] Comparisons may also be made with 23 S rRNA sequences against reference sequences.

[0208] Unculturable microbes often cannot be assigned to a definite species in the absence of a phenotype determination, the microbes can be given a candidatus designation within a genus provided their 16S or 18S rRNA sequences or ITS sequences subscribes to the principles of identity with known species.

[0209] One approach is to observe the distribution of a large number of strains of closely related species in sequence space and to identify clusters of strains that are well resolved from other clusters. This approach has been developed by using the concatenated sequences of multiple core (house-keeping) genes to assess clustering patterns, and has been called multilocus sequence analysis (MLSA) or multilocus sequence phylogenetic analysis. MLSA has been used successfully to explore clustering patterns among large numbers of strains assigned to very closely related species by current taxonomic methods, to look at the relationships between small numbers of strains within a genus, or within a broader taxonomic grouping, and to address specific taxonomic questions. More generally, the method can be used to ask whether bacterial species exist - that is, to observe whether large populations of similar strains invariably fall into well-resolved clusters, or whether in some cases there is a genetic continuum in which clear separation into clusters is not observed.

[0210] In order to more accurately make a determination of genera, a determination of phenotypic traits, such as morphological, biochemical, and physiological characteristics are made for comparison with a reference genus archetype. The colony morphology can include color, shape, pigmentation, production of slime, etc. Features of the cell are described as to shape, size, Gram reaction, extracellular material, presence of endospores, flagella presence and location, motility, and inclusion bodies. Biochemical and physiological features describegrowth of the organism at different ranges of temperature, pH, salinity and atmospheric conditions, growth in presence of different sole carbon and nitrogen sources. One of ordinary skill in the art would be reasonably apprised as to the phenotypic traits that define the genera of the present disclosure.

[0211] In one embodiment, the microbes taught herein were identified utilizing 16S rRNA gene sequences and ITS sequences. It is known in the art that 16S rRNA contains hypervariable regions that can provide species / strain-specific signature sequences useful for bacterial identification, and that ITS sequences can also provide species / strain-specific signature sequences useful for fungal identification.

[0212] Phylogenetic analysis using the rRNA genes and / or ITS sequences are used to define “substantially similar” species belonging to common genera and also to define “substantially similar” strains of a given taxonomic species. Furthermore, physiological and / or biochemical properties of the isolates can be utilized to highlight both minor and significant differences between strains that could lead to advantageous behavior in ruminants.

[0213] Compositions of the present disclosure may include combinations of fungal spores and bacterial spores, fungal spores and bacterial vegetative cells, fungal vegetative cells and bacterial spores, fungal vegetative cells and bacterial vegetative cells. In some embodiments, compositions of the present disclosure comprise bacteria only in the form of spores. In some embodiments, compositions of the present disclosure comprise bacteria only in the form of vegetative cells. In some embodiments, compositions of the present disclosure comprise bacteria in the absence of fungi. In some embodiments, compositions of the present disclosure comprise fungi in the absence of bacteria. In some embodiments, compositions of the present disclosure comprise VBNC bacteria and / or fungi. In some embodiments, compositions of the present disclosure comprise bacteria and / or fungi in a quiescent state. In some embodiments, compositions of the present disclosure include dormant bacteria and / or fungi.

[0214] Bacterial spores may include endospores and akinetes. Fungal spores may include statismospores, ballistospores, autospores, aplanospores, zoospores, mitospores, megaspores, microspores, meiospores, chlamydospores, urediniospores, teliospores, oospores, carpospores, tetraspores, sporangiospores, zygospores, ascospores, basidiospores, ascospores, and asciospores.

[0215] In some embodiments, spores of the composition germinate upon administration to animals of the present disclosure. In some embodiments, spores of the composition germinate only upon administration to animals of the present disclosure.Microbial Compositions

[0216] In some embodiments, the microbes of the disclosure are combined into microbial compositions.

[0217] In some embodiments, the microbial compositions include ruminant feed, such as cereals, grain, or grain byproducts (barley, maize, oats, sorghum, wheat, distillers grains, sweet bran, and the like); roughage (alfalfa, silage, fescue, clover, ryegrass, and the like); starches (tapioca and the like); protein (oilseed cake, vegetable wastes, com by-products, wheat by products, and the like); liquid feeds (condensed com distillers solubles, molasses, tallow, yellow grease, com oil, and the like); or non-nitrogen protein. In some embodiments, the microbial compositions include vitamins and / or metabolites thereof, minerals, amino acids, enzymes, urea, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickening agents, antibiotics, and the like. In some embodiments, the microbial compositions include one or more include one or more of an ionophore; vaccine, antibiotic; antihelmintic; virucide; nematicide; amino acids such as methionine, glutamine, valine, glycine, cysteine, homocysteine, aspartic acid, and arginine; fish oil; oregano; carnitine, pantoate, pantothenate, aspartate, and biologically active molecules such as enzymes.

[0218] In some embodiments, the vitamins include vitamin B5, Bl, B2, B3, B6, B9, B12, H,C, A, D, E, or K; and combinations thereof. In some embodiments, the microbial compositions include microbes that synthesize vitamin B5, Bl, B2, B3, B6, B9, B12, H, C, A, D, E, and / or K. In some embodiments, the microbial compositions include microbes that synthesize vitamin B5. In some embodiments, the metabolites of vitamin B5, Bl, B2, B3, B6, B9, B12, H, C, A,D, E, or K are contemplated as one or more components of a microbial composition of the present disclosure. In one embodiment, pantothenate is a component of a microbial composition of the present disclosure. In one embodiment, a component of a microbial composition of the present disclosure includes one or more precursors utilized by mammalian or microbial biosynthesis of vitamins.

[0219] In some embodiments, the microbial compositions of the present disclosure are solid. Where solid compositions are used, it may be desired to include one or more carrier materials including, but not limited to: mineral earths such as silicas, talc, kaolin, limestone, chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesium sulfate; magnesium oxide; zeolites, calcium carbonate; magnesium carbonate; trehalose; chitosan; shellac; albumins; starch; skim milk powder; sweet whey powder; maltodextrin; lactose; inulin; dextrose; and products of vegetable origin such as cereal meals, tree bark meal, wood meal, and nutshell meal.

[0220] In some embodiments, the microbial compositions of the present disclosure are liquid. In further embodiments, the liquid comprises a solvent that may include water, alcohol, saline, carbohydrate solution, and other animal-safe solvents. In some embodiments, the microbial compositions of the present disclosure include binders such as animal-safe polymers, carboxymethylcellulose, starch, polyvinyl alcohol, and the like.

[0221] In some embodiments, the microbial compositions of the present disclosure comprise thickening agents such as silica, clay, natural extracts of seeds or seaweed, synthetic derivatives of cellulose, guar gum, locust bean gum, alginates, and methylcelluloses. In some embodiments, the microbial compositions comprise anti-settling agents such as modified starches, polyvinyl alcohol, xanthan gum, and the like.

[0222] In some embodiments, the microbial compositions of the present disclosure comprise colorants including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. In some embodiments, the microbial compositions of the present disclosure comprise trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc. In some embodiments, the microbial compositions comprise dyes, both natural and artificial. In some embodiments, the dye is green in color.

[0223] In some embodiments, the microbial compositions of the present disclosure comprise an animal-safe virucide, parasiticide, bacteriocide, fungicide, or nematicide.

[0224] In some embodiments, microbial compositions of the present disclosure comprise saccharides (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, oligosaccharides, and the like), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts and combinations thereof. In a further embodiment, microbial compositions comprise polymers of agar, agarose, gelrite, gellan gum, and the like. In some embodiments, microbial compositions comprise plastic capsules, emulsions (e.g., water and oil), membranes, and artificial membranes. In some embodiments, emulsions or linked polymer solutions may comprise microbial compositions of the present disclosure. See Harel and Bennett (US Patent 8,460,726 B2).

[0225] In some embodiments, microbial compositions of the present disclosure comprise one or more oxygen scavengers, denitrifies, nitrifiers, heavy metal chelators, and / or dechlorinators; and combinations thereof. In one embodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active once themicrobial compositions are mixed with food and / or water to be administered to the animal. In one embodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active when administered to the animal.

[0226] In some embodiments, microbial compositions of the present disclosure occur in a solid form (e.g., dispersed lyophilized spores) or a liquid form (microbes interspersed in a storage medium). In some embodiments, microbial compositions of the present disclosure are added in dry form to a liquid to a liquid to form a suspension immediately prior to administration.

[0227] In some embodiments, microbial compositions of the present disclosure comprise one or more preservatives. The preservatives may be in liquid or gas formulations. The preservatives may be selected from one or more of monosaccharide, disaccharide, trisaccharide, polysaccharide, acetic acid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbic acid, erythrobic acid, potassium nitrate, sodium ascorbate, sodium erythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite, nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate, methyl- / ?-hydroxy benzoate, methyl paraben, potassium acetate, potassium benzoiate, potassium bisulphite, potassium diacetate, potassium lactate, potassium metabisulphite, potassium sorbate, propyl - -hydroxy benzoate, propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite, sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite, sodium salt of methyl- / ?-hydroxy benzoic acid, sodium salt of propyl- -hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodium dithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate, natamycin, potassium sorbate, potassium bisulfite, potassium metabisulfite, propionic acid, sodium diacetate, sodium propionate, sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT), butylated hydroxyl anisole (BHA), citric acid, citric acid esters of mono- and / or diglycerides, L-cysteine, L-cysteine hydrochloride, gum guaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate, monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaric acid, tertiary butyl hydroquinone, stannous chloride, thiodipropionic acid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin, sulfur dioxide, formic acid, or tocopherol(s).

[0228] In some embodiments, microbial compositions of the present disclosure comprise one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators; and combinations thereof. In one embodiment, the one or more oxygen scavengers, denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active once the microbial compositions are mixed with food and / or water to be administered to the beef cattle. In one embodiment, the one or more oxygen scavengers,denitrifiers, nitrifiers, heavy metal chelators, and / or dechlorinators are not chemically active when administered to the beef cattle.

[0229] In some embodiments, microbial compositions of the present disclosure include bacterial and / or fungal cells in spore form, vegetative cell form, and / or lysed cell form. In one embodiment, the lysed cell form acts as a mycotoxin binder, e.g. mycotoxins binding to dead cells.

[0230] In some embodiments, the microbial compositions are shelf stable in a refrigerator (35- 40°F) for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable in a refrigerator (35-40°F) for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0231] In some embodiments, the microbial compositions are shelf stable at room temperature (68-72°F) or between 50-77°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at room temperature (68-72°F) or between 50-77°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0232] In some embodiments, the microbial compositions are shelf stable at -23-35°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at -23-35°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0233] In some embodiments, the microbial compositions are shelf stable at 77-100°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbialcompositions are shelf stable at 77-100°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0234] In some embodiments, the microbial compositions are shelf stable at 101-213 °F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at 101-213°F for a period of 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

[0235] In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40°F), at room temperature (68-72°F), between 50- 77°F, between -23-35°F, between 70-100°F, or between 101-213°F for a period of about 1 to 100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1 to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about 1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to 5, about 5 to 100, about 5 to 95, about 5 to 90, about 5 to 85, about 5 to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about 5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to 10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85, about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10 to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85, about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15 to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40, about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20 to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80, about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20 to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35, about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about 25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to 70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50, about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30 to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80, about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30 to 55, about 30 to 50, about 30 to 45, about 30to 40, about 30 to 35, about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about 35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to 60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40, about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about 40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to 60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100, about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45 to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55, about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about 50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to 65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95, about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55 to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95, about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60 to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90, about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70 to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80, about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about 75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to 90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90, about 90 to 100, about 90 to 95, or 95 to 100 weeks.

[0236] In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40°F), at room temperature (68-72°F), between 50- 77°F, between -23-35°F, between 70-100°F, or between 101-213°F for a period of 1 to 100, 1 to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5 to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95, 10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to 65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 100, 40 to 95, 40 to 90, 40 to 85,40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95,45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50, 50 to 100,50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100,55 to 95, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to 100, 60 to 95,60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65, 65 to 100, 65 to 95, 65 to 90, 65 to 85,65 to 80, 65 to 75, 65 to 70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to 90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95 to 100 weeks.

[0237] In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40°F), at room temperature (68-72°F), between 50- 77°F, between -23-35°F, between 70-100°F, or between 101-213°F for a period of about 1 to 36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1 to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about 1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8, about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34, about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to 24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4 to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6 to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about 6 to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 18, about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to 8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8 to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about 8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10, about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10 to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20, about10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12 to 36, about 12 to 34, about12 to 32, about 12 to 30, about 12 to 28, about 12 to 26, about 12 to 24, about 12 to 22, about12 to 20, about 12 to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34, about14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14 to 24, about 14 to 22, about14 to 20, about 14 to 18, about 14 to 16, about 16 to 36, about 16 to 34, about 16 to 32, about16 to 30, about 16 to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20, about16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18 to 30, about 18 to 28, about18 to 26, about 18 to 24, about 18 to 22, about 18 to 20, about 20 to 36, about 20 to 34, about20 to 32, about 20 to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22, about22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22 to 28, about 22 to 26, about22 to 24, about 24 to 36, about 24 to 34, about 24 to 32, about 24 to 30, about 24 to 28, about24 to 26, about 26 to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28, about28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30 to 36, about 30 to 34, about30 to 32, about 32 to 36, about 32 to 34, or about 34 to 36 months.

[0238] In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40°F), at room temperature (68-72°F), between 50- 77°F, between -23-35°F, between 70-100°F, or between 101-213°F for a period of 1 to 36, 1 to 34, 1 to 32, 1 to 30, 1 to 28, 1 to 26, 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 one 4, 1 to 2, 4 to 36, 4 to 34, 4 to 32, 4 to 30, 4 to 28, 4 to 26, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 36, 6 to 34, 6 to 32, 6 to 30, 6 to 28, 6 to 26, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 36, 8 to 34, 8 to 32, 8 to 30, 8 to 28, 8 to 26, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 14, 8 to 12, 8 to 10, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 36, 12 to 34, 12 to 32, 12 to30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 36, 14 to34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 36, 20 to34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 36„ 22 to 34, 22 to 32, 22 to30, 22 to 28, 22 to 26, 22 to 24, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 36, 28 to 34, 28 to 32, 28 to 30, 30 to 36, 30 to34, 30 to 32, 32 to 36, 32 to 34, or about 34 to 36 months.

[0239] In some embodiments, the microbial compositions of the present disclosure are shelf stable at any of the disclosed temperatures and / or temperature ranges and spans of time at a relative humidity of 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, 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, or 98%.

[0240] In some embodiments, the microbial composition of the present disclosure possesses a water activity (aw) of less than 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.150, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005.

[0241] In some embodiments, the microbial composition of the present disclosure possesses a water activity (aw) of less than 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.150,0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005.

[0242] The water activity values are determined by the method of Saturated Aqueous Solutions (Multon, “Techniques d’ Analyse E De Controle Dans Les Industries Agroalimentaires” APRIA (1981)) or by direct measurement using a viable Robotronic BT hygrometer or other hygrometer or hygroscope.

[0243] In some embodiments, the microbial composition comprises at least two different microbes, and wherein the at least two microbes are present in the composition at a ratio of 1 :2, 1:3, 1:3, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:40, 1:50, 1:60, 1:100, 1:125, 1:150, 1:175, or 1:200 or the inverse thereof. In some embodiments, the microbial composition comprises at least three different microbes, and wherein the three microbes are present in the composition at a ratio of 1:2:1, 1:1:2, 2:2:1, 1:3:1, 1:1:3, 3:1:1, 3:3:1, 1:5:1, 1:1:5, 5:1:1, 5:5:1, or 1:5:5.Encapsulation Compositions

[0244] In some embodiments, the microbes or microbial compositions of the disclosure are encapsulated in an encapsulating composition. An encapsulating composition protects the microbes from external stressors prior to entering the gastrointestinal tract of ruminants. In some embodiments, external stressors include thermal and physical stressors associated with pelleting and extrusion. In some embodiments, external stressors include chemicals present in the compositions. Encapsulating compositions further create an environment that may be beneficial to the microbes, such as minimizing the oxidative stresses of an aerobic environment on anaerobic microbes. See Kalsta et al. (US 5,104,662A), Ford (US 5,733,568A), and Mosbach and Nilsson (US 4,647,536A) for encapsulation compositions of microbes, and methods of encapsulating microbes.

[0245] In one embodiment, the compositions of the present disclosure exhibit a thermal tolerance, which is used interchangeably with heat tolerance and heat resistance. In one embodiment, thermal tolerant compositions of the present disclosure are tolerant of the high temperatures associated with feed manufacturing, mixing of feed and compositions of the present disclosure, storage in high heat environments, etc. In one embodiment, thermal tolerant compositions of the present disclosure are resistant to heat-killing and denaturation of the cell wall components and the intracellular environment.

[0246] In one embodiments, the encapsulation is a reservoir-type encapsulation. In one embodiment, the encapsulation is a matrix-type encapsulation. In one embodiment, the encapsulation is a coated matrix-type encapsulation. Burgain et al. (2011. J. Food Eng. 104:467-483) discloses numerous encapsulation embodiments and techniques, all of which are incorporated by reference.

[0247] In some embodiments, the compositions of the present disclosure are encapsulated in one or more of the following: gellan gum, xanthan gum, K-Carrageenan, cellulose acetate phthalate, chitosan, starch, milk fat, whey protein, Ca-alginate, raftilose, raftiline, pectin, saccharide, glucose, maltodextrin, gum arabic, guar, seed flour, alginate, dextrins, dextrans, celluloase, gelatin, gelatin, albumin, casein, gluten, acacia gum, tragacanth, wax, paraffin, stearic acid, monodiglycerides, and diglycerides. In some embodiments, the compositions of the present disclosure are encapsulated by one or more of a polymer, carbohydrate, sugar, plastic, glass, polysaccharide, lipid, wax, oil, fatty acid, or glyceride. In one embodiment, the microbial composition is encapsulated by glucose. In one embodiment, the microbial composition is encapsulated by a glucose-containing composition. In one embodiment, formulations of the microbial composition comprise a glucose encapsulant. In one embodiment, formulations of the microbial composition comprise a glucose-encapsulated composition. In some embodiments, the microbial composition comprises at least one encapsulation material. In some embodiments, the microbial composition comprises a primary encapsulation material and a secondary encapsulation material.

[0248] In some embodiments, the encapsulation of the compositions of the present disclosure is carried out by an extrusion, emulsification, coating, agglomeration, lyophilization, vitrification, foam drying, preservation by vaporization, vacuum-drying, or spray-drying.

[0249] In some embodiments, the encapsulated compositions of the present disclosure are vitrified. In some embodiments, encapsulation involves a process of drying a composition of the present disclosure in the presence of a substance which forms a glassy, amorphous solid state, a process known as vitrification, and in doing so encapsulates the composition. In some embodiments, the vitrified composition is protected from degradative conditions that would typically destroy or degrade microbes. Many common substances have the property of vitrification; that is, they will form a glassy solid state under certain conditions. Among these substances are several sugars, including sucrose and maltose, and other more complex compounds, such as polyvinyl pyrolidone (PVP). As any solution dries down, the molecules in the solution can either crystalize, or they can vitrify. A solute which has an extensive asymmetry may be a superior vitrifier, because of the hindrances to nucleation of crystalsduring drying. A substance that inhibits the crystallization of another substance may result in the combined substances forming a superior vitrification, such as raffinose in the presence of sucrose. See U.S. Patent Nos. 5,290,765 and 9,469,835.

[0250] In some embodiments, a microbial composition is produced that is encapsulated in a vitrified substance. The vitrified composition may be created by selecting a mixture including cells; combining said mixture with sufficient quantity of one or more vitrifying solutes to protect said mixture during drying and to inhibit destructive reactions; and drying said combination by exposing said combination to a desiccant, or desiccating conditions, at a temperature above that which said combination will freeze and below that at which said vitrifying solutes achieve the vitrified state, at approximately normal atmospheric pressure, until said combination is substantially dry.

[0251] In one embodiment, the encapsulating composition comprises microcapsules having a multiplicity of liquid cores encapsulated in a solid shell material. For purposes of the disclosure, a "multiplicity" of cores is defined as two or more.

[0252] A first category of useful fusible shell materials is that of normally solid fats, including fats which are already of suitable hardness and animal or vegetable fats and oils which are hydrogenated until their melting points are sufficiently high to serve the purposes of the present disclosure. Depending on the desired process and storage temperatures and the specific material selected, a particular fat can be either a normally solid or normally liquid material. The terms "normally solid" and "normally liquid" as used herein refer to the state of a material at desired temperatures for storing the resulting microcapsules. Since fats and hydrogenated oils do not, strictly speaking, have melting points, the term "melting point" is used herein to describe the minimum temperature at which the fusible material becomes sufficiently softened or liquid to be successfully emulsified and spray cooled, thus roughly corresponding to the maximum temperature at which the shell material has sufficient integrity to prevent release of the choline cores. "Melting point" is similarly defined herein for other materials which do not have a sharp melting point.

[0253] Specific examples of fats and oils useful herein (some of which require hardening) are as follows: animal oils and fats, such as beef tallow, mutton tallow, lamb tallow, lard or pork fat, fish oil, and sperm oil; vegetable oils, such as canola oil, cottonseed oil, peanut oil, com oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, and castor oil; fatty acid monoglycerides and diglycerides; free fatty acids, such as stearic acid, palmitic acid, and oleic acid; and mixtures thereof. The above listing of oils and fats is not meant to be exhaustive, but only exemplary.

[0254] Specific examples of fatty acids include linoleic acid, y-linoleic acid, dihomo-y- linolenic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid, elaidic acid, oleic acid, palitoleic acid, stearidonic acid, eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecyclic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatri acontanoic acid, and octatri acontanoic acid.

[0255] Another category of fusible materials useful as encapsulating shell materials is that of waxes. Representative waxes contemplated for use herein are as follows: animal waxes, such as beeswax, lanolin, shell wax, and Chinese insect wax; vegetable waxes, such as carnauba, candelilla, bayberry, and sugar cane; mineral waxes, such as paraffin, microcrystalline petroleum, ozocerite, ceresin, and montan; synthetic waxes, such as low molecular weight polyolefin (e.g., CARBOWAX), and polyol ether-esters (e.g., sorbitol); Fischer-Tropsch process synthetic waxes; and mixtures thereof. Water-soluble waxes, such as CARBOWAX and sorbitol, are not contemplated herein if the core is aqueous.

[0256] Still other fusible compounds useful herein are fusible natural resins, such as rosin, balsam, shellac, and mixtures thereof.

[0257] In some embodiments, the microbes or microbial composition is embedded in a wax, such as the waxes described in the present disclosure.

[0258] In some embodiments, the microbes or microbial composition is embedded in wax balls. In some embodiments, the microbes or microbial composition is already encapsulated prior to being embedded in wax balls. In some embodiments, the wax balls are 10 microbes, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, 550 microns, 600 microns, 650 microns, 700 microns, 750 microns, 800 microns, 850 microns, 900 microns, 950 microns, or 1,000 microns.

[0259] In some embodiments, the wax balls are about 10 microbes, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, or about 1,000 microns.

[0260] In some embodiments, the wax balls are between 10-20 microns, 10-30 microns, 10-40 microns, 10-50 microns, 10-60 microns, 10-70 microns, 10-80 microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500 microns, 10-750 microns, 10-1,000 microns, 20-30 microns, 20-40 microns, 20-50 microns, 20-60 microns, 20-70 microns, 20-80 microns, 20-90 microns, 20-100 microns, 20-250 microns, 20-500 microns, 20-750 microns, 20-1,000 microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70 microns, 30-80 microns, 30-90 microns, 30-100 microns, 30-250 microns, 30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60 microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns, 40-250 microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60 microns, 50-70 microns, 50-80 microns, 50-90 microns, 50-100 microns, 50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70 microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns, 60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90 microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns, 70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250 microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000 microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns, 90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns, 100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns, 500-750 microns, 500-1,000 microns, or 750-1,000 microns.

[0261] In some embodiments, the wax balls are between about 10-20 microns, about 10-30 microns, about 10-40 microns, about 10-50 microns, about 10-60 microns, about 10-70 microns, about 10-80 microns, about 10-90 microns, about 10-100 microns, about 10-250 microns, about 10-500 microns, about 10-750 microns, about 10-1,000 microns, about 20-30 microns, about 20-40 microns, about 20-50 microns, about 20-60 microns, about 20-70 microns, about 20-80 microns, about 20-90 microns, about 20-100 microns, about 20-250 microns, about 20-500 microns, about 20-750 microns, about 20-1,000 microns, about 30-40 microns, about 30-50 microns, about 30-60 microns, about 30-70 microns, about 30-80 microns, about 30-90 microns, about 30-100 microns, about 30-250 microns, about 30-500 microns, about 30-750 microns, about 30-1,000 microns, about 40-50 microns, about 40-60 microns, about 40-70 microns, about 40-80 microns, about 40-90 microns, about 40-100 microns, about 40-250 microns, about 40-500 microns, about 40-750 microns, about 40-1,000 microns, about 50-60 microns, about 50-70 microns, about 50-80 microns, about 50-90 microns, about 50-100 microns, about 50-250 microns, about 50-500 microns, about 50-750microns, about 50-1,000 microns, about 60-70 microns, about 60-80 microns, about 60-90 microns, about 60-100 microns, about 60-250 microns, about 60-500 microns, about 60-750 microns, about 60-1,000 microns, about 70-80 microns about 70-90 microns, about 70-90 microns, about 70-100 microns, about 70-250 microns, about 70-500 microns, about 70-750 microns, about 70-1,000 microns, about 80-90 microns, about 80-100 microns, about 80-250 microns, about 80-500 microns, about 80-500 microns, about 80-750 microns, about 80-1,000 microns, about 90-100 microns, about 90-250 microns, about 90-500 microns, about 90-750 microns, about 90-1,000 microns, about 100-250 microns, about 100-500 microns, about 100- 750 microns, about 100-1,000 microns, about 250-500 microns, about 250-750 microns, about 250-1,000 microns, about 500-750 microns, about 500-1,000 microns, or about 750-1,000 microns.

[0262] Various adjunct materials are contemplated for incorporation in fusible materials according to the present disclosure. For example, antioxidants, light stabilizers, dyes and lakes, flavors, essential oils, anti-caking agents, fillers, pH stabilizers, sugars (monosaccharides, disaccharides, trisaccharides, and polysaccharides) and the like can be incorporated in the fusible material in amounts which do not diminish its utility for the present disclosure.

[0263] The core material contemplated herein constitutes from about 0.1% to about 50%, about 1% to about 35%. or about 5% to about 30% by weight of the microcapsules. In some embodiments, the core material contemplated herein constitutes no more than about 30% by weight of the microcapsules. In some embodiments, the core material contemplated herein constitutes about 5% by weight of the microcapsules. The core material is contemplated as either a liquid or solid at contemplated storage temperatures of the microcapsules.

[0264] The cores may include other additives well-known in the pharmaceutical art, including edible sugars, such as sucrose, glucose, maltose, fructose, lactose, cellobiose, monosaccharides, disaccharides, tri saccharides, polysaccharides, and mixtures thereof; artificial sweeteners, such as aspartame, saccharin, cyclamate salts, and mixtures thereof; edible acids, such as acetic acid (vinegar), citric acid, ascorbic acid, tartaric acid, and mixtures thereof; edible starches, such as com starch; hydrolyzed vegetable protein; water-soluble vitamins, such as Vitamin C; water-soluble medicaments; water-soluble nutritional materials, such as ferrous sulfate; flavors; salts; monosodium glutamate; antimicrobial agents, such as sorbic acid; antimycotic agents, such as potassium sorbate, sorbic acid, sodium benzoate, and benzoic acid; food grade pigments and dyes; and mixtures thereof. Other potentially useful supplemental core materials will be apparent to those of ordinary skill in the art.

[0265] Emulsifying agents may be employed to assist in the formation of stable emulsions. Representative emulsifying agents include glyceryl monostearate, polysorbate esters, ethoxylated mono- and diglycerides, and mixtures thereof.

[0266] For ease of processing, and particularly to enable the successful formation of a reasonably stable emulsion, the viscosities of the core material and the shell material should be similar at the temperature at which the emulsion is formed. In particular, the ratio of the viscosity of the shell to the viscosity of the core, expressed in centipoise or comparable units, and both measured at the temperature of the emulsion, should be from about 22: 1 to about 1 : 1, desirably from about 8: 1 to about 1 : 1, and preferably from about 3: 1 to about 1 : 1. A ratio of 1 : 1 would be ideal, but a viscosity ratio within the recited ranges is useful.

[0267] Encapsulating compositions are not limited to microcapsule compositions as disclosed above. In some embodiments encapsulating compositions encapsulate the microbial compositions in an adhesive polymer that can be natural or synthetic without toxic effect. In some embodiments, the encapsulating composition may be a matrix selected from sugar matrix, gelatin matrix, polymer matrix, silica matrix, starch matrix, foam matrix, glass / glassy matrix, etc. See Pirzio et al. (U.S. Patent 7,488,503). In some embodiments, the encapsulating composition may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; monosaccharides; fats; fatty acids, including oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.

[0268] In some embodiments, the encapsulating compositions comprise at least one layer of encapsulation. In some embodiments, the encapsulating compositions comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 layers of encapsulation / encapsulants.

[0269] In some embodiments, the encapsulating compositions comprise at least two layers of encapsulation. In some embodiments, each layer of encapsulation confers a differentcharacteristic to the composition. In some embodiments, no two consecutive layers confer the same characteristic. In some embodiments, at least one layer of the at least two layers of encapsulation confers thermostability, shelf stability, ultraviolet resistance, moisture resistance, hydrophobicity, hydrophilicity, lipophobicity, lipophilicity, pH stability, acid resistance, and base resistance.

[0270] In some embodiments, the encapsulating compositions comprise two layers of encapsulation; the first layer confers thermostability and / or shelf stability, and the second layer provides pH resistance.

[0271] In some embodiments, the encapsulating layers confer a timed release of the microbial composition held in the center of the encapsulating layers. In some embodiments, the greater the number of layers confers a greater amount of time before the microbial composition is exposed, post administration.

[0272] In some embodiments, the encapsulating shell of the present disclosure can be up to 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 210 pm, 220 pm, 230 pm, 240 pm, 250 pm, 260 pm, 270 pm, 280 pm, 290 pm, 300 pm, 310 pm, 320 pm, 330 pm, 340 pm, 350 pm, 360 pm, 370 pm, 380 pm, 390 pm, 400 pm, 410 pm, 420 pm, 430 pm, 440 pm, 450 pm, 460 pm, 470 pm, 480 pm, 490 pm, 500 pm, 510 pm, 520 pm, 530 pm, 540 pm, 550 pm, 560 pm, 570 pm, 580 pm, 590 pm, 600 pm, 610 pm, 620 pm, 630 pm, 640 pm, 650 pm, 660 pm, 670 pm, 680 pm, 690 pm, 700 pm, 710 pm, 720 pm, 730 pm, 740 pm, 750 pm, 760 pm, 770 pm, 780 pm, 790 pm, 800 pm, 810 pm, 820 pm, 830 pm, 840 pm, 850 pm, 860 pm, 870 pm, 880 pm, 890 pm, 900 pm, 910 pm, 920 pm, 930 pm, 940 pm, 950 pm, 960 pm,970 pm, 980 pm, 990 pm, 1000 pm, 1010 pm, 1020 pm, 1030 pm, 1040 pm, 1050 pm, 1060 pm, 1070 pm, 1080 pm, 1090 pm, 1100 pm, 1110 pm, 1120 pm, 1130 pm, 1140 pm, 1150 pm, 1160 pm, 1170 pm, 1180 pm, 1190 pm, 1200 pm, 1210 pm, 1220 pm, 1230 pm, 1240 pm, 1250 pm, 1260 pm, 1270 pm, 1280 pm, 1290 pm, 1300 pm, 1310 pm, 1320 pm, 1330 pm, 1340 pm, 1350 pm, 1360 pm, 1370 pm, 1380 pm, 1390 pm, 1400 pm, 1410 pm, 1420 pm, 1430 pm, 1440 pm, 1450 pm, 1460 pm, 1470 pm, 1480 pm, 1490 pm, 1500 pm, 1510 pm, 1520 pm, 1530 pm, 1540 pm, 1550 pm, 1560 pm, 1570 pm, 1580 pm, 1590 pm, 1600 pm, 1610 pm, 1620 pm, 1630 pm, 1640 pm, 1650 pm, 1660 pm, 1670 pm, 1680 pm, 1690 pm, 1700 pm, 1710 pm, 1720 pm, 1730 pm, 1740 pm, 1750 pm, 1760 pm, 1770 pm, 1780 pm, 1790 pm, 1800 pm, 1810 pm, 1820 pm, 1830 pm, 1840 pm, 1850 pm, 1860 pm, 1870 pm, 1880 pm, 1890 pm, 1900 pm, 1910 pm, 1920 pm, 1930 pm, 1940 pm, 1950 pm, 1960pm, 1970 pm, 1980 pm, 1990 pm, 2000 pm, 2010 pm, 2020 pm, 2030 pm, 2040 pm, 2050 pm, 2060 pm, 2070 pm, 2080 pm, 2090 pm, 2100 pm, 2110 pm, 2120 pm, 2130 pm, 2140 pm, 2150 pm, 2160 pm, 2170 pm, 2180 pm, 2190 pm, 2200 pm, 2210 pm, 2220 pm, 2230 pm, 2240 pm, 2250 pm, 2260 pm, 2270 pm, 2280 pm, 2290 pm, 2300 pm, 2310 pm, 2320 pm, 2330 pm, 2340 pm, 2350 pm, 2360 pm, 2370 pm, 2380 pm, 2390 pm, 2400 pm, 2410 pm, 2420 pm, 2430 pm, 2440 pm, 2450 pm, 2460 pm, 2470 pm, 2480 pm, 2490 pm, 2500 pm, 2510 pm, 2520 pm, 2530 pm, 2540 pm, 2550 pm, 2560 pm, 2570 pm, 2580 pm, 2590 pm, 2600 pm, 2610 pm, 2620 pm, 2630 pm, 2640 pm, 2650 pm, 2660 pm, 2670 pm, 2680 pm, 2690 pm, 2700 pm, 2710 pm, 2720 pm, 2730 pm, 2740 pm, 2750 pm, 2760 pm, 2770 pm, 2780 pm, 2790 pm, 2800 pm, 2810 pm, 2820 pm, 2830 pm, 2840 pm, 2850 pm, 2860 pm, 2870 pm, 2880 pm, 2890 pm, 2900 pm, 2910 pm, 2920 pm, 2930 pm, 2940 pm, 2950 pm, 2960 pm, 2970 pm, 2980 pm, 2990 pm, or 3000 pm thick.

[0273] In some embodiments, the encapsulation composition of the present disclosure possesses a water activity (aw) of less than 0.750, 0.700, 0.650, 0.600, 0.550, 0.500, 0.475, 0.450, 0.425, 0.400, 0.375, 0.350, 0.325, 0.300, 0.275, 0.250, 0.225, 0.200, 0.190, 0.180, 0.170, 0.160, 0.150, 0.140, 0.130, 0.120, 0.110, 0.100, 0.095, 0.090, 0.085, 0.080, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050, 0.045, 0.040, 0.035, 0.030, 0.025, 0.020, 0.015, 0.010, or 0.005.

[0274] In some embodiments, the encapsulation composition of the present disclosure possesses a water activity (aw) of less than about 0.750, about 0.700, about 0.650, about 0.600, about 0.550, about 0.500, about 0.475, about 0.450, about 0.425, about 0.400, about 0.375, about 0.350, about 0.325, about 0.300, about 0.275, about 0.250, about 0.225, about 0.200, about 0.190, about 0.180, about 0.170, about 0.160, about 0.150, about 0.140, about 0.130, about 0.120, about 0.110, about 0.100, about 0.095, about 0.090, about 0.085, about 0.080, about 0.075, about 0.070, about 0.065, about 0.060, about 0.055, about 0.050, about 0.045, about 0.040, about 0.035, about 0.030, about 0.025, about 0.020, about 0.015, about 0.010, or about 0.005.

[0275] In one embodiment, the microbe(s) are first dried by spray dry, lyophilization, or foam drying along with excipients that may include one or more sugars, sugar alcohols, disaccharides, trisaccharides, polysaccharides, salts, amino acids, amino acid salts, or polymers.

[0276] In some embodiments, the microbes or compositions comprising the microbes are milled to a size of 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 150 microns, 200 microns, 250 microns,300 microns, 350 microns, 400 microns, 450 microns, 500 microns, 550 microns, 600 microns, 650 microns, 700 microns, 750 microns, 800 microns, 850 microns, 900 microns, 950 microns, or 1,000 microns.

[0277] In some embodiments, the microbes or compositions comprising the microbes are milled to a size of about 10 microbes, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, or about 1,000 microns.

[0278] In some embodiments, the microbes or compositions comprising the microbes are milled to a size of between 10-20 microns, 10-30 microns, 10-40 microns, 10-50 microns, 10- 60 microns, 10-70 microns, 10-80 microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500 microns, 10-750 microns, 10-1,000 microns, 20-30 microns, 20-40 microns, 20-50 microns, 20-60 microns, 20-70 microns, 20-80 microns, 20-90 microns, 20-100 microns, 20- 250 microns, 20-500 microns, 20-750 microns, 20-1,000 microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70 microns, 30-80 microns, 30-90 microns, 30-100 microns, 30- 250 microns, 30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60 microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns, 40-250 microns, 40- 500 microns, 40-750 microns, 40-1,000 microns, 50-60 microns, 50-70 microns, 50-80 microns, 50-90 microns, 50-100 microns, 50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70 microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns, 60-500 microns, 60-750 microns, 60-1,000 microns, 70-80 microns 70-90 microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns, 70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250 microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000 microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns, 90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns, 100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns, 500-750 microns, 500-1,000 microns, or 750-1,000 microns.

[0279] In some embodiments, the microbes or compositions comprising the microbes are milled to a size of between about 10-20 microns, about 10-30 microns, about 10-40 microns, about 10-50 microns, about 10-60 microns, about 10-70 microns, about 10-80 microns, about 10-90 microns, about 10-100 microns, about 10-250 microns, about 10-500 microns, about 10-750 microns, about 10-1,000 microns, about 20-30 microns, about 20-40 microns, about 20-50 microns, about 20-60 microns, about 20-70 microns, about 20-80 microns, about 20-90 microns, about 20-100 microns, about 20-250 microns, about 20-500 microns, about 20-750 microns, about 20-1,000 microns, about 30-40 microns, about 30-50 microns, about 30-60 microns, about 30-70 microns, about 30-80 microns, about 30-90 microns, about 30-100 microns, about 30-250 microns, about 30-500 microns, about 30-750 microns, about 30-1,000 microns, about 40-50 microns, about 40-60 microns, about 40-70 microns, about 40-80 microns, about 40-90 microns, about 40-100 microns, about 40-250 microns, about 40-500 microns, about 40-750 microns, about 40-1,000 microns, about 50-60 microns, about 50-70 microns, about 50-80 microns, about 50-90 microns, about 50-100 microns, about 50-250 microns, about 50-500 microns, about 50-750 microns, about 50-1,000 microns, about 60-70 microns, about 60-80 microns, about 60-90 microns, about 60-100 microns, about 60-250 microns, about 60-500 microns, about 60-750 microns, about 60-1,000 microns, about 70-80 microns about 70-90 microns, about 70-90 microns, about 70-100 microns, about 70-250 microns, about 70-500 microns, about 70-750 microns, about 70-1,000 microns, about 80-90 microns, about 80-100 microns, about 80-250 microns, about 80-500 microns, about 80-500 microns, about 80-750 microns, about 80-1,000 microns, about 90-100 microns, about 90-250 microns, about 90-500 microns, about 90-750 microns, about 90-1,000 microns, about 100-250 microns, about 100-500 microns, about 100-750 microns, about 100-1,000 microns, about 250- 500 microns, about 250-750 microns, about 250-1,000 microns, about 500-750 microns, about 500-1,000 microns, or about 750-1,000 microns.

[0280] In some embodiments, the microbes or compositions comprising the microbes are combined with a wax, fat, oil, fatty acid, or fatty alcohol, and spray congealed into beads of about 10 microbes, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, or about 1,000 microns.

[0281] In some embodiments, the microbes or compositions comprising the microbes are combined with a wax, fat, oil, fatty acid, or fatty alcohol, and spray congealed into beads of between 10-20 microns, 10-30 microns, 10-40 microns, 10-50 microns, 10-60 microns, 10-70 microns, 10-80 microns, 10-90 microns, 10-100 microns, 10-250 microns, 10-500 microns, 10- 750 microns, 10-1,000 microns, 20-30 microns, 20-40 microns, 20-50 microns, 20-60 microns,20-70 microns, 20-80 microns, 20-90 microns, 20-100 microns, 20-250 microns, 20-500 microns, 20-750 microns, 20-1,000 microns, 30-40 microns, 30-50 microns, 30-60 microns, 30-70 microns, 30-80 microns, 30-90 microns, 30-100 microns, 30-250 microns, 30-500 microns, 30-750 microns, 30-1,000 microns, 40-50 microns, 40-60 microns, 40-70 microns, 40-80 microns, 40-90 microns, 40-100 microns, 40-250 microns, 40-500 microns, 40-750 microns, 40-1,000 microns, 50-60 microns, 50-70 microns, 50-80 microns, 50-90 microns, 50- 100 microns, 50-250 microns, 50-500 microns, 50-750 microns, 50-1,000 microns, 60-70 microns, 60-80 microns, 60-90 microns, 60-100 microns, 60-250 microns, 60-500 microns, 60- 750 microns, 60-1,000 microns, 70-80 microns 70-90 microns, 70-90 microns, 70-100 microns, 70-250 microns, 70-500 microns, 70-750 microns, 70-1,000 microns, 80-90 microns, 80-100 microns, 80-250 microns, 80-500 microns, 80-500 microns, 80-750 microns, 80-1,000 microns, 90-100 microns, 90-250 microns, 90-500 microns, 90-750 microns, 90-1,000 microns, 100-250 microns, 100-500 microns, 100-750 microns, 100-1,000 microns, 250-500 microns, 250-750 microns, 250-1,000 microns, 500-750 microns, 500-1,000 microns, or 750- 1,000 microns.

[0282] In some embodiments, the microbes or compositions comprising the microbes are combined with a wax, fat, oil, fatty acid, or fatty alcohol, and spray congealed into beads of between about 10-20 microns, about 10-30 microns, about 10-40 microns, about 10-50 microns, about 10-60 microns, about 10-70 microns, about 10-80 microns, about 10-90 microns, about 10-100 microns, about 10-250 microns, about 10-500 microns, about 10-750 microns, about 10-1,000 microns, about 20-30 microns, about 20-40 microns, about 20-50 microns, about 20-60 microns, about 20-70 microns, about 20-80 microns, about 20-90 microns, about 20-100 microns, about 20-250 microns, about 20-500 microns, about 20-750 microns, about 20-1,000 microns, about 30-40 microns, about 30-50 microns, about 30-60 microns, about 30-70 microns, about 30-80 microns, about 30-90 microns, about 30-100 microns, about 30-250 microns, about 30-500 microns, about 30-750 microns, about 30-1,000 microns, about 40-50 microns, about 40-60 microns, about 40-70 microns, about 40-80 microns, about 40-90 microns, about 40-100 microns, about 40-250 microns, about 40-500 microns, about 40-750 microns, about 40-1,000 microns, about 50-60 microns, about 50-70 microns, about 50-80 microns, about 50-90 microns, about 50-100 microns, about 50-250 microns, about 50-500 microns, about 50-750 microns, about 50-1,000 microns, about 60-70 microns, about 60-80 microns, about 60-90 microns, about 60-100 microns, about 60-250 microns, about 60-500 microns, about 60-750 microns, about 60-1,000 microns, about 70-80 microns about 70-90 microns, about 70-90 microns, about 70-100 microns, about 70-250microns, about 70-500 microns, about 70-750 microns, about 70-1,000 microns, about 80-90 microns, about 80-100 microns, about 80-250 microns, about 80-500 microns, about 80-500 microns, about 80-750 microns, about 80-1,000 microns, about 90-100 microns, about 90-250 microns, about 90-500 microns, about 90-750 microns, about 90-1,000 microns, about 100-250 microns, about 100-500 microns, about 100-750 microns, about 100-1,000 microns, about 250- 500 microns, about 250-750 microns, about 250-1,000 microns, about 500-750 microns, about 500-1,000 microns, or about 750-1,000 microns.

[0283] In some embodiments, the microbes or compositions comprising the microbes are combined with a wax, fat, oil, fatty acid, or fatty alcohol as well as a water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol and spray congealed into beads, the size of which are described herein. In some embodiments, the water-soluble polymer, salt, polysaccharide, sugar, or sugar alcohol serves as a disintegrant. In some embodiments, the disintegrant forms pores once the beads are dispersed in the rumen of the animal.

[0284] In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves within 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes of being administered. In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves within about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes of being administered.

[0285] In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves within 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 hours of being administered. In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves within about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12 hours of being administered.

[0286] In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves at a temperature of at least 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 °C. In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves at a temperature of at least about 10, least about 11, least about 12, least about 13, least about 14, least about 15, least about 16, least about 17, least about 18, least about 19, least about 20, least about 21, least about 22, least about 23, least about 24, least about 25, least about 26, least about 27, least about 28, least about 29, least about 30, least about 31, least about 32, least about 33, least about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, least about 45, least about 46, least about 47, least about 48, least about 49, or least about 50 °C.

[0287] In some embodiments, the composition of the water-soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves at a pH of at least 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some embodiments, the composition of the water- soluble polymer, salt, polysaccharide, sugar, polypeptide, protein, or sugar alcohol is modified such that the disintegrant dissolves at a pH of at least about 3.8, least about 3.9, least about 4.0, least about 4.1, least about 4.2, least about 4.3, least about 4.4, least about 4.5, least about 4.6, least about 4.7, least about 4.8, least about 4.9, least about 5.0, least about 5.1, least about 5.2, least about 5.3, least about 5.4, least about 5.5, least about 5.6, least about 5.7, least about 5.8, least about 5.9, least about 6.0, least about 6.2, least about 6.3, least about 6.4, least about 6.5, least about 6.6, least about 6.7, least about 6.8, least about 6.9, least about 7.0, least about 7.1, least about 7.2, least about 7.3, least about 7.4, least about 7.5, least about 7.6, least about 7.7, least about 7.8, least about 7.9, least about 8.0, least about 8.1, least about 8.2, least about 8.3, least about 8.4, least about 8.5, least about 8.6, least about 8.7, least about 8.8, least about 8.9, least about 9.0, least about 9.1, least about 9.2, least about 9.3, least about 9.4, least about 9.5, least about 9.6, least about 9.7, least about 9.8, least about 9.9, or least about 10.0.

[0288] In some embodiments, the microbes or compositions comprising the microbes are coated with a polymer, a polysaccharide, sugar, sugar alcohol, gel, wax, fat, fatty alcohol, or fatty acid.

[0289] In some embodiments, the microbes or compositions comprising the microbes are coated with a polymer, a polysaccharide, sugar, sugar alcohol, gel, wax, fat, fatty alcohol, or fatty acid.

[0290] In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves within 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes of being administered. In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves within about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes of being administered.

[0291] In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves within 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 hours of being administered. In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves within about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, or about 12 hours of being administered.

[0292] In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves at a temperature of at least 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 °C. In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves at a temperature of at least about 10, least about 11, least about 12, least about 13, least about 14, least about 15, least about 16, least about 17, least about 18, least about 19, least about 20, least about 21, least about 22, least about 23, least about 24, least about 25, least about 26, least about 27, least about 28, least about 29, least about 30, least about 31, least about 32, least about 33, least about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, least about 45, least about 46, least about 47, least about 48, least about 49, or least about 50 °C.

[0293] In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves at a pH of at least 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0. In some embodiments, the coating of the microbes or compositions comprising the microbes is modified such that the coating dissolves at a pH of at least about 3.8, least about 3.9, least about 4.0, least about 4.1, least about 4.2, least about 4.3, least about 4.4, least about 4.5, least about 4.6, least about 4.7,least about 4.8, least about 4.9, least about 5.0, least about 5.1, least about 5.2, least about 5.3, least about 5.4, least about 5.5, least about 5.6, least about 5.7, least about 5.8, least about 5.9, least about 6.0, least about 6.2, least about 6.3, least about 6.4, least about 6.5, least about 6.6, least about 6.7, least about 6.8, least about 6.9, least about 7.0, least about 7.1, least about 7.2, least about 7.3, least about 7.4, least about 7.5, least about 7.6, least about 7.7, least about 7.8, least about 7.9, least about 8.0, least about 8.1, least about 8.2, least about 8.3, least about 8.4, least about 8.5, least about 8.6, least about 8.7, least about 8.8, least about 8.9, least about 9.0, least about 9.1, least about 9.2, least about 9.3, least about 9.4, least about 9.5, least about 9.6, least about 9.7, least about 9.8, least about 9.9, or least about 10.0.Animal Feed

[0294] In some embodiments, compositions of the present disclosure are mixed with animal feed. In some embodiments, animal feed may be present in various forms such as pellets, capsules, granulated, powdered, mash, liquid, semi-liquid, or mixed-rations.

[0295] In some embodiments, compositions of the present disclosure are mixed into the premix at at the feed mill (e.g., Carghill or Western Millin), alone as a standalone premix, and / or alongside other feed additives such as MONENSIN, vitamins, etc. In one embodiment, compositions of the present disclosure are mixed into the feed itself. In some embodiment, the compositions of the present disclosure are mixed into the feed at the feed mill.

[0296] In some embodiments, feed of the present disclosure may be supplemented with water, premix or premixes, forage, beans (e.g., whole, cracked, or ground), grains (e.g., whole, cracked, or ground), bean- or grain-based oils, bean- or grain-based meals, bean- or grain-based haylage or silage, bean- or grain-based syrups, fatty acids, sugar alcohols (e.g., polyhydric alcohols), commercially available formula feeds, oyster shells and those of other bivalves, and mixtures thereof.

[0297] In some embodiments, forage encompasses hay, haylage, and silage. In some embodiments, hays include grass hays (e.g., sudangrass, orchardgrass, or the like), alfalfa hay, and clover hay. In some embodiments, haylages include grass haylages, sorghum haylage, and alfalfa haylage. In some embodiments, silages include maize, oat, wheat, alfalfa, clover, and the like.

[0298] In some embodiments, premix or premixes may be utilized in the feed. Premixes may comprise micro-ingredients such as vitamins, minerals, amino acids; chemical preservatives; pharmaceutical compositions such as antibiotics, ionophores, and other medicaments;fermentation products, and other ingredients. In some embodiments, premixes are blended into the feed.

[0299] In some embodiments, the feed may include feed concentrates such as soybean hulls, soyboy oils, sugar beet pulp, molasses, high protein soybean meal, ground com, shelled corn, cornflakes, wheat midds, distiller grain, cottonseed hulls, rumen-bypass protein, rumen-bypass fat, and grease. See Luhman (U.S. Publication US20150216817A1), Anderson et al. (U.S. Patent 3,484,243) and Porter and Luhman (U.S. Patent 9,179,694B2), Iritani et al. (U.S. Patent 6,090,416), Axelrod et al. (U.S. Publication US20060127530A1), and Katsumi et al. (U.S. Patent 5,741,508) for animal feed and animal feed supplements capable of use in the present compositions and methods.

[0300] In some embodiments, feed occurs as a compound, which includes, in a mixed composition capable of meeting the basic dietary needs, the feed itself, vitamins, minerals, amino acids, and other necessary components. Compound feed may further comprise premixes.

[0301] In some embodiments, microbial compositions of the present disclosure may be mixed with animal feed, premix, and / or compound feed. Individual components of the animal feed may be mixed with the microbial compositions prior to feeding to ruminants. The microbial compositions of the present disclosure may be applied into or on a premix, into or on a feed, and / or into or on a compound feed.Administration of Microbial Compositions

[0302] In some embodiments, microbial compositions of the present disclosure may be mixed with animal feed, premix, and / or compound feed at various stages of animal adaptation to the step-up or finishing diet.

[0303] In some embodiments, microbial compositions of the present disclosure are mixed with feed and microingredients. Microingredients include liquid fat blends, glycerin, rumensin, monensin, vitamins, tylan, optaflex, melengesterol acetate, minerals, and amino acids. In some embodiments, the mixing of feed, microbial compositions of the present disclosure, and microingredients is performed at the feedlot.

[0304] In some embodiments, cattle begin a step up or a starting ration. As used herein, a “step- up diet” or “starting ration” is a diet fed to feedlot cattle as a transition to the high grain content of the grower or finishing diet. In some embodiments, the step-up diet may involve one or more step-up diets that ease the cattle into the transition to the diet with more concentrate. In some embodiments, the step-up diet is formulated to slowly increase the amount of high energy feed in the diet while mitigating gastrointestinal distress and the effects of rapid onset acidosis. Insome embodiments, the cattle are fed a single type of step-up diet. In some embodiments, the cattle are fed multiple varieties of step-up diets, increasing the amount of high energy feed with each iteration of the step up diet variety. In some embodiments, the cattle are fed at least one step up diet, wherein the subsequent diets are different from each of those step up diets that follow. In some embodiments, the cattle are fed at least two different step up diets. In some embodiments, the cattle are fed at least three different step up diets.

[0305] As used herein, a “grower diet” is a high-energy diet (often high-grain) that contains a significant portion of silage, hay, or other forage ingredient. This diet is fed to cattle to support growth prior to shifting to a finishing diet.

[0306] As used herein, a “finishing diet” is a concentrated high-energy diet (often high-grain) fed to cattle on a feedlot to rapidly bring the cattle up to get them to market weight by the time the cattle are rendered. In some embodiments, the finishing diet may result in liver disease, liver abscesses, and / or acidosis.

[0307] In some embodiments, the microbial compositions of the present disclosure are mixed with step-up diets. In some embodiments, the microbial compositions of the present disclosure are mixed with grower and / or finisher diets.

[0308] In some embodiments, the microbial compositions of the present disclosure are administered to ruminants. In some embodiments, the microbial compositions of the present disclosure are administered to cattle such as steers, bulls, cows, heifers, or calves. In some embodiments, the microbial compositions of the present disclosure are administered to cows. In some embodiments, the microbial compositions of the present disclosure are administered to calves.Administration of Microbial Compositions

[0309] In some embodiments, the microbial compositions of the present disclosure are administered to ruminants via the oral route. In some embodiments the microbial compositions are administered via a direct injection route into the gastrointestinal tract. In further embodiments, the direct injection administration delivers the microbial compositions directly to the rumen. In some embodiments, the microbial compositions of the present disclosure are administered to animals anally. In further embodiments, anal administration is in the form of an inserted suppository.

[0310] In some embodiments, the microbial composition is administered in a dose volume comprising a total of, or at least, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1,000 mL.

[0311] In some embodiments, the microbial composition is administered in a dose comprising a total of, or at least, 1018, 1017, 1016, 1015, 1014, 1013, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102microbial cells.

[0312] In some embodiments, the microbial compositions are mixed with feed, and the administration occurs through the ingestion of the microbial compositions along with the feed. In some embodiments, the dose of the microbial composition is administered such that there exists 102to 1012, 103to 1012, 104to 1012, 105to 1012, 106to 1012, 107to 1012, 108to 1012, 109to 1012, 1010to 1012, 10nto 1012, 102to 1011, 103to 1011, 104to 1011, 105to 1011, 106to 1011, 107to 1011, 108to 1011, 109to 1011, 1010to 1011, 102to 1010, 103to 1010, 104to 1010, 105to 1010, 106to 1010, 107to 1010, 108to 1010, 109to 1010, 102to 109, 103to 109, 104to 109, 105to 109, 106to 109, 107to 109, 108to 109, 102to 108, 103to 108, 104to 108, 105to 108, 106to 108, 107to 108, 102to 107, 103to 107, 104to 107, 105to 107, 106to 107, 102to 106, 103to 106, 104to 106, 105to 106,102to 105, 103to 105, 104to 105, 102to 104, 103to 104, 102to 103, 1012, 1011, IO10, 109, 108, 107, 106, 105, 104, 103, or 102total microbial cells per gram or milliliter of the composition.

[0313] In some embodiments, the microbial compositions are mixed with feed, and the administration occurs through the ingestion of the microbial compositions along with the feed. In some embodiments, the dose of the microbial composition is administered such that there exists 102to 1012, 103to 1012, 104to 1012, 105to 1012, 106to 1012, 107to 1012, 108to 1012, 109to 1012, 1010to 1012, 10nto 1012, 102to 1011, 103to 1011, 104to 1011, 105to 1011, 106to 1011, 107to 1011, 108to 1011, 109to 1011, 1010to 1011, 102to IO10, 103to IO10, 104to IO10, 105to IO10, 106to IO10, 107to IO10, 108to IO10, 109to IO10, 102to 109, 103to 109, 104to 109, 105to 109, 106to 109, 107to 109, 108to 109, 102to 108, 103to 108, 104to 108, 105to 108, 106to 108, 107to 108, 102to 107, 103to 107, 104to 107, 105to 107, 106to 107, 102to 106, 103to 106, 104to 106, 105to 106,102to 105, 103to 105, 104to 105, 102to 104, 103to 104, 102to 103, 1012, 1011, IO10, 109, 108, 107, 106, 105, 104, 103, or 102colony forming units per gram or milliliter of the composition.

[0314] In some embodiments, the administered dose of the microbial composition comprises 102to 1018, 103to 1018, 104to 1018, 105to 1018, 106to 1018, 107to 1018, 108to 1018, 109to 1018, 1010to 1018, 10nto 1018, 1012to 1018, 1013to 1018, 1014to 1018, 1015to 1018, 1016to 1018, 1017to 1018, 102to 1012, 103to 1012, 104to 1012, 105to 1012, 106to 1012, 107to 1012, 108to 1012, 109to 1012, 1010to 1012, 10nto 1012, 102to 1011, 103to 1011, 104to 1011, 105to 1011, 106to 1011, 107to 1011, 108to 1011, 109to 1011, 1010to 1011, 102to IO10, 103to IO10, 104to IO10, 105to IO10, 106to IO10, 107to IO10, 108to IO10, 109to IO10, 102to 109, 103to 109, 104to 109, 105to 109, 106to 109, 107to 109, 108to 109, 102to 108, 103to 108, 104to 108, 105to 108, 106to 108, 107to 108, 102to 107, 103to 107, 104to 107, 105to IO7, 106to 107, 102to 106, 103to 106, 104to 106, 105to 106,102to IO5, 103to IO5, 104to IO5, 102to 104, 103to IO4, 102to 103, 1018, 1017, 1016, IO15, 1014, 1013, 1012, IO11, IO10, 109, 108, 107, 106, IO5, 104, 103, or 102total microbial cells.

[0315] In some embodiments, the administered dose of each microbe in the microbial composition is at least about, at least about 103colony forming units (CFU), at least about 104CFU, at least about 105CFU, at least about 106CFU, at least about 107CFU, at least about 108CFU, at least about 109CFU, at least about IO10CFU, at least about 1011CFU, at least about 1012CFU, at least about 1013CFU, at least about 1014CFU, at least about 1015CFU, at least about 1016CFU, at least about 1017CFU, at least about 1018CFU, at least about 1019CFU, or at least about IO20CFU.

[0316] In some embodiments, the composition is administered 1 or more times per day. In some aspects, the composition is administered with food each time the animal is fed. In some embodiments, the composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 times per day.

[0317] In some embodiments, the microbial composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.

[0318] In some embodiments, the microbial composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

[0319] In some embodiments, the microbial composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.

[0320] In some embodiments, the microbial composition is administered to animals throughout the entire time they are on the feedlot. In some embodiments, the microbial composition is administered to animals only during a portion of time while they are on the feedlot. In some embodiments, the microbial composition is administered only during the grower phase. In some embodiments, the microbial composition is administered only during the time when animals are in the receiving pen. In some embodiments, the microbial composition is administered only when the animals are receiving vaccinations and / or treatments. In some embodiments, the microbial composition is administered only when the animals are on a step up diet or when being adapted to a high grain diet. In some embodiments, the microbial composition is administered only when the animals are on a finisher diet or a high grain diet.

[0321] In some embodiments, the microbial composition is administered during the grower phase, when animals are in the receiving pen, when animals are receiving vaccinations and / or treatments, when animals are being adapted to a high grain diet or are on a step up diet, and / or when the animals are on a finisher diet or a high grain diet.

[0322] In some embodiments, an animal entering the feed lot receives at least one microbial composition prior to entering the feed lot. In some embodiments, an animal on the feed lot receives a microbial composition that is different from the first at least one microbial composition. In further embodiments, an animal on the feed lot receives a microbial composition that is different from the first and second at least one microbial composition.

[0323] In some embodiments, the type of diet fed to the animal corresponds with the type of microbial composition administered to the animal. In some embodiments, a grazing or grass / hay-fed animal will receive a first microbial composition. In further embodiments, the same animal fed a different diet will receive a second microbial composition, wherein the first microbial composition is different from the second microbial composition. In some embodiments, the same animal fed yet a different diet will receive a third microbial composition, wherein the first microbial composition is different from the second and third microbial compositions. In some embodiments, the same animal fed yet a different diet will receive a fourth microbial composition, wherein the first microbial composition is different from the second, third, and fourth microbial compositions. In some embodiments, the same animal fed yet a different diet will receive a fifth microbial composition, wherein the first microbial composition is different from the second, third, fourth, and fifth microbial compositions.

[0324] In some embodiments, the feed can be uniformly coated with one or more layers of the microbes and / or microbial compositions disclosed herein, using conventional methods ofmixing, spraying, or a combination thereof through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply coatings. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists, or a combination thereof. Liquid treatments such as those of the present disclosure can be applied via either a spinning “atomizer” disk or a spray nozzle, which evenly distributes the microbial composition onto the feed as it moves though the spray pattern. In some aspects, the feed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying.

[0325] In some embodiments, the feed coats of the present disclosure can be up to 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 110 pm, 120 pm, 130 pm, 140 pm, 150 pm, 160 pm, 170 pm, 180 pm, 190 pm, 200 pm, 210 pm, 220 pm, 230 pm, 240 pm, 250 pm, 260 pm, 270 pm, 280 pm, 290 pm, 300 pm, 310 pm, 320 pm, 330 pm, 340 pm, 350 pm, 360 pm, 370 pm, 380 pm, 390 pm, 400 pm, 410 pm, 420 pm, 430 pm, 440 pm, 450 pm, 460 pm, 470 pm, 480 pm, 490 pm, 500 pm, 510 pm, 520 pm, 530 pm, 540 pm, 550 pm, 560 pm, 570 pm, 580 pm, 590 pm, 600 pm, 610 pm, 620 pm, 630 pm, 640 pm, 650 pm, 660 pm, 670 pm, 680 pm, 690 pm, 700 pm, 710 pm, 720 pm, 730 pm, 740 pm, 750 pm, 760 pm, 770 pm, 780 pm, 790 pm, 800 pm, 810 pm, 820 pm, 830 pm, 840 pm, 850 pm, 860 pm, 870 pm, 880 pm, 890 pm, 900 pm, 910 pm, 920 pm, 930 pm, 940 pm, 950 pm, 960 pm, 970 pm, 980 pm, 990 pm, 1000 pm, 1010 pm, 1020 pm, 1030 pm, 1040 pm, 1050 pm, 1060 pm, 1070 pm, 1080 pm, 1090 pm, 1100 pm, 1110 pm, 1120 pm, 1130 pm, 1140 pm, 1150 pm, 1160 pm, 1170 pm, 1180 pm, 1190 pm, 1200 pm, 1210 pm, 1220 pm, 1230 pm, 1240 pm, 1250 pm, 1260 pm, 1270 pm, 1280 pm, 1290 pm, 1300 pm, 1310 pm, 1320 pm, 1330 pm, 1340 pm, 1350 pm, 1360 pm, 1370 pm, 1380 pm, 1390 pm, 1400 pm, 1410 pm, 1420 pm, 1430 pm, 1440 pm, 1450 pm, 1460 pm, 1470 pm, 1480 pm, 1490 pm, 1500 pm, 1510 pm, 1520 pm, 1530 pm, 1540 pm, 1550 pm, 1560 pm, 1570 pm, 1580 pm, 1590 pm, 1600 pm, 1610 pm, 1620 pm, 1630 pm, 1640 pm, 1650 pm, 1660 pm, 1670 pm, 1680 pm, 1690 pm, 1700 pm, 1710 pm, 1720 pm, 1730 pm, 1740 pm, 1750 pm, 1760 pm, 1770 pm, 1780 pm, 1790 pm, 1800 pm, 1810 pm, 1820 pm, 1830 pm, 1840 pm, 1850 pm, 1860 pm, 1870 pm, 1880 pm, 1890 pm, 1900 pm, 1910 pm, 1920 pm, 1930 pm, 1940 pm, 1950 pm, 1960 pm, 1970 pm, 1980 pm, 1990 pm, 2000 pm, 2010 pm, 2020 pm, 2030 pm, 2040 pm, 2050 pm, 2060 pm, 2070 pm, 2080 pm, 2090 pm, 2100 pm, 2110 pm, 2120 pm, 2130 pm, 2140 pm, 2150 pm, 2160 pm, 2170 pm, 2180 pm, 2190 pm, 2200 pm, 2210 pm, 2220 pm, 2230 pm, 2240 pm, 2250 pm, 2260 pm, 2270 pm, 2280 pm, 2290 pm, 2300 pm, 2310 pm, 2320 pm, 2330 pm, 2340 pm, 2350 pm, 2360 pm, 2370 pm, 2380 pm, 2390 pm, 2400 pm, 2410 pm, 2420pm, 2430 pm, 2440 pm, 2450 pm, 2460 pm, 2470 pm, 2480 pm, 2490 pm, 2500 pm, 2510 pm, 2520 pm, 2530 pm, 2540 pm, 2550 pm, 2560 pm, 2570 pm, 2580 pm, 2590 pm, 2600 pm, 2610 pm, 2620 pm, 2630 pm, 2640 pm, 2650 pm, 2660 pm, 2670 pm, 2680 pm, 2690 pm, 2700 pm, 2710 pm, 2720 pm, 2730 pm, 2740 pm, 2750 pm, 2760 pm, 2770 pm, 2780 pm, 2790 pm, 2800 pm, 2810 pm, 2820 pm, 2830 pm, 2840 pm, 2850 pm, 2860 pm, 2870 pm, 2880 pm, 2890 pm, 2900 pm, 2910 pm, 2920 pm, 2930 pm, 2940 pm, 2950 pm, 2960 pm, 2970 pm, 2980 pm, 2990 pm, or 3000 pm thick.

[0326] In some embodiments, the microbial cells can be coated freely onto any number of compositions or they can be formulated in a liquid or solid composition before being coated onto a composition. For example, a solid composition comprising the microorganisms can be prepared by mixing a solid carrier with a suspension of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.

[0327] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents e.g., activated carbon, minerals, vitamins, and other agents capable of improving the quality of the products or a combination thereof.

[0328] Methods of coating and compositions in use of said methods that are known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present disclosure. Such coating methods and apparatus for their application are disclosed in, for example: U.S. Pat. Nos. 8,097,245, and 7,998,502; and PCT Pub. App. Publication Nos. WO 2008 / 076975, WO 2010 / 138522, WO 2011 / 094469, WO 2010 / 111347, and WO 2010 / 111565 each of which is incorporated by reference herein.

[0329] In some embodiments, the microbial compositions of the present disclosure exhibit a synergistic effect, on one or more of the traits described herein, in the presence of one or more of the microbial compositions coming into contact with one another. The synergistic effect obtained by the taught methods can be quantified, for example, according to Colby’s formula (i.e., (E) = X+Y - (X*Y / 100)). See Colby, R.S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967. Weeds. Vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, “synergistic” is intended to reflect an outcome / parameter / effect that has been increased by more than an additive amount.

[0330] In some embodiments, the microbial compositions of the present disclosure may be administered via bolus. In one embodiment, a bolus (e.g., capsule containing the composition) is inserted into a bolus gun, and the bolus gun is inserted into the buccal cavity and / oresophagus of the animal, followed by the release / inj ection of the bolus into the animal’s digestive tract. In one embodiment, the bolus gun / applicator is a BOVIKALC bolus gun / applicator. In another embodiment, the bolus gun / applicator is a QUADRICAL gun / applicator.

[0331] In some embodiments, the microbial compositions of the present disclosure may be administered via drench. In one embodiment, the drench is an oral drench. A drench administration comprises utilizing a drench kit / applicator / syringe that injects / releases a liquid comprising the microbial compositions into the buccal cavity and / or esophagas of the animal.

[0332] In some embodiments, the microbial compositions of the present disclosure may be administered in a time-released fashion. The composition may be coated in a chemical composition, or may be contained in a mechanical device or capsule that releases the microbes or microbial consortia over a period of time instead all at once. In one embodiment, the microbial compositions are administered to an animal in a time-release capsule. In one embodiment, the composition may be coated in a chemical composition, or may be contained in a mechanical device or capsul that releases the mcirobes or microbial consortia all at once a period of time hours post ingestion.

[0333] In some embodiments, one microbe composition is administered one or more times when the animals are on a step up diet, and a different microbe composition is administered one or more times when the animals are on a finishing diet. In some embodiments, one microbe composition is administered one or more times when the animals are on a step up diet, a different microbe composition is administered one or more times when the animals are on the first thirty days of the finishing diet, and yet a different microbe composition is administered one or more times when the animals have been on the finishing diet for greater than thirty days.

[0334] In some embodiments, one microbe composition is administered one or more times while the animals exhibit signs of acidosis, and different microbe composition is administered one or more times once the signs of acidosis have abated. In some embodiments, a microbe composition is administered to animals that do not exhibit signs of acidosis, and a different microbe composition is administered if the animals exhibit signs of acidosis.

[0335] In some embodiments, the microbial compositions are administered in a time-released fashion between 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, or 1 to 100 hours.

[0336] In some embodiments, the microbial compositions are administered in a time-released fashion between 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, or 1 to 30 days.Microorganisms

[0337] As used herein the term “microorganism” should be taken broadly. It includes, but is not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as eukaryotic fungi, protozoa, and viruses.

[0338] In certain embodiments, the microorganism is unculturable. This should be taken to mean that the microorganism is not known to be culturable or is difficult to culture using methods known to one skilled in the art.

[0339] In one embodiment, the microbes are obtained from animals (e.g., mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air, water (e.g., marine, freshwater, wastewater sludge), sediment, oil, plants (e.g., roots, leaves, stems), agricultural products, and extreme environments (e.g., acid mine drainage or hydrothermal systems). In a further embodiment, microbes obtained from marine or freshwater environments such as an ocean, river, or lake. In a further embodiment, the microbes can be from the surface of the body of water, or any depth of the body of water (e.g., a deep sea sample).

[0340] The microorganisms of the disclosure may be isolated in substantially pure or mixed cultures. They may be concentrated, diluted, or provided in the natural concentrations in which they are found in the source material. For example, microorganisms from saline sediments may be isolated for use in this disclosure by suspending the sediment in fresh water and allowing the sediment to fall to the bottom. The water containing the bulk of the microorganisms may be removed by decantation after a suitable period of settling and either administered to the GI tract of a ruminant, or concentrated by filtering or centrifugation, diluted to an appropriate concentration and administered to the GI tract of a ruminant with the bulk of the salt removed. By way of further example, microorganisms from mineralized or toxic sources may be similarly treated to recover the microbes for application to the ruminant to minimize the potential for damage to the animal.

[0341] In another embodiment, the microorganisms are used in a crude form, in which they are not isolated from the source material in which they naturally reside. For example, the microorganisms are provided in combination with the source material in which they reside; for example, fecal matter, rumen content, rumen fluid, or other composition found in the gastrointestinal tract. In this embodiment, the source material may include one or more species of microorganisms.

[0342] In some embodiments, a mixed population of microorganisms is used in the methods of the disclosure.

[0343] In embodiments of the disclosure where the microorganisms are isolated from a source material (for example, the material in which they naturally reside), any one or a combination of a number of standard techniques which will be readily known to skilled persons may be used. However, by way of example, these in general employ processes by which a solid or liquid culture of a single microorganism can be obtained in a substantially pure form, usually by physical separation on the surface of a solid microbial growth medium or by volumetric dilutive isolation into a liquid microbial growth medium. These processes may include isolation from dry material, liquid suspension, slurries or homogenates in which the material is spread in a thin layer over an appropriate solid gel growth medium, or serial dilutions of the material made into a sterile medium and inoculated into liquid or solid culture media.

[0344] While not essential, in one embodiment, the material containing the microorganisms may be pre-treated prior to the isolation process in order to either multiply all microorganisms in the material, remove certain microorganisms in the material, and / or shift the distribution of microorganisms in the material. Microorganisms can then be isolated from the enriched materials as disclosed above.

[0345] In certain embodiments, as mentioned herein before, the microorganism(s) may be used in crude form and need not be isolated from an animal or a media. For example, feces, or growth media which includes the microorganisms identified to be of benefit to increased milk production, increased feed efficiency, or reduced methane emissions in ruminants may be obtained and used as a crude source of microorganisms for the next round of the method or as a crude source of microorganisms at the conclusion of the method. For example, fresh feces could be obtained and optionally processed.Microbiome Shift and Abundance of Microbes

[0346] In some embodiments, the microbiome of a ruminant, including the rumen microbiome, comprises a diverse arrive of microbes with a wide variety of metabolic capabilities. The microbiome is influenced by a range of factors including diet, variations in animal metabolism, and breed, among others. Most bovine diets are plant-based and rich in complex polysaccharides that enrich the gastrointestinal microbial community for microbes capable of breaking down specific polymeric components in the diet such as cellulose, hemicellulose, lignin, etc. The end products of primary degradation sustain a chain of microbes that ultimately produce a range of organic acids together with hydrogen and carbon dioxide. Because of thecomplex and interlinked nature of the microbiome, changing the diet and thus substrates for primary degradation may have a cascading effect on gut microbial metabolism, with changes in both the organic acid profiles and the methane levels produced, thus impacting the quality and quantity of animal production and or the products produced by the animal. See Menezes et al. (2011. FEMS Microbiol. Ecol. 78(2):256-265.)

[0347] In some aspects, the present disclosure is drawn to administering microbial compositions described herein to modulate or shift the microbiome of a ruminant.

[0348] In some embodiments, the microbiome is shifted through the administration of one or more microbes to one or more sections of the gastrointestinal tract. . In some embodiments, the microbiome is shifted through the administration of one or more microbes to the rumen. In further embodiments, the one or more microbes are those selected from Table 1 and Tables 3-6. In some embodiments, the microbiome shift or modulation includes a decrease or loss of specific microbes that were present prior to the administration of one or more microbes of the present disclosure. In some embodiments, the microbiome shift or modulation includes an increase in microbes that were present prior to the administration of one or more microbes of the present disclosure. In some embodiments, the microbiome shift or modulation includes a gain of one or more microbes that were not present prior to the administration of one or more microbes of the present disclosure. In a further embodiment, the gain of one or more microbes is a microbe that was not specifically included in the administered microbial composition.

[0349] In some embodiments, the administration of microbes of the present disclosure results in a sustained modulation of the microbiome such that the administered microbes are present in the microbiome for a period of at least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 days.

[0350] In some embodiments, the administration of microbes of the present disclosure results in a sustained modulation of the microbiome such that the administered microbes are present in the microbiome for a period of at least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0351] In some embodiments, the administration of microbes of the present disclosure results in a sustained modulation of the microbiome such that the administered microbes are present in the microbiome for a period of at least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8,8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0352] In some embodiments, the presence of the administered microbes are detected by sampling the gastrointestinal tract and using primers to amplify the 16S or 18S rDNA sequences, or the ITS rDNA sequences of the administered microbes. In some embodiments, the administered microbes are one or more of those selected from Table 1 and Tables 3-6. In some embodiments, the administered microbes are any one of SEQ ID NOs: 1-10940.

[0353] In some embodiments, the microbiome of a ruminant is measured by amplifying polynucleotides collected from gastrointestinal samples, wherein the polynucleotides may be 16S or 18S rDNA fragments, or ITS rDNA fragments of microbial rDNA. In one embodiment, the microbiome is fingerprinted by a method of denaturing gradient gel electrophoresis (DGGE) wherein the amplified rDNA fragments are sorted by where they denature, and form a unique banding pattern in a gel that may be used for comparing the microbiome of the same ruminant over time or the microbiomes of multiple ruminants. In another embodiment, the microbiome is fingerprinted by a method of terminal restriction fragment length polymorphism (T-RFLP), wherein labelled PCR fragments are digested using a restriction enzyme and then sorted by size. In a further embodiment, the data collected from the T-RFLP method is evaluated by nonmetric multidimensional scaling (nMDS) ordination and PERMANOVA statistics identify differences in microbiomes, thus allowing for the identification and measurement of shifts in the microbiome. See also Shanks et al. (2011. AppL Environ. Microbiol. 77(9):2992-3001), Petri et al. (2013. PLOS one. 8(12):e83424), and Menezes et al. (2011. FEMS Microbiol. Ecol. 78(2):256-265.)

[0354] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that increases the number and / or type of carbon dioxide fixing microbes. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of carbon dioxide fixing microbes by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of carbon dioxide fixing microbes by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0355] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that decreases the number and / or type of methanogenic microbes. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that reduces the number and / or type of methanogenic microbes by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that decreases the number and / or type of methanogenic microbes by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0356] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that decreases the number and / or type of lactate producing microbes. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that reduces the number and / or type of lactate producing microbes by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that decreases the number and / or type of lactate producing microbes by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0357] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that increases the number and / or type of lactate degrading microbes. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of lactate degrading microbes by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of lactate degrading microbes by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0358] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that increases the number and / or type of volatile fatty acid (VFA) - producing microbes. In some embodiments, the VFAs include acetate, butyrate, propionate, isobutyrate, isovalerate, and valerate. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of VFA-producing microbes by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of VFA-producing microbes by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0359] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that increases the number and / or type of microbes that are utilized as protein sources for the animal. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of microbes that are utilized as protein sources for the animal by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of microbes that are utilized as protein sources for the animal by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0360] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that increases the number and / or type of vitamin synthesizing microbes. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of vitamin synthesizing microbes by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that increases the number and / or type of vitamin synthesizing microbes by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0361] In some embodiments, administration of one or more microbial compositions results in a shift in the microbiome that reduces the overall alpha diversity of the microbial community. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that reduces the overall alpha diversity of the microbial community by at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, or at least 700%. In some embodiments, administration of one or more microbial composition results in a shift in the microbiome that reduces the overall alpha diversity of the microbial community by at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or at least about 700%.

[0362] In some embodiments, the administration of microbes of the present disclosure results in a modulation or shift of the microbiome which further results in a desired phenotype or improved trait.Cattle Microbial Compositional Diversity

[0363] Bovine in a commercial setting have been found to exhibit a high degree of animal-to- animal variability in terms of the microbial diversity of the rumen. The increased variability of the microbial compositions of the rumen may lead to a lower ability to reach a stable microbial composition. Lower variability in turn results in a considerable difference in health, weight, and other attributes that affect commercial viability of the animal. See Shabat SKB et al. (ISME J 10:2958-2972.)

[0364] In some embodiments, the administration of one or more microbes and / or bioensembles of the present disclosure during feed transition in beef cattle decreases the variability of the rumen microbiome in cattle and further establishes a stable rumen microbiome.

[0365] In some embodiments, the variability of the rumen microbiome is measured as the total number of species present in the rumen at one or more locations.

[0366] In some embodiments, the administration of one or more microbes and / or bioensembles of the present disclosure reduces the amount of time required for the rumen microbiome to reach a stabilized state.

[0367] In some embodiments, the administration of one or more microbes and / or bioensembles of the present disclosure results in beef cattle of the present disclosure reaching a stabilized state of the rumen microbiome; a reduction in the variability of the rumen microbiome.

[0368] In some embodiments, the stabilized state of the rumen microbiome is reached when the rumen microbiome of beef cattle contains about 10, about 20, about, 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, or about 10,000 different species.

[0369] In some embodiments, the stabilized state of the rumen microbiome is reached when the rumen microbiome of beef cattle contains between about 10 to about 50, about 10 to about 100, about 50 to about 100, about 50 to about 200, about 100 to about 150, about 100 to about 200, about 100 to about 400, about 200 to about 500, about 200 to about 700, about 400 to about 800, about 500 to about 1,000, about 500 to about 2,000, about 1,000 to about 2,000, about 1,000 to about 5,000, about 5,000 to about 7,000, about 5,000 to about 10,000, or about 8,000 to about 10,000 different species.

[0370] In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the beef cattle in a feed transition reach a stabilized state after administration of one or more microbes and / or bioensembles of the present disclosure.MIC Scoring

[0371] According to the methods provided herein, a sample is processed to detect the presence of one or more microorganism types in the sample (Fig. 1, 1001; Fig. 2, 2001). The absolute number of one or more microorganism organism type in the sample is determined (Fig. 1, 1002; Fig. 2, 2002). The determination of the presence of the one or more organism types and the absolute number of at least one organism type can be conducted in parallel or serially. Forexample, in the case of a sample comprising a microbial community comprising bacteria (z.e., one microorganism type) and fungi (z.e., a second microorganism type), the user in one embodiment detects the presence of one or both of the organism types in the sample (Fig. 1, 1001; Fig. 2, 2001). The user, in a further embodiment, determines the absolute number of at least one organism type in the sample - in the case of this example, the number of bacteria, fungi or combination thereof, in the sample (Fig. 1, 1002; Fig. 2, 2002).

[0372] In one embodiment, the sample, or a portion thereof is subjected to flow cytometry (FC) analysis to detect the presence and / or number of one or more microorganism types (Fig. 1, 1001, 1002; Fig. 2, 2001, 2002). In one flow cytometer embodiment, individual microbial cells pass through an illumination zone, at a rate of at least about 300 *s-1, or at least about 500 *s' or at least about 1000 *s-1. However, one of ordinary skill in the art will recognize that this rate can vary depending on the type of instrument is employed. Detectors which are gated electronically measure the magnitude of a pulse representing the extent of light scattered. The magnitudes of these pulses are sorted electronically into “bins” or “channels,” permitting the display of histograms of the number of cells possessing a certain quantitative property (e.g., cell staining property, diameter, cell membrane) versus the channel number. Such analysis allows for the determination of the number of cells in each “bin” which in embodiments described herein is an “microorganism type” bin, e.g., a bacteria, fungi, nematode, protozoan, archaea, algae, dinoflagellate, virus, viroid, etc.

[0373] In one embodiment, a sample is stained with one or more fluorescent dyes wherein a fluorescent dye is specific to a particular microorganism type, to enable detection via a flow cytometer or some other detection and quantification method that harnesses fluorescence, such as fluorescence microscopy. The method can provide quantification of the number of cells and / or cell volume of a given organism type in a sample. In a further embodiment, as described herein, flow cytometry is harnessed to determine the presence and quantity of a unique first marker and / or unique second marker of the organism type, such as enzyme expression, cell surface protein expression, etc. Two- or three-variable histograms or contour plots of, for example, light scattering versus fluorescence from a cell membrane stain (versus fluorescence from a protein stain or DNA stain) may also be generated, and thus an impression may be gained of the distribution of a variety of properties of interest among the cells in the population as a whole. A number of displays of such multiparameter flow cytometric data are in common use and are amenable for use with the methods described herein.

[0374] In one embodiment of processing the sample to detect the presence and number of one or more microorganism types, a microscopy assay is employed (Fig. 1, 1001, 1002). In oneembodiment, the microscopy is optical microscopy, where visible light and a system of lenses are used to magnify images of small samples. Digital images can be captured by a chargecouple device (CCD) camera. Other microscopic techniques include, but are not limited to, scanning electron microscopy and transmission electron microscopy. Microorganism types are visualized and quantified according to the aspects provided herein.

[0375] In another embodiment of the disclosure, in order to detect the presence and number of one or more microorganism types, each sample, or a portion thereof is subjected to fluorescence microscopy. Different fluorescent dyes can be used to directly stain cells in samples and to quantify total cell counts using an epifluorescence microscope as well as flow cytometry, described above. Useful dyes to quantify microorganisms include but are not limited to acridine orange (AO), 4,6-di-amino-2 phenylindole (DAPI) and 5-cyano-2,3 Dytolyl Tetrazolium Chloride (CTC). Viable cells can be estimated by a viability staining method such as the LIVE / DEAD® Bacterial Viability Kit (Bac-Light™) which contains two nucleic acid stains: the green-fluorescent SYTO 9™ dye penetrates all membranes and the red-fluorescent propidium iodide (PI) dye penetrates cells with damaged membranes. Therefore, cells with compromised membranes will stain red, whereas cells with undamaged membranes will stain green. Fluorescent in situ hybridization (FISH) extends epifluorescence microscopy, allowing for the fast detection and enumeration of specific organisms. FISH uses fluorescent labelled oligonucleotides probes (usually 15-25 basepairs) which bind specifically to organism DNA in the sample, allowing the visualization of the cells using an epifluorescence or confocal laser scanning microscope (CLSM). Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) improves upon the FISH method by using oligonucleotide probes labelled with a horse radish peroxidase (HRP) to amplify the intensity of the signal obtained from the microorganisms being studied. FISH can be combined with other techniques to characterize microorganism communities. One combined technique is high affinity peptide nucleic acid (PNA)-FISH, where the probe has an enhanced capability to penetrate through the Extracellular Polymeric Substance (EPS) matrix. Another example is LIVE / DEAD-FISH which combines the cell viability kit with FISH and has been used to assess the efficiency of disinfection in drinking water distribution systems.

[0376] In another embodiment, each sample, or a portion thereof is subjected to Raman microspectroscopy in order to determine the presence of a microorganism type and the absolute number of at least one microorganism type (Fig. 1, 1001-1002; Fig. 2, 2001-2002). Raman micro-spectroscopy is a non-destructive and label-free technology capable of detecting and measuring a single cell Raman spectrum (SCRS). A typical SCRS provides an intrinsicbiochemical “fingerprint” of a single cell. A SCRS contains rich information of the biomolecules within it, including nucleic acids, proteins, carbohydrates and lipids, which enables characterization of different cell species, physiological changes and cell phenotypes. Raman microscopy examines the scattering of laser light by the chemical bonds of different cell biomarkers. A SCRS is a sum of the spectra of all the biomolecules in one single cell, indicating a cell’s phenotypic profile. Cellular phenotypes, as a consequence of gene expression, usually reflect genotypes. Thus, under identical growth conditions, different microorganism types give distinct SCRS corresponding to differences in their genotypes and can thus be identified by their Raman spectra.

[0377] In yet another embodiment, the sample, or a portion thereof is subjected to centrifugation in order to determine the presence of a microorganism type and the number of at least one microorganism type (Fig. 1, 1001-1002; Fig. 2, 2001-2002). This process sediments a heterogeneous mixture by using the centrifugal force created by a centrifuge. More dense components of the mixture migrate away from the axis of the centrifuge, while less dense components of the mixture migrate towards the axis. Centrifugation can allow fractionation of samples into cytoplasmic, membrane and extracellular portions. It can also be used to determine localization information for biological molecules of interest. Additionally, centrifugation can be used to fractionate total microbial community DNA. Different prokaryotic groups differ in their guanine-plus-cytosine (G+C) content of DNA, so densitygradient centrifugation based on G+C content is a method to differentiate organism types and the number of cells associated with each type. The technique generates a fractionated profile of the entire community DNA and indicates abundance of DNA as a function of G+C content. The total community DNA is physically separated into highly purified fractions, each representing a different G+C content that can be analyzed by additional molecular techniques such as denaturing gradient gel electrophoresis (DGGE) / amplified ribosomal DNA restriction analysis (ARDRA) (see discussion herein) to assess total microbial community diversity and the presence / quantity of one or more microorganism types.

[0378] In another embodiment, the sample, or a portion thereof is subjected to staining in order to determine the presence of a microorganism type and the number of at least one microorganism type (Fig. 1, 1001-1002; Fig. 2, 2001-2002). Stains and dyes can be used to visualize biological tissues, cells or organelles within cells. Staining can be used in conjunction with microscopy, flow cytometry or gel electrophoresis to visualize or mark cells or biological molecules that are unique to different microorganism types. In vivo staining is the process of dyeing living tissues, whereas in vitro staining involves dyeing cells or structures that havebeen removed from their biological context. Examples of specific staining techniques for use with the methods described herein include, but are not limited to: gram staining to determine gram status of bacteria, endospore staining to identify the presence of endospores, Ziehl- Neelsen staining, haematoxylin and eosin staining to examine thin sections of tissue, papanicolaou staining to examine cell samples from various bodily secretions, periodic acid- Schiff staining of carbohydrates, Masson’s trichome employing a three-color staining protocol to distinguish cells from the surrounding connective tissue, Romanowsky stains (or common variants that include Wright's stain, Jenner's stain, May-Grunwald stain, Leishman stain and Giemsa stain) to examine blood or bone marrow samples, silver staining to reveal proteins and DNA, Sudan staining for lipids and Conklin’s staining to detect true endospores. Common biological stains include acridine orange for cell cycle determination; bismarck brown for acid mucins; carmine for glycogen; carmine alum for nuclei; Coomassie blue for proteins; Cresyl violet for the acidic components of the neuronal cytoplasm; Crystal violet for cell walls; DAPI for nuclei; eosin for cytoplasmic material, cell membranes, some extracellular structures and red blood cells; ethidium bromide for DNA; acid fuchsine for collagen, smooth muscle or mitochondria; haematoxylin for nuclei; Hoechst stains for DNA; iodine for starch; malachite green for bacteria in the Gimenez staining technique and for spores; methyl green for chromatin; methylene blue for animal cells; neutral red for Nissl substance; Nile blue for nuclei; Nile red for lipohilic entities; osmium tetroxide for lipids; rhodamine is used in fluorescence microscopy; safranin for nuclei. Stains are also used in transmission electron microscopy to enhance contrast and include phosphotungstic acid, osmium tetroxide, ruthenium tetroxide, ammonium molybdate, cadmium iodide, carbohydrazide, ferric chloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate, lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate, sodium chloroaurate, thallium nitrate, thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate.

[0379] In another embodiment, the sample, or a portion thereof is subjected to mass spectrometry (MS) in order to determine the presence of a microorganism type and the number of at least one microorganism type (Fig. 1, 1001-1002; Fig. 2, 2001-2002). MS, as discussed below, can also be used to detect the presence and expression of one or more unique markers in a sample (Fig. 1, 1003-1004; Fig. 2, 2003-2004). MS is used for example, to detect the presence and quantity of protein and / or peptide markers unique to microorganism types and therefore to provide an assessment of the number of the respective microorganism type in the sample. Quantification can be either with stable isotope labelling or label-free. De novosequencing of peptides can also occur directly from MS / MS spectra or sequence tagging (produce a short tag that can be matched against a database). MS can also reveal post- translational modifications of proteins and identify metabolites. MS can be used in conjunction with chromatographic and other separation techniques (such as gas chromatography, liquid chromatography, capillary electrophoresis, ion mobility) to enhance mass resolution and determination.

[0380] In another embodiment, the sample, or a portion thereof is subjected to lipid analysis in order to determine the presence of a microorganism type and the number of at least one microorganism type (Fig. 1, 1001-1002; Fig. 2, 2001-2002). Fatty acids are present in a relatively constant proportion of the cell biomass, and signature fatty acids exist in microbial cells that can differentiate microorganism types within a community. In one embodiment, fatty acids are extracted by saponification followed by derivatization to give the respective fatty acid methyl esters (FAMEs), which are then analyzed by gas chromatography. The FAME profile in one embodiment is then compared to a reference FAME database to identify the fatty acids and their corresponding microbial signatures by multivariate statistical analyses.

[0381] In the aspects of the methods provided herein, the number of unique first makers in the sample, or portion thereof (e.g., sample aliquot) is measured, as well as the abundance of each of the unique first markers (Fig. 1, 1003; Fig. 2, 2003). A unique marker is a marker of a microorganism strain. It should be understood by one of ordinary skill in the art that depending on the unique marker being probed for and measured, the entire sample need not be analyzed. For example, if the unique marker is unique to bacterial strains, then the fungal portion of the sample need not be analyzed. As described above, in some embodiments, measuring the absolute abundance of one or more organism types in a sample comprises separating the sample by organism type, e.g., via flow cytometry.

[0382] Any marker that is unique to an organism strain can be employed herein. For example, markers can include, but are not limited to, small subunit ribosomal RNA genes (16S / 18S rDNA), large subunit ribosomal RNA genes (23S / 25S / 28S rDNA), intercalary 5.8S gene, cytochrome c oxidase, beta-tubulin, elongation factor, RNA polymerase and internal transcribed spacer (ITS).

[0383] Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNA genes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16S rRNA (16S rDNA) in the case of prokaryotes, have been the predominant target for the assessment of organism types and strains in a microbial community. However, the large subunit ribosomal RNA genes, 28S rDNAs, have been also targeted. rDNAs are suitable for taxonomic identification because: (i)they are ubiquitous in all known organisms; (ii) they possess both conserved and variable regions; (iii) there is an exponentially expanding database of their sequences available for comparison. In community analysis of samples, the conserved regions serve as annealing sites for the corresponding universal PCR and / or sequencing primers, whereas the variable regions can be used for phylogenetic differentiation. In addition, the high copy number of rDNA in the cells facilitates detection from environmental samples.

[0384] The internal transcribed spacer (ITS), located between the 18S rDNA and 28S rDNA, has also been targeted. The ITS is transcribed but spliced away before assembly of the ribosomes The ITS region is composed of two highly variable spacers, ITS1 and ITS2, and the intercalary 5.8S gene. This rDNA operon occurs in multiple copies in genomes. Because the ITS region does not code for ribosome components, it is highly variable.

[0385] In one embodiment, the unique RNA marker can be an mRNA marker, an siRNA marker or a ribosomal RNA marker.

[0386] Protein-coding functional genes can also be used herein as a unique first marker. Such markers include but are not limited to: the recombinase A gene family (bacterial RecA, archaea RadA and RadB, eukaryotic Rad51 and Rad57, phage UvsX); RNA polymerase P subunit (RpoB) gene, which is responsible for transcription initiation and elongation; chaperonins. Candidate marker genes have also been identified for bacteria plus archaea: ribosomal protein S2 (rpsB), ribosomal protein S10 (rpsJ), ribosomal protein LI rplA), translation elongation factor EF-2, translation initiation factor IF-2, metalloendopeptidase, ribosomal protein L22, ffh signal recognition particle protein, ribosomal protein L4 / Lle (rplD), ribosomal protein L2 (rplB), ribosomal protein S9 (rpsl), ribosomal protein L3 (rplC), phenylalanyl-tRNA synthetase beta subunit, ribosomal protein L14b / L23e (rplN), ribosomal protein S5, ribosomal protein S19 (rpsS), ribosomal protein S7, ribosomal protein L16 / L10E (rplP), ribosomal protein S13 (rpsM), phenylalanyl-tRNA synthetase a subunit, ribosomal protein L15, ribosomal protein L25 / L23, ribosomal protein L6 (rplF), ribosomal protein Li l (rplK), ribosomal protein L5 (rplE), ribosomal protein S12 / S23, ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal protein SI 1 (rpsK), ribosomal protein LIO, ribosomal protein S8, tRNA pseudouridine synthase B, ribosomal protein L18P / L5E, ribosomal protein S15P / S13e, Porphobilinogen deaminase, ribosomal protein SI 7, ribosomal protein LI 3 (rplM), phosphoribosylformylglycinamidine cyclo-ligase (rpsE), ribonuclease HII and ribosomal protein L24. Other candidate marker genes for bacteria include: transcription elongation protein NusA (nusA), rpoB DNA-directed RNA polymerase subunit beta (rpoB), GTP -binding protein EngA, rpoC DNA-directed RNA polymerase subunit beta', priA primosome assemblyprotein, transcription-repair coupling factor, CTP synthase (pyrG), secY preprotein translocase subunit SecY, GTP -binding protein Obg / CgtA, DNA polymerase I, rpsF 30S ribosomal protein S6, poA DNA-directed RNA polymerase subunit alpha, peptide chain release factor 1, rpll 50S ribosomal protein L9, polyribonucleotide nucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 50S ribosomal protein L17, tRNA (guanine-N(l)-)-m ethyltransferase (rplS), rplY probable 50S ribosomal protein L25, DNA repair protein RadA, glucose-inhibited division protein A, ribosome-binding factor A, DNA mismatch repair protein MutL, smpB SsrA- binding protein (smpB), N-acetylglucosaminyl transferase, S-adenosyl-methyltransferase MraW, UDP-N-acetylmuramoylalanine— D-glutamate ligase, rplS 50S ribosomal protein L19, rplT 50S ribosomal protein L20 (rplT), ruvA Holliday junction DNA helicase, ruvB Holliday junction DNA helicase B, serS seryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30S ribosomal protein SI 8, DNA mismatch repair protein MutS, rpsT 30S ribosomal protein S20, DNA repair protein RecN, frr ribosome recycling factor (firr), recombination protein RecR, protein of unknown function UPF0054, miaA tRNA isopentenyltransf erase, GTP- binding protein YchF, chromosomal replication initiator protein DnaA, dephospho-CoA kinase, 16S rRNA processing protein RimM, ATP-cone domain protein, 1 -deoxy -D-xylulose 5-phosphate reductoisom erase, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, fatty acid / phospholipid synthesis protein PlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvC Holliday junction resolvase, rpsP 30S ribosomal protein S16, Recombinase A recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetase beta subunit, trmU tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpml 50S ribosomal protein L35, hemE uroporphyrinogen decarboxylase, Rod shape-determining protein, rpmA 50S ribosomal protein L27 (rpmA), peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC), UDP- N-acetylmuramyl-tripeptide synthetase, rpmF 50S ribosomal protein L32, rpIL 50S ribosomal protein L7 / L12 (rpIL), leuS leucyl-tRNA synthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA, GTP -binding protein TypA, ATP-dependent Clp protease, ATP -binding subunit ClpX, DNA replication and repair protein RecF and UDP-N- acetylenolpyruvoylglucosamine reductase.

[0387] Phospholipid fatty acids (PLFAs) may also be used as unique first markers according to the methods described herein. Because PLFAs are rapidly synthesized during microbial growth, are not found in storage molecules and degrade rapidly during cell death, it provides an accurate census of the current living community. All cells contain fatty acids (FAs) that can be extracted and esterified to form fatty acid methyl esters (FAMEs). When the FAMEs are analyzed using gas chromatography-mass spectrometry, the resulting profile constitutes a‘fingerprint’ of the microorganisms in the sample. The chemical compositions of membranes for organisms in the domains Bacteria and Eukarya are comprised of fatty acids linked to the glycerol by an ester-type bond (phospholipid fatty acids (PLFAs)). In contrast, the membrane lipids of Archaea are composed of long and branched hydrocarbons that are joined to glycerol by an ether-type bond (phospholipid ether lipids (PLELs)). This is one of the most widely used non-genetic criteria to distinguish the three domains. In this context, the phospholipids derived from microbial cell membranes, characterized by different acyl chains, are excellent signature molecules, because such lipid structural diversity can be linked to specific microbial taxa.

[0388] As provided herein, in order to determine whether an organism strain is active, the level of expression of one or more unique second markers, which can be the same or different as the first marker, is measured (Fig. 1, 1004; Fig. 2, 2004). Unique first unique markers are described above. The unique second marker is a marker of microorganism activity. For example, in one embodiment, the mRNA or protein expression of any of the first markers described above is considered a unique second marker for the purposes of this invention.

[0389] In one embodiment, if the level of expression of the second marker is above a threshold level (e.g., a control level) or at a threshold level, the microorganism is considered to be active (Fig. 1, 1005; Fig. 2, 2005). Activity is determined in one embodiment, if the level of expression of the second marker is altered by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30%, as compared to a threshold level, which in some embodiments, is a control level.

[0390] Second unique markers are measured, in one embodiment, at the protein, RNA or metabolite level. A unique second marker is the same or different as the first unique marker.

[0391] As provided above, a number of unique first markers and unique second markers can be detected according to the methods described herein. Moreover, the detection and quantification of a unique first marker is carried out according to methods known to those of ordinary skill in the art (Fig. 1, 1003-1004, Fig. 2, 2003-2004).

[0392] Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodiment is used to determine absolute cell count of a unique first marker and / or unique second marker. Sequencing platforms include, but are not limited to, Sanger sequencing and high-throughput sequencing methods available from Roche / 454 Life Sciences, Illumina / Solexa, Pacific Biosciences, Ion Torrent and Nanopore. The sequencing can be amplicon sequencing of particular DNA or RNA sequences or whole metagenome / transcriptome shotgun sequencing.

[0393] Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing with chainterminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp. 5463-5467, incorporated byreference herein in its entirety) relies on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication and is amenable for use with the methods described herein.

[0394] In another embodiment, the sample, or a portion thereof is subjected to extraction of nucleic acids, amplification of DNA of interest (such as the rRNA gene) with suitable primers and the construction of clone libraries using sequencing vectors. Selected clones are then sequenced by Sanger sequencing and the nucleotide sequence of the DNA of interest is retrieved, allowing calculation of the number of unique microorganism strains in a sample.

[0395] 454 pyrosequencing from Roche / 454 Life Sciences yields long reads and can be harnessed in the methods described herein (Margulies et al. (2005) Nature, 437, pp. 376-380; U.S. Patents Nos. 6,274,320; 6,258,568; 6,210,891, each of which is herein incorporated in its entirety for all purposes). Nucleic acid to be sequenced (e.g., amplicons or nebulized genomic / metagenomic DNA) have specific adapters affixed on either end by PCR or by ligation. The DNA with adapters is fixed to tiny beads (ideally, one bead will have one DNA fragment) that are suspended in a water-in-oil emulsion. An emulsion PCR step is then performed to make multiple copies of each DNA fragment, resulting in a set of beads in which each bead contains many cloned copies of the same DNA fragment. Each bead is then placed into a well of a fiber-optic chip that also contains enzymes necessary for the sequencing-by- synthesis reactions. The addition of bases (such as A, C, G, or T) trigger pyrophosphate release, which produces flashes of light that are recorded to infer the sequence of the DNA fragments in each well. About 1 million reads per run with reads up to 1,000 bases in length can be achieved. Paired-end sequencing can be done, which produces pairs of reads, each of which begins at one end of a given DNA fragment. A molecular barcode can be created and placed between the adapter sequence and the sequence of interest in multiplex reactions, allowing each sequence to be assigned to a sample bioinformatically.

[0396] Illumina / Solexa sequencing produces average read lengths of about 25 basepairs (bp) to about 300 bp (Bennett et al. (2005) Pharmacogenomics, 6:373-382; Lange et al. (2014). BMC Genomics 15, p. 63; Fadrosh et al. (2014) Microbiome 2, p. 6; Caporaso et a\. (2012) ISME J, 6, p. 1621-1624; Bentley et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature, 456:53-59). This sequencing technology is also sequencing-by-synthesis but employs reversible dye terminators and a flow cell with a field of oligos attached. DNA fragments to be sequenced have specific adapters on either end and are washed over a flow cell filled with specific oligonucleotides that hybridize to the ends of the fragments. Each fragment is then replicated to make a cluster of identical fragments.Reversible dye-terminator nucleotides are then washed over the flow cell and given time to attach. The excess nucleotides are washed away, the flow cell is imaged, and the reversible terminators can be removed so that the process can repeat and nucleotides can continue to be added in subsequent cycles. Paired-end reads that are 300 bases in length each can be achieved. An Illumina platform can produce 4 billion fragments in a paired-end fashion with 125 bases for each read in a single run. Barcodes can also be used for sample multiplexing, but indexing primers are used.

[0397] The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) process is a “sequencing-by-ligation” approach, and can be used with the methods described herein for detecting the presence and abundance of a first marker and / or a second marker (Fig. 1, 1003-1004; Fig. 2, 2003-2004) (Peckham et al. SOLiD™ Sequencing and 2-Base Encoding. San Diego, CA: American Society of Human Genetics, 2007; Mitra et al. (2013) Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing. BMC Genomics, 14(Suppl 5): S16; Mardis (2008) Nextgeneration DNA sequencing methods. Annu Rev Genomics Hum Genet, 9:387-402; each incorporated by reference herein in its entirety). A library of DNA fragments is prepared from the sample to be sequenced, and are used to prepare clonal bead populations, where only one species of fragment will be present on the surface of each magnetic bead. The fragments attached to the magnetic beads will have a universal Pl adapter sequence so that the starting sequence of every fragment is both known and identical. Primers hybridize to the Pl adapter sequence within the library template. A set of four fluorescently labelled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length. The SOLiD platform can produce up to 3 billion reads per run with reads that are 75 bases long. Paired-end sequencing is available and can be used herein, but with the second read in the pair being only 35 bases long. Multiplexing of samples is possible through a system akin to the one used by Illumina, with a separate indexing run.

[0398] The Ion Torrent system, like 454 sequencing, is amenable for use with the methods described herein for detecting the presence and abundance of a first marker and / or a second marker (Fig. 1, 1003-1004; Fig. 2, 2003-2004). It uses a plate of microwells containing beads to which DNA fragments are attached. It differs from all of the other systems, however, in the manner in which base incorporation is detected. When a base is added to a growing DNA strand, a proton is released, which slightly alters the surrounding pH. Microdetectors sensitiveto pH are associated with the wells on the plate, and they record when these changes occur. The different bases (A, C, G, T) are washed sequentially through the wells, allowing the sequence from each well to be inferred. The Ion Proton platform can produce up to 50 million reads per run that have read lengths of 200 bases. The Personal Genome Machine platform has longer reads at 400 bases. Bidirectional sequencing is available. Multiplexing is possible through the standard in-line molecular barcode sequencing.

[0399] Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule, real-time sequencing approach and in one embodiment, is used with the methods described herein for detecting the presence and abundance of a first marker and / or a second marker (Fig. 1, 1003- 1004; Fig. 2, 2003-2004). The PacBio sequencing system involves no amplification step, setting it apart from the other major next-generation sequencing systems. In one embodiment, the sequencing is performed on a chip containing many zero-mode waveguide (ZMW) detectors. DNA polymerases are attached to the ZMW detectors and phospholinked dye- labeled nucleotide incorporation is imaged in real time as DNA strands are synthesized. The PacBio system yields very long read lengths (averaging around 4,600 bases) and a very high number of reads per run (about 47,000). The typical “paired-end” approach is not used with PacBio, since reads are typically long enough that fragments, through CCS, can be covered multiple times without having to sequence from each end independently. Multiplexing with PacBio does not involve an independent read, but rather follows the standard “in-line” barcoding model.

[0400] In one embodiment, where the first unique marker is the ITS genomic region, automated ribosomal intergenic spacer analysis (ARISA) is used in one embodiment to determine the number and identity of microorganism strains in a sample (Fig. 1, 1003, Fig. 2, 2003) (Ranjard et al. (2003). Environmental Microbiology 5, pp. 1111-1120, incorporated by reference in its entirety for all purposes). The ITS region has significant heterogeneity in both length and nucleotide sequence. The use of a fluorescence-labeled forward primer and an automatic DNA sequencer permits high resolution of separation and high throughput. The inclusion of an internal standard in each sample provides accuracy in sizing general fragments.

[0401] In another embodiment, fragment length polymorphism (RFLP) of PCR-amplified rDNA fragments, otherwise known as amplified ribosomal DNA restriction analysis (ARDRA), is used to characterize unique first markers and the abundance of the same in samples (Fig. 1, 1003, Fig. 2, 2003) (for additional detail, see Massol-Deya et al. (1995). Mol. Microb. Ecol. Manual. 3.3.2, pp. 1-18, the entirety of which is herein incorporated by reference). rDNA fragments are generated by PCR using general primers, digested withrestriction enzymes, electrophoresed in agarose or acrylamide gels, and stained with ethidium bromide or silver nitrate.

[0402] One fingerprinting technique used in detecting the presence and abundance of a unique first marker is single-stranded-conformation polymorphism (SSCP) (see Lee et al. (1996). Appl Environ Microbiol 62, pp. 3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp. 103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp. 4870-4876, each of which is incorporated by reference herein in its entirety). In this technique, DNA fragments such as PCR products obtained with primers specific for the 16S rRNA gene, are denatured and directly electrophoresed on a non-denaturing gel. Separation is based on differences in size and in the folded conformation of single-stranded DNA, which influences the electrophoretic mobility. Reannealing of DNA strands during electrophoresis can be prevented by a number of strategies, including the use of one phosphorylated primer in the PCR followed by specific digestion of the phosphorylated strands with lambda exonuclease and the use of one biotinylated primer to perform magnetic separation of one single strand after denaturation. To assess the identity of the predominant populations in a given microbial composition, in one embodiment, bands are excised and sequenced, or SSCP-patterns can be hybridized with specific probes. Electrophoretic conditions, such as gel matrix, temperature, and addition of glycerol to the gel, can influence the separation.

[0403] In addition to sequencing based methods, other methods for quantifying expression (e.g., gene, protein expression) of a second marker are amenable for use with the methods provided herein for determining the level of expression of one or more second markers (Fig. 1, 1004; Fig. 2, 2004). For example, quantitative RT-PCR, microarray analysis, linear amplification techniques such as nucleic acid sequence based amplification (NASBA) are all amenable for use with the methods described herein, and can be carried out according to methods known to those of ordinary skill in the art.

[0404] In another embodiment, the sample, or a portion thereof is subjected to a quantitative polymerase chain reaction (PCR) for detecting the presence and abundance of a first marker and / or a second marker (Fig. 1, 1003-1004; Fig. 2, 2003-2004). Specific microorganism strains activity is measured by reverse transcription of transcribed ribosomal and / or messenger RNA (rRNA and mRNA) into complementary DNA (cDNA), followed by PCR (RT-PCR).

[0405] In another embodiment, the sample, or a portion thereof is subjected to PCR-based fingerprinting techniques to detect the presence and abundance of a first marker and / or a second marker (Fig. 1, 1003-1004; Fig. 2, 2003-2004). PCR products can be separated by electrophoresis based on the nucleotide composition. Sequence variation among the differentDNA molecules influences the melting behavoir, and therefore molecules with different sequences will stop migrating at different positions in the gel. Thus electrophoretic profiles can be defined by the position and the relative intensity of different bands or peaks and can be translated to numerical data for calculation of diversity indices. Bands can also be excised from the gel and subsequently sequenced to reveal the phylogenetic affiliation of the community members. Electrophoresis methods include, but are not limited to: denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), single- stranded-conformation polymorphism (SSCP), restriction fragment length polymorphism analysis (RFLP) or amplified ribosomal DNA restriction analysis (ARDRA), terminal restriction fragment length polymorphism analysis (T-RFLP), automated ribosomal intergenic spacer analysis (ARISA), randomly amplified polymorphic DNA (RAPD), DNA amplification fingerprinting (DAF) and Bb-PEG electrophoresis.

[0406] In another embodiment, the sample, or a portion thereof is subjected to a chip-based platform such as microarray or microfluidics to determine the abundance of a unique first marker and / or presence / abundance of a unique second marker (Fig. 1, 1003-1004, Fig. 2, 2003- 2004). The PCR products are amplified from total DNA in the sample and directly hybridized to known molecular probes affixed to microarrays. After the fluorescently labeled PCR amplicons are hybridized to the probes, positive signals are scored by the use of confocal laser scanning microscopy. The microarray technique allows samples to be rapidly evaluated with replication, which is a significant advantage in microbial community analyses. In general, the hybridization signal intensity on microarrays can be directly proportional to the abundance of the target organism. The universal high-density 16S microarray (e.g., PHYLOCHIP) contains about 30,000 probes of 16SrRNA gene targeted to several cultured microbial species and “candidate divisions”. These probes target all 121 demarcated prokaryotic orders and allow simultaneous detection of 8,741 bacterial and archaeal taxa. Another microarray in use for profiling microbial communities is the Functional Gene Array (FGA). Unlike PHYLOCHIPs, FGAs are designed primarily to detect specific metabolic groups of bacteria. Thus, FGA not only reveal the community structure, but they also shed light on the in situ community metabolic potential. FGA contain probes from genes with known biological functions, so they are useful in linking microbial community composition to ecosystem functions. An FGA termed GeoChip contains >24,000 probes from all known metabolic genes involved in various biogeochemical, ecological, and environmental processes such as ammonia oxidation, methane oxidation, and nitrogen fixation.

[0407] A protein expression assay, in one embodiment, is used with the methods described herein for determining the level of expression of one or more second markers (Fig. 1, 1004; Fig. 2, 2004). For example, in one embodiment, mass spectrometry or an immunoassay such as an enzyme-linked immunosorbant assay (ELISA) is utilized to quantify the level of expression of one or more unique second markers, wherein the one or more unique second markers is a protein.

[0408] In one embodiment, the sample, or a portion thereof is subjected to Bromodeoxyuridine (BrdU) incorporation to determine the level of a second unique marker (Fig. 1, 1004; Fig. 2, 2004). BrdU, a synthetic nucleoside analog of thymidine, can be incorporated into newly synthesized DNA of replicating cells. Antibodies specific for BRdU can then be used for detection of the base analog. Thus BrdU incorporation identifies cells that are actively replicating their DNA, a measure of activity of a microorganism according to one embodiment of the methods described herein. BrdU incorporation can be used in combination with FISH to provide the identity and activity of targeted cells.

[0409] In one embodiment, the sample, or a portion thereof is subjected to microautoradiography (MAR) combined with FISH to determine the level of a second unique marker (Fig. 1, 1004; Fig. 2, 2004). MAR-FISH is based on the incorporation of radioactive substrate into cells, detection of the active cells using autoradiography and identification of the cells using FISH. The detection and identification of active cells at single-cell resolution is performed with a microscope. MAR-FISH provides information on total cells, probe targeted cells and the percentage of cells that incorporate a given radiolabelled substance. The method provides an assessment of the in situ function of targeted microorganisms and is an effective approach to study the in vivo physiology of microorganisms. A technique developed for quantification of cell-specific substrate uptake in combination with MAR-FISH is known as quantitative MAR (QMAR).

[0410] In one embodiment, the sample, or a portion thereof is subjected to stable isotope Raman spectroscopy combined with FISH (Raman-FISH) to determine the level of a second unique marker (Fig. 1, 1004; Fig. 2, 2004). This technique combines stable isotope probing, Raman spectroscopy and FISH to link metabolic processes with particular organisms. The proportion of stable isotope incorporation by cells affects the light scatter, resulting in measurable peak shifts for labelled cellular components, including protein and mRNA components. Raman spectroscopy can be used to identify whether a cell synthesizes compounds including, but not limited to: oil (such as alkanes), lipids (such as triacylglycerols (TAG)), specific proteins (such as heme proteins, metalloproteins), cytochrome (such as P450,cytochrome c), chlorophyll, chromophores (such as pigments for light harvesting carotenoids and rhodopsins), organic polymers (such as polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB)), hopanoids, steroids, starch, sulfide, sulfate and secondary metabolites (such as vitamin B 12).

[0411] In one embodiment, the sample, or a portion thereof is subjected to DNA / RNA stable isotope probing (SIP) to determine the level of a second unique marker (Fig. 1, 1004; Fig. 2, 2004). SIP enables determination of the microbial diversity associated with specific metabolic pathways and has been generally applied to study microorganisms involved in the utilization of carbon and nitrogen compounds. The substrate of interest is labelled with stable isotopes (such as13C or15N) and added to the sample. Only microorganisms able to metabolize the substrate will incorporate it into their cells. Subsequently,13C-DNA and15N-DNA can be isolated by density gradient centrifugation and used for metagenomic analysis. RNA-based SIP can be a responsive biomarker for use in SIP studies, since RNA itself is a reflection of cellular activity.

[0412] In one embodiment, the sample, or a portion thereof is subjected to isotope array to determine the level of a second unique marker (Fig. 1, 1004; Fig. 2, 2004). Isotope arrays allow for functional and phylogenetic screening of active microbial communities in a high- throughput fashion. The technique uses a combination of SIP for monitoring the substrate uptake profiles and microarray technology for determining the taxonomic identities of active microbial communities. Samples are incubated with a14C-labeled substrate, which during the course of growth becomes incorporated into microbial biomass. The14C-labeled rRNA is separated from unlabeled rRNA and then labeled with fluorochromes. Fluorescent labeled rRNA is hybridized to a phylogenetic microarray followed by scanning for radioactive and fluorescent signals. The technique thus allows simultaneous study of microbial community composition and specific substrate consumption by metabolically active microorganisms of complex microbial communities.

[0413] In one embodiment, the sample, or a portion thereof is subjected to a metabolomics assay to determine the level of a second unique marker (Fig. 1, 1004; Fig. 2, 2004). Metabolomics studies the metabolome which represents the collection of all metabolites, the end products of cellular processes, in a biological cell, tissue, organ or organism. This methodology can be used to monitor the presence of microorganisms and / or microbial mediated processes since it allows associating specific metabolite profiles with different microorganisms. Profiles of intracellular and extracellular metabolites associated with microbial activity can be obtained using techniques such as gas chromatography-massspectrometry (GC-MS). The complex mixture of a metabolomic sample can be separated by such techniques as gas chromatography, high performance liquid chromatography and capillary electrophoresis. Detection of metabolites can be by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, ion-mobility spectrometry, electrochemical detection (coupled to HPLC) and radiolabel (when combined with thin-layer chromatography).

[0414] According to the embodiments described herein, the presence and respective number of one or more active microorganism strains in a sample are determined (Fig. 1, 1006; Fig. 2, 2006). For example, strain identity information obtained from assaying the number and presence of first markers is analyzed to determine how many occurrences of a unique first marker are present, thereby representing a unique microorganism strain (e.g., by counting the number of sequence reads in a sequencing assay). This value can be represented in one embodiment as a percentage of total sequence reads of the first maker to give a percentage of unique microorganism strains of a particular microorganism type. In a further embodiment, this percentage is multiplied by the number of microorganism types (obtained at step 1002 or 2002, see Fig. 1 and Fig. 2) to give the absolute abundance of the one or more microorganism strains in a sample and a given volume.

[0415] The one or more microorganism strains are considered active, as described above, if the level of second unique marker expression at a threshold level, higher than a threshold value, e.g., higher than at least about 5%, at least about 10%, at least about 20% or at least about 30% over a control level.

[0416] In another aspect of the disclosure, a method for determining the absolute abundance of one or more microorganism strains is determined in a plurality of samples (Fig. 2, see in particular, 2007). For a microorganism strain to be classified as active, it need only be active in one of the samples. The samples can be taken over multiple time points from the same source, or can be from different environmental sources (e.g., different animals).

[0417] The absolute abundance values over samples are used in one embodiment to relate the one or more active microorganism strains, with an environmental parameter (Fig. 2, 2008). In one embodiment, the environmental parameter is the presence of a second active microorganism strain. Relating the one or more active microorganism strains to the environmental parameter, in one embodiment, is carried out by determining the co-occurrence of the strain and parameter by correlation or by network analysis.

[0418] In one embodiment, determining the co-occurrence of one or more active microorganism strains with an environmental parameter comprises a network and / or cluster analysis method to measure connectivity of strains or a strain with an environmental parameterwithin a network, wherein the network is a collection of two or more samples that share a common or similar environmental parameter. In another embodiment, the network and / or cluster analysis method may be applied to determining the co-occurrence of two or more active microorganism strains in a sample (Fig. 2, 2008). In another embodiment, the network analysis comprises nonparametric approaches including mutual information to establish connectivity between variables. In another embodiment, the network analysis comprises linkage analysis, modularity analysis, robustness measures, betweenness measures, connectivity measures, transitivity measures, centrality measures or a combination thereof (Fig. 2, 2009). In another embodiment, the cluster analysis method comprises building a connectivity model, subspace model, distribution model, density model, or a centroid model and / or using community detection algorithms such as the Louvain, Bron-Kerbosch, Girvan-Newman, Clauset-Newman- Moore, Pons-Latapy, and Wakita-Tsurumi algorithms (Fig. 2, 2010).

[0419] In one embodiment, the cluster analysis method is a heuristic method based on modularity optimization. In a further embodiment, the cluster analysis method is the Louvain method. See, e.g., the method described by Blondel et al. (2008). Fast unfolding of communities in large networks. Journal of Statistical Mechanics: Theory and Experiment, Volume 2008, October 2008, incorporated by reference herein in its entirety for all purposes.

[0420] In another embodiment, the network analysis comprises predictive modeling of network through link mining and prediction, collective classification, link-based clustering, relational similarity, or a combination thereof. In another embodiment, the network analysis comprises differential equation based modeling of populations. In another embodiment, the network analysis comprises Lotka- Vol terra modeling.

[0421] In one embodiment, relating the one or more active microorganism strains to an environmental parameter (e.g., determining the co-occurrence) in the sample comprises creating matrices populated with linkages denoting environmental parameter and microorganism strain associations.

[0422] In one embodiment, the multiple sample data obtained at step 2007 (e.g., over two or more samples which can be collected at two or more time points where each time point corresponds to an individual sample), is compiled. In a further embodiment, the number of cells of each of the one or more microorganism strains in each sample is stored in an association matrix (which can be in some embodiments, an abundance matrix). In one embodiment, the association matrix is used to identify associations between active microorganism strains in a specific time point sample using rule mining approaches weighted with association (e.g., abundance) data. Filters are applied in one embodiment to remove insignificant rules.

[0423] In one embodiment, the absolute abundance of one or more, or two or more active microorganism strains is related to one or more environmental parameters (Fig. 2, 2008), e.g., via co-occurrence determination. Environmental parameters are chosen by the user depending on the sample(s) to be analyzed and are not restricted by the methods described herein. The environmental parameter can be a parameter of the sample itself, e.g., pH, temperature, amount of protein in the sample. Alternatively, the environmental parameter is a parameter that affects a change in the identity of a microbial community (z.e., where the “identity” of a microbial community is characterized by the type of microorganism strains and / or number of particular microorganism strains in a community), or is affected by a change in the identity of a microbial community. For example, an environmental parameter in one embodiment, is the food intake of an animal or the amount of milk (or the protein or fat content of the milk) produced by a lactating ruminant. In one embodiment, the environmental parameter is the presence, activity and / or abundance of a second microorganism strain in the microbial community, present in the same sample.

[0424] In some embodiments described herein, an environmental parameter is referred to as a metadata parameter.

[0425] Other examples of metadata parameters include but are not limited to genetic information from the host from which the sample was obtained (e.g., DNA mutation information), sample pH, sample temperature, expression of a particular protein or mRNA, nutrient conditions (e.g, level and / or identity of one or more nutrients) of the surrounding environment / ecosystem), susceptibility or resistance to disease, onset or progression of disease, susceptibility or resistance of the sample to toxins, efficacy of xenobiotic compounds (pharmaceutical drugs), biosynthesis of natural products, or a combination thereof.

[0426] For example, according to one embodiment, microorganism strain number changes are calculated over multiple samples according to the method of Fig. 2 (i.e., at 2001-2007). Strain number changes of one or more active strains over time is compiled (e.g, one or more strains that have initially been identified as active according to step 2006), and the directionality of change is noted (i.e., negative values denoting decreases, positive values denoting increases). The number of cells over time is represented as a network, with microorganism strains representing nodes and the abundance weighted rules representing edges. Markov chains and random walks are leveraged to determine connectivity between nodes and to define clusters. Clusters in one embodiment are filtered using metadata in order to identify clusters associated with desirable metadata (Fig. 2, 2008).

[0427] In a further embodiment, microorganism strains are ranked according to importance by integrating cell number changes over time and strains present in target clusters, with the highest changes in cell number ranking the highest.Network Analysis

[0428] Network and / or cluster analysis method in one embodiment, is used to measure connectivity of the one or more strains within a network, wherein the network is a collection of two or more samples that share a common or similar environmental parameter. In one embodiment, network analysis comprises linkage analysis, modularity analysis, robustness measures, betweenness measures, connectivity measures, transitivity measures, centrality measures or a combination thereof. In another embodiment, network analysis comprises predictive modeling of network through link mining and prediction, social network theory, collective classification, link-based clustering, relational similarity, or a combination thereof. In another embodiment, network analysis comprises differential equation based modeling of populations. In yet another embodiment, network analysis comprises Lotka- Vol terra modeling.

[0429] Cluster analysis method comprises building a connectivity model, subspace model, distribution model, density model, or a centroid model.

[0430] Network and cluster based analysis, for example, to carry out method step 2008 of Fig. 2, can be carried out via a module. As used herein, a component and / or module can be, for example, any assembly, instructions and / or set of operatively-coupled electrical components, and can include, for example, a memory, a processor, electrical traces, optical connectors, software (executing in hardware) and / or the like.Ruminant Pathogen Resistance and Clearance

[0431] In some aspects, the present disclosure is drawn to administering one or more microbial compositions described herein to ruminants to clear the gastrointestinal tract of pathogenic microbes. In some embodiments, the present disclosure is further drawn to administering microbial compositions described herein to prevent colonization of pathogenic microbes in the gastrointestinal tract. In some embodiments, the administration of microbial compositions described herein further clear pathogens from the integument and the respiratory tract of ruminants, and / or prevent colonization of pathogens on the integument and in the respiratory tract. In some embodiments, the administration of microbial compositions described herein reduce leaky gut / intestinal permeability, levels of histamine, production of lipopolysaccharides(LPS), inflammation, ketosis, laminitis, respiratory and metabolic acidosis, rumen acidosis, bloat, abomasal dysplasia, liver abscesses, and / or incidence of liver disease.

[0432] In some embodiments, the microbial compositions of the present disclosure comprise one or more microbes that are present in the gastrointestinal tract of ruminant...

Claims

CLAIMSWhat is claimed is:

1. A method for reducing enteric methane emission (EME) and / or EME intensity by a ruminant, the method comprising: administering a microbial supplement to the ruminant, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067; and e. a carrier suitable for ruminant administration.

2. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

3. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1; and / ord. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

4. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

5. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; b. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1; and c. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067.

6. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; b. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1; and c. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

7. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; b. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1; and c. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

8. The method of claim 1, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; b. a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO:1; and c. a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

9. The method of any one of claims 1-8, wherein the microbial supplement is a feed supplement.

10. The method of any one of claims 1-8, wherein the microbial supplement is administered orally to the ruminant.

11. The method of any one of claims 1-8, wherein the carrier is calcium bicarbonate.

12. The method of any one of claims 1-11, wherein the carrier is zeolite.

13. The method of any one of claims 1-12, wherein the microbial supplement is encapsulated.

14. The method of any one of claims 1-8, wherein the Clostridium sp., the Pichia sp., the Ruminococcus sp. and / or the Butyrivibrio sp. is a spore.

15. The method of any one of claims 1-13, wherein the ruminant is a cow, a steer, a heifer, or a calf.

16. The method of claim 15, wherein the ruminant is a dairy cow.

17. The method of any one of claims 1-16, wherein the abundance of methanogenic microbial populations is reduced in the ruminant administered the microbial supplement compared to a ruminant not administered the microbial supplement.

18. The method of any one of claims 1-16, wherein EME is reduced in the ruminant administered the microbial supplement compared to a ruminant not administered the microbial supplement.

19. The method of any one of claims 1-16, wherein EME intensity is reduced in the ruminant administered the microbial supplement compared to a ruminant not administered the microbial supplement.

20. A method of quantifying enteric methane emissions in a ruminant, the method comprising: a. collecting a sample from the ruminant; b. extracting DNA from the sample; c. performing next generation sequencing on the DNA to determine a microbiome of the ruminant; and d. quantifying enteric methane emissions in the ruminant using a biomarker from step (c) that is determined using a machine learning model.

21. The method of claim 20, wherein the sample is an oral swab sample, a rumen sample, or a saliva sample.

22. The method of claim 20 or 21, wherein the machine learning model associates the microbiome with EME.

23. The method of any one of claims 20-22, wherein the machine learning model uses random forest regression.

24. The method of any one of claims 20-22, wherein the machine learning model uses variational autoencoders or dynamical variational autoencoders.

25. The method of any one of claims 20-24, wherein the machine learning model associates microbial composition, microbial diversity, specific taxonomic groups, and / or core members of the microbiome with EME.

26. The method of any one of claims 20-24, wherein the machine learning model associates microbial composition, microbial diversity, specific taxonomic groups, and / or core members of the microbiome and additional animal characteristics with EME.

27. A method of claim 26, wherein the additional animal characteristics comprise milk production, dry matter intake, intake, energy corrected milk production, feed efficiency, fat percentage, protein percentage.

28. The method of any one of claims 20-27, wherein the machine learning model associates microbial composition, microbial diversity, specific taxonomic groups, and / or core members of the microbiome with EME, and wherein the microbial composition, microbial diversity, specific taxonomic groups, and / or core members are represented as a ratio.

29. The method of any one of claims 20-28, wherein the next generation sequencing is 16S rRNA gene amplicon sequencing.

30. The method of any one of claims 20-28, wherein the next generation sequencing is mcrA gene amplicon sequencing.

31. The method of any one of claims 20-30, wherein the ruminant is a cow, a steer, a heifer, or a calf.

32. The method of claim 31, wherein the ruminant is a dairy cow.

33. A machine learning model to predict EME by a cow, wherein the machine learning model associates oral microbiome of the cow with EME.

34. A method of quantifying enteric methane emissions in a ruminant, the method comprising: a. collecting a sample from the ruminant; b. extracting DNA from the sample; c. performing qPCR on the DNA to determine the copy number of a particular marker gene; and d. quantifying enteric methane emissions in the ruminant based on copy number.

35. The method of claim 34, wherein the sample is an oral swab sample, a rumen sample or a saliva sample.

36. The method of claim 34, wherein the marker gene is selected from genes specific to methanogens.

37. The method of claim 36, wherein the marker gene encodes a subunit of methyl coenzyme M or methyltransferase.

38. The method of claim 36, wherein the marker gene is mcrA, mcrB, mcrG, mtaB, or mtbA gene.

39. The method of any one of claims 30-38, wherein the ruminant is a cow, a steer, a heifer, or a calf.

40. The method of claim 39, wherein the ruminant is a dairy cow.

41. A machine learning model to predict EME by a cow, wherein the machine learning model associates oral microbiome of the cow with EME.

42. A microbial supplement for reducing EME and / or EME intensity by a ruminant, comprising: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 97% sequence identity to SEQ ID NO: 2067; and e. a carrier suitable for ruminant administration.

43. The microbial supplement of claim 42, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 32;c. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 98% sequence identity to SEQ ID NO: 2067.

44. The microbial supplement of claim 42, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 2067.

45. The microbial supplement of claim 42, wherein the microbial supplement comprises: a. a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; b. a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; c. a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO:1; and / or d. a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.

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