METHODS FOR IMPROVING MILK PRODUCTION BY ADMINISTRATION OF MICROBIAL CONSORTIA

Abstract
The disclosure relates to isolated microorganisms—including novel strains of the microorganisms—microbial consortia, and compositions comprising the same. Furthermore, the disclosure teaches methods of utilizing the described microorganisms, microbial consortia, and compositions comprising the same, in methods for modulating the production and yield of milk and milk components in ruminants. In particular aspects, the disclosure provides methods of increasing desirable components of milk in ruminants. Furthermore, the disclosure provides for methods of modulating the rumen microbiome.
Description
FIELD

The present disclosure relates to isolated and biologically pure microorganisms that have applications, inter alia, in dairy production. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into compositions. Furthermore, the disclosure provides microbial consortia, containing at least two members of the disclosed microorganisms, as well as methods of utilizing said consortia. Furthermore, the disclosure provides for methods of modulating the rumen microbiome.


STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is ASBI_002_02US_SeqList_ST25.txt. The text file is 893 kb, was created on Oct. 31, 2016, and is being submitted electronically via EFS-Web.


BACKGROUND

The global population is predicted to increase to over 9 billion people by the year 2050 with a concurrent reduction in the quantity of land, water, and other natural resources available per capita. Projections indicate that the average domestic income will also increase, with the projected rise in the GDP of China and India. The desire for a diet richer in animal-source proteins rises in tandem with increasing income, thus the global livestock sector will be charged with the challenge of producing more milk using fewer resources. The Food and Agriculture Organization of the United Nations predict that 70% more food will have to be produced, yet the area of arable land available will decrease. It is clear that the food output per unit of resource input will have to increase considerably in order to support the rise in population.


Milk and milk components from lactating ruminants are predominantly utilized in the preparation of foodstuffs in many different forms. Nevertheless, milk and milk components find numerous alternative applications in non-food areas such as the manufacture of glues, textile fibers, plastic materials, or in the production of ethanol or methane. There have been many strategies to improve milk production and content in ruminants through nutritional modulations, hormone treatments, changes in animal management, and selective breeding; however, the need for more efficient production of milk and milk components per animal is required.


Identifying compositions and methods for sustainably increasing milk production and modulating milk components of interest while balancing animal health and wellbeing have become imperative to satisfy the needs of every day humans in an expanding population. Increasing the worldwide production of milk and further modulating desirable milk components by scaling up the total number of livestock on dairy farms would not only be economically infeasible for many parts of the world, but would further result in negative environmental consequences.


Thus, meeting global milk and milk component yield expectations, by simply scaling up current high-input agricultural systems—utilized in most of the developed world—is simply not feasible.


There is therefore an urgent need in the art for improved methods of increasing milk production and further increasing yield of desirable milk components.


SUMMARY OF THE DISCLOSURE

In some aspects, the present disclosure provides isolated microbes, including novel strains of microbes, presented in Table 1 and/or Table 3.


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


In some aspects, the disclosure provides for utilizing one or more microbes selected from Table 1 and/or Table 3 to increase a phenotypic trait of interest in a ruminant. Furthermore, the disclosure provides for methods of modulating the rumen microbiome by utilizing one or more microbes selected from Table 1 and/or Table 3.


In some embodiments, a microbial consortium comprises at least two microbial strains selected from Table 1 and/or Table 3. In some embodiments, a microbial consortium comprises at least one microbial strain selected from Table 1 and/or Table 3. In a further embodiment, a microbial consortium comprises at least two microbial strains, wherein each microbe comprise a 16S rRNA sequence encoded by a sequence selected from SEQ ID NOs:1-30 and 2045-2103 or an ITS sequence selected from SEQ ID NOs:31-60 and 2104-2107. In an additional embodiment, a microbial consortium comprises at least one microbial strain, wherein each microbe comprise a 16S rRNA sequence encoded by a sequence selected from SEQ ID NOs:1-30 and 2045-2103, or an ITS sequence selected from SEQ ID NOs:31-60 and 2104-2107.


In some embodiments, the microbial consortia of the present disclosure comprise at least two microbial strains, wherein each microbe comprises a 16S rRNA sequence encoded by a sequence selected from SEQ ID NOs:1-30, SEQ ID NOs:61-1988, or SEQ ID NOs:2045-2103; or an ITS sequences selected from SEQ ID NOs:31-60, SEQ ID NOs:1989-2044, or SEQ ID NOs:2104-2107.


In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_24. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_24. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_24. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_24. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_1801, Ascusf_45, and Ascusf_24. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_1801, Ascusf_45, and Ascusf_24. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_268, Ascusf_45, and Ascusf_24. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_268, Ascusf_45, and Ascusf_24. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_232, Ascusf_45, and Ascusf_24. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_232, Ascusf_45, and Ascusf_24. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_249. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_249. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_353. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_353. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_23. In a further embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_23. In one embodiment, the microbial consortium comprises at least two microbial strains comprising Ascusb_3138 and Ascusf_15. In a further embodiment, the microbial consortium comprises at least one microbial strain comprising Ascusb_3138 and Ascusf_15. In one embodiment, the at least one microbial strain comprises Ascusb_3138. In another embodiment, the at least one microbial strain comprises Ascusf_15.


In one embodiment, a composition comprises a microbial consortium of the present disclosure and an acceptable carrier. In a further embodiment, a composition comprises a microbial consortium of the present disclosure and acceptable carrier. In a further embodiment, the microbial consortium is encapsulated. In a further embodiment, the encapsulated microbial consortium comprises a polymer. In a further embodiment, the polymer may be selected from a saccharide polymer, agar polymer, agarose polymer, protein polymer, sugar polymer, and lipid polymer.


In some embodiments, the acceptable carrier is selected from the group consisting of edible feed grade material, mineral mixture, water, glycol, molasses, and corn oil. In some embodiments, the at least two microbial strains forming the microbial consortium are present in the composition at 102 to 1015 cells per gram of said composition.


In some embodiments, the composition may be mixed with livestock feed.


In some embodiments, a method of imparting at least one improved trait upon an animal comprises administering the composition to the animal. In further embodiments, the animal is a ruminant, which may further be a cow.


In some embodiments, the composition is administered at least once per day. In a further embodiment, the composition is administered at least once per month. In a further embodiment, the composition is administered at least once per week. In a further embodiment, the composition is administered at least once per hour.


In some embodiments, the administration comprises injection of the composition into the rumen. In some embodiments, the composition is administered anally. In further embodiments, anal administration comprises inserting a suppository into the rectum. In some embodiments, the composition is administered orally. In some aspects, the oral administration comprises administering the composition in combination with the animal's feed, water, medicine, or vaccination. In some aspects, the oral administration comprises applying the composition in a gel or viscous solution to a body part of the animal, wherein the animal ingests the composition by licking. In some embodiments, the administration comprises spraying the composition onto the animal, and wherein the animal ingests the composition. In some embodiments, the administration occurs each time the animal is fed. In some embodiments, the oral administration comprises administering the composition in combination with the animal feed.


In some embodiments, the at least one improved trait is selected from the group consisting of: an increase of fat in milk, an increase of carbohydrates in milk, an increase of protein in milk, an increase of vitamins in milk, an increase of minerals in milk, an increase in milk volume, an improved efficiency in feed utilization and digestibility, an increase in polysaccharide and lignin degradation, an increase in fatty acid concentration in the rumen, pH balance in the rumen, a reduction in methane emissions, a reduction in manure production, improved dry matter intake, an increase in energy corrected milk (ECM) by weight and/or volume, an improved efficiency of nitrogen utilization, and any combination thereof; wherein said increase or reduction is determined by comparing against an animal not having been administered said composition.


In some embodiments, the increase in fat in milk is an increase in triglycerides, triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, cholesterol, glycolipids, and/or fatty acids. In some embodiments, an increase of carbohydrates is an increase in oligosaccharides, lactose, glucose, and/or glucose. In some embodiments, an increase in polysaccharide degradation is an increase in the degradation of cellulose, lignin, and/or hemicellulose. In some embodiments, an increase in fatty acid concentration is an increase in acetic acid, propionic acid, and/or butyric acid.


In some embodiments, the at least two microbial strains or the at least one microbial strain present in a composition, or consortia, of the disclosure exhibit an increased utility that is not exhibited when said strains occur alone or when said strains are present at a naturally occurring concentration. In some embodiments, compositions of the disclosure, comprising at least two microbial strains as taught herein, exhibit a synergistic effect on imparting at least one improved trait in an animal. In some embodiments, the compositions of the disclosure—comprising one or more isolated microbes as taught herein—exhibit markedly different characteristics/properties compared to their closest naturally occurring counterpart. That is, the compositions of the disclosure exhibit markedly different functional and/or structural characteristics/properties, as compared to their closest naturally occurring counterpart. For instance, the microbes of the disclosure are structurally different from a microbe as it naturally exists in a rumen, for at least the following reasons: said microbe can be isolated and purified, such that it is not found in the milieu of the rumen, said microbe can be present at concentrations that do not occur in the rumen, said microbe can be associated with acceptable carriers that do not occur in the rumen, said microbe can be formulated to be shelf-stable and exist outside the rumen environment, and said microbe can be combined with other microbes at concentrations that do not exist in the rumen. Further, the microbes of the disclosure are functionally different from a microbe as it naturally exists in a rumen, for at least the following reasons: said microbe when applied in an isolated and purified form can lead to modulation of the rumen microbiome, increased milk production, and/or improved milk compositional characteristics, said microbe can be formulated to be shelf-stable and able to exist outside the rumen environment, such that the microbe now has a new utility as a supplement capable of administration to a ruminant, wherein the microbe could not have such a utility in it's natural state in the rumen, as the microbe would be unable to survive outide the rumen without the intervention of the hand of man to formulate the microbe into a shelf-stable state and impart this new utility that has the aforementioned functional characteristics not possessed by the microbe in it's natural state of existence in the rumen.


In one embodiment, the disclosure provides for a ruminant feed supplement capable of increasing a desirable phenotypic trait in a ruminant. In a particular embodiment, the ruminant feed supplement comprises: a microbial consortium of the present disclosure at a concentration that does not occur naturally, and an acceptable carrier. In one aspect, the microbial consortium is encapsulated.


In one embodiment, an isolated microbial strain is selected from any one of the microbial strains in Table 1 and/or Table 3. In one embodiment, an isolated microbial strain is selected from the group consisting of: Ascusb_7 deposited as Bigelow Accession Deposit No. Patent201612011; Ascusb_32 deposited as Bigelow Accession Deposit No. Patent201612007; Ascusb_82 deposited as Bigelow Accession Deposit No. Patent201612012; Ascusb_119 deposited as Bigelow Accession Deposit No. Patent201612009; Ascusb_1801 deposited as Bigelow Accession Deposit No. Patent201612009; Ascusf_206 deposited as Bigelow Accession Deposit No. Patent201612003; Ascusf_23 deposited as Bigelow Accession Deposit No. Patent201612014; Ascusf_24 deposited as Bigelow Accession Deposit No. Patent201612004; Ascusf_45 deposited as Bigelow Accession Deposit No. Patent201612002; Ascusf_208 deposited as Bigelow Accession Deposit No. Patent201612003; Ascusb_3138 deposited as NRRL Accession Deposit No. B-67248; and Ascusf_15 deposited as NRRL Accession Deposit No. Y-67249.


In one embodiment, an isolated microbial strain of the present disclosure comprises a polynucleotide sequence sharing at least 90% sequence identity with any one of SEQ ID NOs:1-2107. In another embodiment, an isolated microbial strain of the present disclosure comprises a polynucleotide sequence sharing at least 90% sequence identity with any one of SEQ ID NOs:1-60 and 2045-2107.


In one embodiment, a substantially pure culture of an isolated microbial strain may comprise any one of the strains or microbes of the present disclosure.


In one embodiment, a method of modulating the microbiome of a ruminant comprises administering a composition of the present disclosure. In a further embodiment, the administration of the composition imparts at least one improved train upon the ruminant. In one embodiment, the at least one improved trait is selected from the group consisting of: an increase of fat in milk, an increase of carbohydrates in milk, an increase of protein in milk, an increase of vitamins in milk, an increase of minerals in milk, an increase in milk volume, an improved efficiency in feed utilization and digestibility, an increase in polysaccharide and lignin degradation, an increase in fatty acid concentration in the rumen, pH balance in the rumen, a reduction in methane emissions, a reduction in manure production, improved dry matter intake, an increase in energy corrected milk (ECM) by weight and/or volume, and an improved efficiency of nitrogen utilization; wherein said increase or reduction is determined by comparing against an animal not having been administered said composition. In an additional embodiment, the modulation of the microbiome is a decrease in the proportion of the microbial strains present in the microbiome prior to the administration of the composition, wherein the decrease is measured relative to the microbiome of the ruminant prior to the administration of the composition.


In one embodiment, the method of increasing fat in milk is an increase in triglycerides, triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, cholesterol, glycolipids, and/or fatty acids.


In one embodiment, the method of increasing carbohydrates is an increase in oligosaccharides, lactose, glucose, and/or galactose.


In one embodiment, the method of increasing polysaccharide degradation is an increase in the degradation of lignin, cellulose, pectin and/or hemicellulose.


In one embodiment, the method of increasing fatty acid concentration is an increase in acetic acid, propionic acid, and/or butyric acid.


In one embodiment, the method of modulation of the microbiome is an increase in the proportion of the at least one microbial strain of the microbiome, wherein the increase is measured relative to a ruminant that did not have the at least one microbial strain administered.


In one embodiment, the method of modulation of the microbiome is a decrease in the proportion of the microbial strains present in the microbiome prior to the administration of the composition, wherein the decrease is measured relative to the microbiome of the ruminant prior to the administration of the composition.


In one embodiment, a method of increasing resistance of cows to the colonization of pathogenic microbes comprises administering a composition of the present disclosure, resulting in the pathogenic microbes being unable to colonize the gastrointestinal tract of a cow. In another embodiment, a method for treating cows for the presence of at least one pathogenic microbe comprises the administration of a microbial consortium of the present disclosure and an acceptable carrier. In a further embodiment, the administration of the microbial consortium or microbial composition results in the relative abundance of the at least one pathogenic microbe to decrease to less than 5% relative abundance in the gastrointestinal tract. In another embodiment, the administration of the microbial consortium or microbial composition results in the relative abundance of the at least one pathogenic microbe to decrease to less than 1% relative abundance in the gastrointestinal tract. In another embodiment, the administration of the microbial consortium or microbial composition results in the pathogenic microbe being undetectable in the gastrointestinal tract.


In one embodiment, the microbial compositions and/or consortium comprise bacteria and/or fungi in spore form. In one embodiment, the microbial compositions and/or consortium of the disclosure comprise bacteria and/or fungi in whole cell form. In one embodiment, the microbial compositions and/or consortium of the disclosure comprise bacteria and/or fungi in lysed cell form. In some aspects of formulating microbes according to the dislcosure, the microbes are: fermented→filtered→centrifuged→lyophilized or spray dried→and optionally coated (i.e. a “fluidized bed step”).


Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures

Some microorganisms described in this Application were deposited on Apr. 25, 20161, with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Culture Collection (NRRL®), located at 1815 N. University St., Peoria, Ill. 61604, USA. Some microorganisms described in this application were deposited with the Bigelow National Center for Marine Algae and Microbiota, located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA. 1 ASC-01 (NRRL B-67248) and ASC-02 (NRRL Y-67249) were deposited on this date


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. The NRRL® and/or Bigelow National Center for Marine Algae and Microbiota accession numbers for the aforementioned Budapest Treaty deposits are provided in Table 3. The accession numbers and corresponding dates of deposit for the microorganisms described in this Application are separately provided in Table 25.


The strains designated in the below tables have been deposited in the labs of Ascus Biosciences, Inc. since at least Dec. 15, 2015.


In Table 1, the closest predicted hits for taxonomy of the microbes are listed in columns 2, and 5. Column 2 is the top taxonomic hit predicted by BLAST, and column 5 is the top taxonomic hit for genus+species predicted by BLAST. The strains designated in the below table have been deposited in the labs of Ascus Biosciences, Inc. since at least Dec. 15, 2015.









TABLE 1







Microbes of the present disclosure, including bacteria (1-89) and fungi (90-123).
























Sequence







BLAST



Identifier



BLAST
Blast

Taxonomic Top
Blast


for


Predicted Taxa of
Taxonomic Top
%
Query
Hit w/Genus +
%
Query
Strain
Associated
MIC


Isolated Microbes
Hit
Ident.
Cover
Species
Identity
Cover
Designation
Marker
Score



















1. Clostridium IV
Clostridiaceae
96%
100% 

Ruminococcus

91%
82%
Ascusb_5
SEQ ID
0.85694


(Cluster)
bacterium



bromii




NO: 1


2. Ruminococcus
Rumen bacterium
93%
84%

Ruminococcus

91%
82%
Ascusb_7
SEQ ID
0.97384


(Genus)




bromii




NO: 2


3. Clostridium IV
Rumen bacterium
89%
97%

Intestinimonas

85%
100% 
Ascusb_26
SEQ ID
0.82051


(Cluster)
NK4A214



butyriciproducens




NO: 3


4. Roseburia (Genus)
Lachno- spiraceae
89%
100% 

Pseudobutyrivibrio

89%
96%
Ascusb_27
SEQ ID
0.87214



bacterium



ruminis




NO: 4


5. Hydrogenoanaerobacterium (Genus)
Lachno- spiraceae
87%
93%

Roseburia

86%
93%
Ascusb_32
SEQ ID
0.81269



bacterium



inulinivorans




NO: 5


6. Clostridium XIVa

Eubacterium

92%
100% 

Eubacterium

92%
100% 
Ascusb_79
SEQ ID
0.82765


(Cluster)

ventriosum




ventriosum




NO: 6


7. Saccharofermentans
Rumen bacterium
87%
100% 

Faecalibacterium

91%
76%
Ascusb_82
SEQ ID
0.93391


(Genus)




prausnitzii




NO: 7


8. Saccharofermentans

Saccharofermentans

100% 
99%

Saccharofermentans

83%
92%
Ascusb_102
SEQ ID
0.82247


(Genus)
sp.



acetigenes




NO: 8


9. Butyricicoccus

Clostridium sp.

87%
100% 

Ruminococcus

86%
99%
Ascusb_89
SEQ ID
0.74361


(Genus)




flavefaciens




NO: 9


10. Papillibacter
Rumen bacterium
91%
99%

Clostridium

88%
82%
Ascusb_111
SEQ ID
0.82772


(Genus)
NK4A214



saccharolyticum




NO: 10


11. Ruminococcus
Ruminococcaceae
100% 
94%

Clostridium

85%
99%
Ascusb_119
SEQ ID
0.8263


(Genus)




lentocellum




NO: 11


12. Hydrogenoanaerobacterium
Rumen bacterium
85%
98%

Ruminococcus

85%
100% 
Ascusb_145
SEQ ID
0.81161


(Genus)
NK4B29



flavefaciens




NO: 12


13. Pelotomaculum

Faecalibacterium

86%
93%

Faecalibacterium

86%
82%
Ascusb_205
SEQ ID
0.81461


(Genus)
sp.



prausnitzii




NO: 13


14. Saccharofermentans
Bacterium MA3003
99%
91%

Saccharofermentans

90%
79%
Ascusb_232
SEQ ID
0.81428


(Genus)




acetigenes




NO: 14


15. Lachnospiraceae
Bacterium
95%
93%

Blautia luti

88%
92%
Ascusb_252
SEQ ID
0.8196


incertae sedis (Family)
VCD3003






NO: 15


16. Butyricicoccus
Ruminococcaceae
91%
77%

Clostridium

83%
99%
Ascusb_268
SEQ ID
0.74813


sensu stricto (Genus)
bacterium



lentocellum




NO: 16


17. Lachnospiraceae
Bacterium
96%
92%

Coprococcus catus

88%
100% 
Ascusb_374
SEQ ID
0.76214


incertae sedis (Family)
YAB2006






NO: 17


18. Anaeroplasma

Anaeroplasma

97%
100% 

Anaeroplasma

97%
100% 
Ascusb_411
SEQ ID
0.76213


(Genus)

varium




varium




NO: 18


19. Clostridium sensu
Clostridiales
100% 
93%

Clostridium

81%
91%
Ascusb_546
SEQ ID
0.83869


stricto (Genus)
bacterium



stercorarium




NO: 19


20. Butyricicoccus
Clostridiales
88%
91%

Aminiphilus

80%
77%
Ascusb_672
SEQ ID
0.74829


(Genus)
bacterium



circumscriptus




NO: 20


21. Butyricicoccus
Clostridiales
89%
89%

Aminiphilus

97%
27%
Ascusb_765
SEQ ID
0.74111


(Genus)
bacterium



circumscriptus




NO: 21


22. Rikenella (Genus)

Bacteroides sp.

93%
64%

Alistipes shahii

93%
64%
Ascusb_812
SEQ ID
0.73874










NO: 22


23. Tannerella (Genus)

Alistipes shahii

86%
100% 

Alistipes shahii

86%
100% 
Ascusb_1295
SEQ ID
0.8365










NO: 23


24. Howardella (Genus)
Clostridiales
85%
100% 

Oscillibacter

89%
41%
Ascusb_1763
SEQ ID
0.75083



bacterium



valericigenes




NO: 24


25. Prevotella (Genus)
Bacteroidetes
97%
95%

Odoribacter

77%
86%
Ascusb_1780
SEQ ID
0.89749



bacterium



splanchnicus




NO: 25


26. Butyricimonas
Bacteroidetes
95%
99%

Tannerella forsythia

83%
92%
Ascusb_1801
SEQ ID
0.89664


(Genus)
bacterium






NO: 26


27. Clostridium sensu
Bacterium
96%
93%

Hydrogenoanaerobacterium

84%
86%
Ascusb_1833
SEQ ID
0.73989


stricto (Genus)
XBB3002



saccharovorans




NO: 27


28. Clostridium sensu

Clostridium

98%
100% 

Clostridium

98%
100% 
Ascusb_3138
SEQ ID
0.76524


stricto (Genus)

butyricum




butyricum




NO: 28


29. Saccharofermentans
Rumen bacterium
87%
99%

Faecalibacterium

90%
76%
Ascusb_6589
SEQ ID
0.76539


(Genus)
NK4A214



prausnitzii




NO: 29


30. Lachnospiraceae

Roseburia

90%
100% 

Roseburia

90%
100% 
Ascusb_7921
SEQ ID
0.86201


incertae sedis (Family)

intestinalis




intestinalis




NO: 30


31. Succinivibrio

Succinivibrio

95%
99%

Succinivibrio

95%
99%
Ascusb_11
SEQ ID
0.50001


(Genus)

dextrinosolvens




dextrinosolvens




NO: 2045


32. Prevotella (Genus)
Bacterium
100% 
93%

Prevotella

91%

Ascusb_36
SEQ ID
0.55431



MB2027



ruminicola




NO: 2046


33. Prevotella (Genus)

Prevotella

100% 
99%

Prevotella

100% 

Ascusb_67
SEQ ID
0.49156




ruminicola




ruminicola




NO: 2047


34. Prevotella (Genus)

Prevotella

97%
100% 

Prevotella

97%
100% 
Ascusb_87
SEQ ID
0.59635




ruminicola




ruminicola




NO: 2048


35. Ruminobacter

Ruminobacter sp.

92%
99%

Ruminobacter

92%
100% 
Ascusb_101
SEQ ID
0.75099


(Genus)




amylophilus




NO: 2049


36. Syntrophococcus

Blautia producta

91%
100% 

Blautia producta

91%
100% 
Ascusb_104
SEQ ID
0.70044


(Genus)







NO: 2050


37. Succinivibrio

Succinivibrio

96%
99%

Succinivibrio

96%
99%
Ascusb_125
SEQ ID
0.44408


(Genus)

dextrinosolvens




dextrinosolvens




NO: 2051


38. Pseudobutyrivibrio

Butyrivibrio

99%
100% 

Butyrivibrio

99%
100% 
Ascusb_149
SEQ ID
0.50676


(Genus)

fibrisolvens




fibrisolvens




NO: 2052


39. Prevotella (Genus)

Prevotella

99%
99%

Prevotella

99%
99%
Ascusb_159
SEQ ID
0.5744




ruminicola




ruminicola




NO: 2053


40. Prevotella (Genus)

Prevotella

96%
99%

Prevotella

96%
99%
Ascusb_183
SEQ ID
0.50204




ruminicola




ruminicola




NO: 2054


41. Prevotella (Genus)

Prevotella

99%
100% 

Prevotella

99%
100% 
Ascusb_187
SEQ ID
0.56688




ruminicola




ruminicola




NO: 2055


42. Prevotella (Genus)
Bacterium XBB2006
100% 
94%

Prevotella albensis

87%
97%
Ascusb_190
SEQ ID
0.56183










NO: 2056


43. Lachnospiraceae
Lachnospiraceae
91%
100% 

Roseburia

89%
100% 
Ascusb_199
SEQ ID
0.62487


incertae sedis (Family)
bacterium



inulinivorans




NO: 2057


44. Syntrophococcus

Ruminococcus

95%
100% 

Ruminococcus

95%
100% 
Ascusb_278
SEQ ID
0.51235


(Genus)

gnavus




gnavus




NO: 2058


45. Ruminobacter

Ruminobacter sp.

100% 
99%

Ruminobacter

99%
100% 
Ascusb_329
SEQ ID
0.4754


(Genus)




amylophilus




NO: 2059


46. Butyrivibrio

Butyrivibrio sp.

100% 
100% 

Butyrivibrio

99%
98%
Ascusb_368
SEQ ID
0.60727


(Genus)




hungatei




NO: 2060


47. Clostridium_XIVa

Eubacterium

100% 
96%

Eubacterium

100% 
96%
Ascusb_469
SEQ ID
0.66345


(Cluster)

oxidoreducens




oxidoreducens




NO: 2061


48. Prevotella (Genus)
Rumen bacterium
99%
99%

Prevotella brevis

91%
100% 
Ascusb_530
SEQ ID
0.44804



NK4A111






NO: 2062


49. Prevotella (Genus)

Prevotella sp.

100% 
93%

Prevotella copri

100% 
93%
Ascusb_728
SEQ ID
0.55431










NO: 2063


50. Lachnospiraceae

Eubacterium

99%
100% 

Eubacterium

99%
100% 
Ascusb_756
SEQ ID
0.72136


incertae sedis (Family)

ruminantium




ruminantium




NO: 2064


51. Roseburia (Genus)
Lachnospiraceae
89%
93%
[Clostridium]
89%
91%
Ascusb_810
SEQ ID
0.65527



bacterium



xylanovorans




NO: 2065


52. Lachnospiraceae

Lachnospira

99%
100% 

Lachnospira

99%
100% 
Ascusb_817
SEQ ID
0.46512


incertae sedis (Family)

pectinoschiza




pectinoschiza




NO: 2066


53. Butyrivibrio

Butyrivibrio

98%
99%

Butyrivibrio

98%
99%
Ascusb_826
SEQ ID
0.65357


(Genus)

fibrisolvens




fibrisolvens




NO: 2067


54. Pseudobutyrivibrio

Pseudobutyrivibrio

100% 
95%

Pseudobutyrivibrio

97%
100% 
Ascusb_880
SEQ ID
0.52295


(Genus)
sp.



ruminis




NO: 2068


55. Turicibacter

Sinimarinibacterium

87%
69%

Sinimarinibacterium

87%
69%
Ascusb_913
SEQ ID
0.55141


(Genus)

flocculans




flocculans




NO: 2069


56. Lachnospiraceae
Bacterium FB3002
100% 
91%

Butyrivibrio

90%
100% 
Ascusb_974
SEQ ID
0.53487


incertae sedis (Family)




fibrisolvens




NO: 2070


57. Pseudobutyrivibrio

Pseudobutyrivibrio

97%
99%

Pseudobutyrivibrio

97%
99%
Ascusb_1069
SEQ ID
0.55299


(Genus)

ruminis




ruminis




NO: 2071


58. Anaerolinea
Chloroflexi
88%
99%

Anaerolinea

90%
57%
Ascusb_1074
SEQ ID
0.50893


(Genus)
bacterium



thermophila




NO: 2072


59. Roseburia (Genus)
Lachnospiraceae
98%
99%

Eubacterium rectale

94%
100% 
Ascusb_1293
SEQ ID
0.61745










NO: 2073


60. Propionibacterium

Propionibacterium

100% 
100% 

Propionibacterium

100% 
100% 
Ascusb_1367
SEQ ID
0.54046


(Genus)

acnes




acnes




NO: 2074


61. Clostridium_XIVa
Lachnospiraceae
88%
100% 

Pseudobutyrivibrio

86%
97%
Ascusb_1632
SEQ ID
0.46826


(Cluster)
bacterium



ruminis




NO: 2075


62. Olsenella (Genus)
Coriobacteriaceae
98%
100% 

Olsenella profusa

97%
100% 
Ascusb_1674
SEQ ID
0.51533



bacterium






NO: 2076


63. Streptococcus

Streptococcus

95%
82%

Streptococcus

95%
82%
Ascusb_1786
SEQ ID
0.48678


(Genus)

dentirousetti




dentirousetti




NO: 2077


64. Clostridium_XIVa

Butyrivibrio sp.

99%
96%

Butyrivibrio

93%
100% 
Ascusb_1812
SEQ ID
0.64367


(Cluster)




proteoclasticus




NO: 2078


65. Clostridium_XIVa
Bacterium
99%
91%

Butyrivibrio

96%
99%
Ascusb_1850
SEQ ID
0.57807


(Cluster)
DAZ2002



hungatei




NO: 2079


66. Roseburia (Genus)
Lachnospiraceae
95%
99%

Eubacterium

89%
100% 
Ascusb_1879
SEQ ID
0.45014



bacterium



oxidoreducens




NO: 2080


67. Clostridium_IV
Ruminococcaceae
87%
99%

Ruminococcus

85%
91%
Ascusb_2090
SEQ ID
0.75266


(Cluster)
bacterium



bromii




NO: 2081


68. Clostridium_XICa
Bacterium MA2020
99%
99%

Clostridium

85%
90%
Ascusb_2124
SEQ ID
0.4673


(Cluster)




algidixylanolyticum




NO: 2082


69. Lachnospiracea
Bacterium YSB2008
94%
94%

Eubacterium

91%
100% 
Ascusb_2198
SEQ ID
0.55249


incertae sedis (Family)




ruminantium




NO: 2083


70. Erysipelotrichaceae

Catenisphaera

90%
91%

Catenisphaera

90%
91%
Ascusb_2511
SEQ ID
0.50619


incertae sedis (Family)

adipataccumulans




adipataccumulans




NO: 2084


71. Solobacterium
Erysipelotrichaceae
92%
99%

Solobacterium

91%
100% 
Ascusb_2530
SEQ ID
0.53735


(Genus)
bacterium



moorei




NO: 2085


72. Lachnospiraceae

Eubacterium

95%
100% 

Eubacterium

95%
100% 
Ascusb_2597
SEQ ID
0.52028


incertae sedis (Genus)

ruminantium




ruminantium




NO: 2086


73. Clostridium XIVa

Butyrivibrio

99%
100% 

Butyrivibrio

99%
100% 
Ascusb_2624
SEQ ID
0.55465


(Cluster)

proteoclasticus




proteoclasticus




NO: 2087


74. Ralstonia (Genus)

Ralstonia sp. 94

100% 
99%

Ralsonia insidiosa

99%
100% 
Ascusb_2667
SEQ ID
0.52371










NO: 2088


75. Clostridium_XIVa

Butyrivibrio sp.

97%
94%

Butyrivibrio

95%
100% 
Ascusb_2836
SEQ ID
0.43374


(Cluster)




proteoclasticus




NO: 2089


76. Eubacterium
Eubacteriaceae
84%
100% 

Casaltella

87%
82%
Ascusb_3003
SEQ ID
0.56301


(Genus)
bacterium



massiliensis




NO: 2090


77. Lachnobacterium
Rumen bacterium
89%
98%

Eubacterium

90%
91%
Ascusb_3504
SEQ ID
0.52856


(Genus)




xylanophilum




NO: 2091


78. Acholeplasma

Acholeplasma

86%
72%

Acholeplasma

86%
72%
Ascusb_3881
SEQ ID
0.4402


(Genus)

brassicae




brassicae




NO: 2092


79. Selenomonas

Mitsuokella

91%
97%

Mitsuokella

91%
97%
Ascusb_4728
SEQ ID
0.4761


(Genus)

jalaludinii




jalaludinii




NO: 2093


80. Prevotella (Genus)

Prevotella

98%
100% 

Prevotella

98%
100% 
Ascusb_4934
SEQ ID
0.56204




ruminicola




ruminicola




NO: 2094


81. Clostridium_XIVa

Butyrivibrio sp.

99%
99%

Butyrivibrio

97%
100% 
Ascusb_4959
SEQ ID
0.42892


(Cluster)




fibrisolvens




NO: 2095


82. Succinivibrio

Succinivibrio

86%
84%

Succinivibrio

86%
84%
Ascusb_5525
SEQ ID
0.51758


(Genus)

dextrinosolvens




dextrinosolvens




NO: 2096


83. Ruminobacter

Ruminobacter sp.

100% 
99%

Ruminobacter

99%
100% 
Ascusb_12103
SEQ ID
0.52909


(Genus)




amylophilus




NO: 2097


84. Sharpea (Genus)
Lachnospiraceae
97%
100% 

Sharpea azabuensis

100% 
91%
Ascusb_14245
SEQ ID
0.61391



bacterium






NO: 2098


85. Prevotella (Genus)

Prevotella

87%
97%

Prevotella

87%
97%
Ascusb_14945
SEQ ID
0.80101




ruminicola




ruminicola




NO: 2099


86. Prevotella (Genus)

Prevotella sp. DJF

88%
89%

Prevotella

87%
945
Ascusb_17461
SEQ ID
0.44777







ruminicola




NO: 2100


87. Prevotella (Genus)
Bacterium MB2027
100% 
93%

Prevotella

91%
99%
Ascusb_20083
SEQ ID
0.52538







ruminicola




NO: 2101


88. Prevotella (Genus)

Prevotella

99%
100% 

Prevotella

99%
100% 
Ascusb_20187
SEQ ID
0.59156




ruminicola




ruminicola




NO: 2102


89. Prevotella (Genus)

Prevotella

100% 
100% 

Prevotella

100% 
100% 
Ascusb_20539
SEQ ID
0.4912




ruminicola




ruminicola




NO: 2103


90. Piromyces (Genus)

Piromyces sp.

93%
100% 

Neocallimastix

84%
100% 
Ascusf_11
SEQ ID
0.81719







frontalis




NO: 31


91. Candida xylopsoc

Pichia kudriavzevii

100% 
100% 

Pichia kudriavzevii

100% 
100% 
Ascusf_15
SEQ ID
0.76088


(Genus + Species)







NO: 32


92. Orpinomyces

Orpinomyces sp.

100% 
100% 

Neocallimastix

86%
100% 
Ascusf_22
SEQ ID
0.76806


(Genus)




frontalis




NO: 33


93. Orpinomycs

Neocallimastix

86%
80%

Neocallimastix

86%
80%
Ascusf_23
SEQ ID
0.85707


(Genus)

frontalis




frontalis




NO: 34


94. Orpinomyces

Orpinomyces sp.

95%
100% 

Neocallimastix

86%
100% 
Ascusf_24
SEQ ID
0.85292


(Genus)




frontalis




NO: 35


95. Candida apicol

Candida apicola

100% 
100% 

Candida apicola

100% 
100% 
Ascusf_25
SEQ ID
0.70561


(Genus + Species)







NO: 36


96. Candida rugosa

Candida

100% 
100% 

Candida

100% 
100% 
Ascusf_38
SEQ ID
0.78246


(Genus + Species)

akabanensis




akabanensis




NO: 37


97. Neocallimastix

Neocallimastix sp.

99%
100% 

Neocallimastix

99%
100% 
Ascusf_45
SEQ ID
0.86185


(Genus)




frontalis




NO: 38


98. Orpinomyces

Orpinomyces sp.

99%
100% 

Orpinomyces

96%
96%
Ascusf_60
SEQ ID
0.72985


(Genus)




joyonii




NO: 39


99. Orpinomyces

Neocallimastix

86%
78%

Neocallimastix

86%
78%
Ascusf_73
SEQ ID
0.76064


(Genus)

frontalis




frontalis




NO: 40


100. Neocallimastix

Neocallimastix sp.

98%
100% 

Neocallimastix

93%
100% 
Ascusf_77
SEQ ID
0.83475


(Genus)




frontalis




NO: 41


101. Neocallimastix

Neocallimastix

97%
100% 

Neocallimastix

97%
100% 
Ascusf_94
SEQ ID
0.77644


(Genus)

frontalis




frontalis




NO: 42


102. Ascomycota (Genus)
Basidiomycota sp.
85%
98%

Sugiyamaella

97%
26%
Ascusf_95
SEQ ID
0.7089







lignohabitans




NO: 43


103. Piromyces (Genus)

Caecomyces sp.

94%
100% 

Cyllamyces

86%
89%
Ascusf_108
SEQ ID
0.68405







aberensis




NO: 44


104. Orpinomyces

Orpinomyces sp.

95%
100% 

Orpinomyces

87%
96%
Ascusf_119
SEQ ID
0.80055


(Genus)




joyonii




NO: 45


105. Cyllamyces (Genus)

Caecomyces sp.

90%
100% 

Caecomyces

90%
83%
Ascusf_127
SEQ ID
0.66812







communis




NO: 46


106. Piromyces (Genus)

Caecomyces sp.

91%
100% 

Caecomyces

92%
83%
Ascusf_136
SEQ ID
0.73201







communis




NO: 47


107. Cyllamyces (Genus)

Cyllamyces sp.

97%
100% 

Cyllamyces

94%
89%
Ascusf_193
SEQ ID
0.7586







aberensis




NO: 48


108. Piromyces (Genus)

Piromyces sp.

92%
100% 

Neocallimastix

84%
100% 
Ascusf_228
SEQ ID
0.83403







frontalis




NO: 49


109. Piromyces (Genus)

Caecomyces sp.

94%
100% 

Cyllamyces

86%
89%
Ascusf_249
SEQ ID
0.78679







aberensis




NO: 50


110. Neocallimastix

Neocallimastix sp.

98%
100% 

Neocallimastix

92%
100% 
Ascusf_307
SEQ ID
0.77859


(Genus)




frontalis




NO: 51


111. Piromyces (Genus)

Piromyces sp.

94%
100% 

Neocallimastix

83%
100% 
Ascusf_315
SEQ ID
0.81028







frontalis




NO: 52


112. Neocallimastix

Neocallimastix sp.

100% 
98%

Neocallimastix

100% 
90%
Ascusf_334
SEQ ID
0.76456


(Genus)




frontalis




NO: 53


113. Saccharomycetales

Candida ethanolica

100% 
100% 

Candida ethanolica

100% 
100% 
Ascusf_353
SEQ ID
0.82628


(Order)







NO: 54


114. Piromyces (Genus)

Piromyces sp.

91%
100% 

Neocallimastix

83%
100% 
Ascusf_448
SEQ ID
0.70021







frontalis




NO: 55


115. Orpinomyces

Neocallimastix sp.

88%
91%

Neocallimastix

96%
88%
Ascusf_786
SEQ ID
0.63201


(Genus)




frontalis




NO: 56


116. Piromyces (Genus)

Piromyces sp.

91%
100% 

Neocallimastix

83%
100% 
Ascusf_836
SEQ ID
0.65492







frontalis




NO: 57


117. Phyllosticta
Tremellales sp.
96%
74%

Tremella giraffa

83%
96%
Ascusf_923
SEQ ID
0.76115



capitalensis (Genus +








NO: 58


Species)


118. Orpinomyces

Neocallimastix

87%
77%

Neocallimastix

87%
77%
Ascusf_1020
SEQ ID
0.68043


(Genus)

frontalis




frontalis




NO: 59


119. Orpinomyces

Neocallimastix

85%
80%

Neocallimastix

85%
80%
Ascusf_1103
SEQ ID
0.73004


(Genus)

frontalis




frontalis




NO: 60


120. Orpinomyces
Fungal sp. Tianzhu-
99%
100% 

Orpinomyces

94%
96%
Ascusf_81
SEQ ID
0.44471


(Genus)
Yak6



joyonii




NO: 2104


121. Piromyces (Genus)

Piromyces sp.

99%
100% 

Neocallimastix

84%
100% 
Ascusf_206
SEQ ID
0.49752







frontalis




NO: 2105


122. Piromyces (Genus)

Piromyces sp.

96%
100% 

Neocallimastix

82%
100% 
Ascusf_208
SEQ ID
0.4176







frontalis




NO: 2106


123. Piromyces (Genus)

Piromyces sp.

99%
100% 

Neocallimastix

82%
100% 
Ascusf_1012
SEQ ID
0.55922







frontalis




NO: 2107
















TABLE 2







Microbial Deposits Corresponding to the Microbes of Table 1
















Sequence



Sequence





Identifier



Identifier


Predicted Taxa

for

Predicted Taxa

for


of Isolated
Strain
Associated

of Isolated
Strain
Associated


Microbes
Designation
Marker
Deposit #
Microbes
Designation
Marker
Deposit #






Clostridium IV (Cluster)

Ascusb_5
SEQ ID
PATENT201612001,

Streptococcus

Ascusb_1786
SEQ ID
PATENT201612011,




NO: 1
PATENT201612007,
(Genus)

NO: 2077
PATENT201612012,





PATENT201612009,



PATENT201612013





PATENT201612010,





PATENT201612011,





PATENT201612012



Ruminococcus (Genus)

Ascusb_7
SEQ ID
PATENT201612005,

Clostridium_XIVa

Ascusb_1812
SEQ ID
PATENT201612011,




NO: 2
PATENT201612007,
(Cluster)

NO: 2078
PATENT201612012





PATENT201612009,





PATENT201612010,





PATENT201612011,





PATENT201612012,





PATENT201612013



Clostridium IV (Cluster)

Ascusb_26
SEQ ID
PATENT201612005,

Clostridium_XIVa

Ascusb_1850
SEQ ID
PATENT201612013




NO: 3
PATENT201612009,
(Cluster)

NO: 2079





PATENT201612011,





PATENT201612012



Roseburia (Genus)

Ascusb_27
SEQ ID
PATENT201612009

Roseburia (Genus)

Ascusb_1879
SEQ ID




NO: 4



NO: 2080



Hydrogenoanaerobacterium

Ascusb_32
SEQ ID
PATENT201612006,

Clostridium_IV

Ascusb_2090
SEQ ID
PATENT201612007,


(Genus)

NO: 5
PATENT201612009,
(Cluster)

NO: 2081
PATENT201612009





PATENT201612012



Clostridium XIVa

Ascusb_79
SEQ ID
PATENT201612011,

Clostridium_XICa

Ascusb_2124
SEQ ID
PATENT201612012


(Cluster)

NO: 6
PATENT201612012
(Cluster)

NO: 2082



Saccharofermentans

Ascusb_82
SEQ ID
PATENT201612005,

Lachnospiracea

Ascusb_2198
SEQ ID
PATENT201612012


(Genus)

NO: 7
PATENT201612006,
incertae sedis

NO: 2083





PATENT201612007,
(Family)





PATENT201612009,





PATENT201612010,





PATENT201612012



Saccharofermentans

Ascusb_102
SEQ ID
PATENT201612005,
Erysipelotrichaceae
Ascusb_2511
SEQ ID
PATENT201612001,


(Genus)

NO: 8
PATENT201612007,
incertae sedis

NO: 2084
PATENT201612007,





PATENT201612010,
(Family)


PATENT201612009





PATENT201612011,





PATENT201612012



Butyricicoccus (Genus)

Ascusb_89
SEQ ID
PATENT201612011,

Solobacterium

Ascusb_2530
SEQ ID
PATENT201612011,




NO: 9
PATENT201612012
(Genus)

NO: 2085
PATENT201612012



Papillibacter (Genus)

Ascusb_111
SEQ ID
PATENT201612005,
Lachnospiraceae
Ascusb_2597
SEQ ID
PATENT201612013




NO: 10
PATENT201612007,
incertae sedis

NO: 2086





PATENT201612012
(Genus)



Ruminococcus (Genus)

Ascusb_119
SEQ ID
PATENT201612011,

Clostridium_XIVa

Ascusb_2624
SEQ ID
PATENT201612009,




NO: 11
PATENT201612012
(Cluster)

NO: 2087
PATENT201612011,









PATENT201612012



Hydrogenoanaerobacterium

Ascusb_145
SEQ ID
PATENT201612011,

Ralstonia (Genus)

Ascusb_2667
SEQ ID
PATENT201612013


(Genus)

NO: 12
PATENT201612012


NO: 2088



Pelotomaculum (Genus)

Ascusb_205
SEQ ID
PATENT201612005,

Clostridium_XIVa

Ascusb_2836
SEQ ID
PATENT201612013




NO: 13
PATENT201612006,
(Cluster)

NO: 2089





PATENT201612011,





PATENT201612012



Saccharofermentans

Ascusb_232
SEQ ID
PATENT201612010,

Eubacterium

Ascusb_3003
SEQ ID
PATENT201612009


(Genus)

NO: 14
PATENT201612011,
(Genus)

NO: 2090





PATENT201612012


Lachnospiraceae incertae
Ascusb_252
SEQ ID


Lachnobacterium

Ascusb_3504
SEQ ID
PATENT201612011,


sedis (Family)

NO: 15

(Genus)

NO: 2091
PATENT201612012



Butyricicoccus sensu

Ascusb_268
SEQ ID
PATENT201612007,

Acholeplasma

Ascusb_3881
SEQ ID
PATENT201612007


stricto (Genus)

NO: 16
PATENT201612011,
(Genus)

NO: 2092





PATENT201612012


Lachnospiraceae incertae
Ascusb_374
SEQ ID
PATENT201612007,

Selenomonas

Ascusb_4728
SEQ ID


sedis (Family)

NO: 17
PATENT201612009,
(Genus)

NO: 2093





PATENT201612010,





PATENT201612011





PATENT201612012



Anaeroplasma (Genus)

Ascusb_411
SEQ ID
PATENT201612007,

Prevotella (Genus)

Ascusb_4934
SEQ ID




NO: 18
PATENT201612011,


NO: 2094





PATENT201612012



Clostridium sensu stricto

Ascusb_546
SEQ ID
PATENT201612013

Clostridium_XIVa

Ascusb_4959
SEQ ID


(Genus)

NO: 19

(Cluster)

NO: 2095



Butyricicoccus (Genus)

Ascusb_672
SEQ ID


Succinivibrio

Ascusb_5525
SEQ ID




NO: 20

(Genus)

NO: 2096



Butyricicoccus (Genus)

Ascusb_765
SEQ ID
PATENT201612013

Ruminobacter

Ascusb_12103
SEQ ID
PATENT201612001




NO: 21

(Genus)

NO: 2097



Rikenella (Genus)

Ascusb_812
SEQ ID
PATENT201612005,

Sharpea (Genus)

Ascusb_14245
SEQ ID
PATENT201612001,




NO: 22
PATENT201612006,


NO: 2098
PATENT201612008,





PATENT201612011,



PATENT201612009,





PATENT201612012



PATENT201612011,









PATENT201612012,









PATENT201612013



Tannerella (Genus)

Ascusb_1295
SEQ ID
PATENT201612007,

Prevotella (Genus)

Ascusb_14945
SEQ ID




NO: 23
PATENT201612009,


NO: 2099





PATENT201612011,





PATENT201612012



Howardella (Genus)

Ascusb_1763
SEQ ID
PATENT201612011,

Prevotella (Genus)

Ascusb_17461
SEQ ID




NO: 24
PATENT201612012


NO: 2100



Prevotella (Genus)

Ascusb_1780
SEQ ID
PATENT201612013

Prevotella (Genus)

Ascusb_20083
SEQ ID
PATENT201612006




NO: 25



NO: 2101



Butyricimonas (Genus)

Ascusb_1801
SEQ ID
PATENT201612005

Prevotella (Genus)

Ascusb_20187
SEQ ID
PATENT201612009,




NO: 26



NO: 2102
PATENT201612011,









PATENT201612012



Clostridium sensu stricto

Ascusb_1833
SEQ ID
PATENT201612006,

Prevotella (Genus)

Ascusb_20539
SEQ ID


(Genus)

NO: 27
PATENT201612007,


NO: 2103





PATENT201612009,





PATENT201612010,





PATENT201612011,





PATENT201612012



Clostridium sensu stricto

Ascusb_3138
SEQ ID
PATENT201612005,

Piromyces (Genus)

Ascusf_11
SEQ ID


(Genus)

NO: 28
PATENT201612006,


NO: 31





PATENT201612008,





PATENT201612009,





PATENT201612010,





PATENT201612011,





PATENT201612012,





PATENT201612013,





NRRL B-67248



Saccharofermentans

Ascusb_6589
SEQ ID
PATENT201612005

Candida xylopsoc

Ascusf_15
SEQ ID
NRRL Y-67249,


(Genus)

NO: 29

(Genus + Species)

NO: 32
PATENT201612014


Lachnospiraceae incertae
Ascusb_7921
SEQ ID


Orpinomyces

Ascusf_22
SEQ ID
PATENT201612002,


sedis (Family)

NO: 30

(Genus)

NO: 33
PATENT201612004



Succinivibrio (Genus)

Ascusb_11
SEQ ID
PATENT201612001,

Orpinomycs

Ascusf_23
SEQ ID
PATENT201612014




NO: 2045
PATENT201612008,
(Genus)

NO: 34





PATENT201612009,





PATENT201612011,





PATENT201612012



Prevotella (Genus)

Ascusb_36
SEQ ID
PATENT201612013

Orpinomyces

Ascusf_24
SEQ ID
PATENT201612002,




NO: 2046

(Genus)

NO: 35
PATENT201612004



Prevotella (Genus)

Ascusb_67
SEQ ID


Candida apicol

Ascusf_25
SEQ ID
PATENT201612014




NO: 2047

(Genus + Species)

NO: 36



Prevotella (Genus)

Ascusb_87
SEQ ID


Candida rugosa

Ascusf_38
SEQ ID
PATENT201612004




NO: 2048

(Genus + Species)

NO: 37



Ruminobacter (Genus)

Ascusb_101
SEQ ID
PATENT201612001,

Neocallimastix

Ascusf_45
SEQ ID
PATENT201612002,




NO: 2049
PATENT201612005,
(Genus)

NO: 38
PATENT201612014





PATENT201612011,





PATENT201612012



Syntrophococcus (Genus)

Ascusb_104
SEQ ID
PATENT201612005,

Orpinomyces

Ascusf_60
SEQ ID




NO: 2050
PATENT201612006
(Genus)

NO: 39



Succinivibrio (Genus)

Ascusb_125
SEQ ID
PATENT201612001,

Orpinomyces

Ascusf_73
SEQ ID




NO: 2051
PATENT201612005,
(Genus)

NO: 40





PATENT201612006,





PATENT201612008,





PATENT201612009,





PATENT201612011,





PATENT201612012



Pseudobutyrivibrio

Ascusb_149
SEQ ID
PATENT201612001,

Neocallimastix

Ascusf_77
SEQ ID
PATENT201612014


(Genus)

NO: 2052
PATENT201612008,
(Genus)

NO: 41





PATENT201612009,





PATENT201612011,





PATENT201612012,





PATENT201612013



Prevotella (Genus)

Ascusb_159
SEQ ID
PATENT201612005,

Neocallimastix

Ascusf_94
SEQ ID
PATENT201612014




NO: 2053
PATENT201612006,
(Genus)

NO: 42





PATENT201612007,





PATENT201612008,





PATENT201612009,





PATENT201612010,





PATENT201612011,





PATENT201612012



Prevotella (Genus)

Ascusb_183
SEQ ID
PATENT201612008,

Ascomycota

Ascusf_95
SEQ ID




NO: 2054
PATENT201612009
(Genus)

NO: 43



Prevotella (Genus)

Ascusb_187
SEQ ID
PATENT201612007,

Piromyces (Genus)

Ascusf_108
SEQ ID
PATENT201612014




NO: 2055
PATENT201612008,


NO: 44





PATENT201612010,





PATENT201612011,





PATENT201612012



Prevotella (Genus)

Ascusb_190
SEQ ID
PATENT201612005,

Orpinomyces

Ascusf_119
SEQ ID




NO: 2056
PATENT201612006,
(Genus)

NO: 45





PATENT201612007,





PATENT201612012


Lachnospiraceae incertae
Ascusb_199
SEQ ID
PATENT201612011,

Cyllamyces

Ascusf_127
SEQ ID


sedis (Family)

NO: 2057
PATENT201612012
(Genus)

NO: 46



Syntrophococcus (Genus)

Ascusb_278
SEQ ID
PATENT201612008

Piromyces (Genus)

Ascusf_136
SEQ ID




NO: 2058



NO: 47



Ruminobacter (Genus)

Ascusb_329
SEQ ID
PATENT201612010

Cyllamyces

Ascusf_193
SEQ ID




NO: 2059

(Genus)

NO: 48



Butyrivibrio (Genus)

Ascusb_368
SEQ ID
PATENT201612011,

Piromyces (Genus)

Ascusf_228
SEQ ID




NO: 2060
PATENT201612012


NO: 49



Clostridium_XIVa

Ascusb_469
SEQ ID


Piromyces (Genus)

Ascusf_249
SEQ ID


(Cluster)

NO: 2061



NO: 50



Prevotella (Genus)

Ascusb_530
SEQ ID


Neocallimastix

Ascusf_307
SEQ ID
PATENT201612002,




NO: 2062

(Genus)

NO: 51
PATENT201612014



Prevotella (Genus)

Ascusb_728
SEQ ID
PATENT201612008,

Piromyces (Genus)

Ascusf_315
SEQ ID




NO: 2063
PATENT201612009,


NO: 52





PATENT201612011,





PATENT201612012,





PATENT201612013


Lachnospiraceae incertae
Ascusb_756
SEQ ID


Neocallimastix

Ascusf_334
SEQ ID
PATENT201612014


sedis (Family)

NO: 2064

(Genus)

NO: 53



Roseburia (Genus)

Ascusb_810
SEQ ID
PATENT201612011,

Saccharomycetales

Ascusf_353
SEQ ID
PATENT201612014




NO: 2065
PATENT201612012
(Order)

NO: 54


Lachnospiraceae incertae
Ascusb_817
SEQ ID
PATENT201612001,

Piromyces (Genus)

Ascusf_448
SEQ ID


sedis (Family)

NO: 2066
PATENT201612006,


NO: 55





PATENT201612009,





PATENT201612012,





PATENT201612013,





NRRL B-67349



Butyrivibrio (Genus)

Ascusb_826
SEQ ID
PATENT201612011,

Orpinomyces

Ascusf_786
SEQ ID




NO: 2067
PATENT201612012,
(Genus)

NO: 56





PATENT201612013,





NRRL B-67347



Pseudobutyrivibrio

Ascusb_880
SEQ ID
PATENT201612008,

Piromyces (Genus)

Ascusf_836
SEQ ID


(Genus)

NO: 2068
PATENT201612009


NO: 57



Turicibacter (Genus)

Ascusb_913
SEQ ID
PATENT201612007,

Phyllosticta

Ascusf_923
SEQ ID




NO: 2069
PATENT201612008,

capitalensis (Genus +


NO: 58





PATENT201612009,
Species)





PATENT201612010,





PATENT201612011,





PATENT201612012


Lachnospiraceae incertae
Ascusb_974
SEQ ID
PATENT201612013

Orpinomyces

Ascusf_1020
SEQ ID


sedis (Family)

NO: 2070

(Genus)

NO: 59



Pseudobutyrivibrio

Ascusb_1069
SEQ ID
PATENT201612011,

Orpinomyces

Ascusf_1103
SEQ ID


(Genus)

NO: 2071
PATENT201612012,
(Genus)

NO: 60





NRRL B-67348



Anaerolinea (Genus)

Ascusb_1074
SEQ ID
PATENT201612005,

Orpinomyces

Ascusf_81
SEQ ID




NO: 2072
PATENT201612007,
(Genus)

NO: 2104





PATENT201612008,





PATENT201612012



Roseburia (Genus)

Ascusb_1293
SEQ ID


Piromyces (Genus)

Ascusf_206
SEQ ID
PATENT201612003




NO: 2073



NO: 2105



Propionibacterium

Ascusb_1367
SEQ ID
PATENT201612007,

Piromyces (Genus)

Ascusf_208
SEQ ID
PATENT201612003


(Genus)

NO: 2074
PATENT201612009,


NO: 2106





PATENT201612012



Clostridium_XIVa

Ascusb_1632
SEQ ID
PATENT201612011,

Piromyces (Genus)

Ascusf_1012
SEQ ID
PATENT201612003


(Cluster)

NO: 2075
PATENT201612012


NO: 2107



Olsenella (Genus)

Ascusb_1674
SEQ ID
PATENT201612001,




NO: 2076
PATENT201612009
















TABLE 3







Bacteria of the present disclosure.









Predicted Closest




Taxa of Isolated
Strain
Sequence


Microbes
Designation
Identifier













Corynebacterium

Ascusb_3
61



Prevotella

Ascusb_50
62



Comamonas

Ascusb_90
63



Clostridium_XIVa

Ascusb_117
64



Hippea

Ascusb_171
65



Anaerovorax

Ascusb_177
66



Clostridium_XIVa

Ascusb_179
67



Rummeliibacillus

Ascusb_224
68



Clostridium_XIVa

Ascusb_234
69


Lachnospiracea_incertae_sedis
Ascusb_274
70



Prevotella

Ascusb_276
71



Anaerovorax

Ascusb_293
72



Pseudoflavonifractor

Ascusb_327
73



Prevotella

Ascusb_337
74



Clostridium_XIVa

Ascusb_357
75



Clostridium_XIVa

Ascusb_357
76



Coprococcus

Ascusb_361
77



Pyramidobacter

Ascusb_388
78



Syntrophococcus

Ascusb_425
79



Prevotella

Ascusb_444
80



Clostridium_XIVa

Ascusb_456
81



Prevotella

Ascusb_492
82



Roseburia

Ascusb_523
83



Clostridium_XIVa

Ascusb_526
84


Lachnospiracea_incertae_sedis
Ascusb_570
85



Clostridium_XIVa

Ascusb_584
86



Acidothermus

Ascusb_605
87



Adlercreutzia

Ascusb_606
88



Prevotella

Ascusb_617
89


Lachnospiracea_incertae_sedis
Ascusb_635
90



Proteiniclasticum

Ascusb_642
91


Lachnospiracea_incertae_sedis
Ascusb_647
92



Anaerovorax

Ascusb_656
93



Prevotella

Ascusb_669
94



Bacteroides

Ascusb_681
95



Clostridium_III

Ascusb_704
96



Prevotella

Ascusb_706
97



Acinetobacter

Ascusb_717
98



Erysipelothrix

Ascusb_752
99



Bacteroides

Ascusb_790
100



Clostridium_XIVa

Ascusb_797
101



Butyrivibrio

Ascusb_802
102



Eubacterium

Ascusb_805
103



Prevotella

Ascusb_828
104



Eubacterium

Ascusb_890
105



Prevotella

Ascusb_909
106


Lachnospiracea_incertae_sedis
Ascusb_924
107



Coprococcus

Ascusb_955
108



Prevotella

Ascusb_958
109



Clostridium_XIVa

Ascusb_980
110



Prevotella

Ascusb_982
111



Catonella

Ascusb_990
112



Methanobrevibacter

Ascusb_993
113



Ruminococcus

Ascusb_1013
114


Lachnospiracea_incertae_sedis
Ascusb_1021
115



Coprococcus

Ascusb_1033
116



Clostridium_XIVa

Ascusb_1090
117


Lachnospiracea_incertae_sedis
Ascusb_1108
118



Prevotella

Ascusb_1113
119



Anaerovorax

Ascusb_1114
120



Asteroleplasma

Ascusb_1116
121



Clostridium_XIVa

Ascusb_1118
122



Caulobacter

Ascusb_1123
123


Lachnospiracea_incertae_sedis
Ascusb_1128
124



Roseburia

Ascusb_1152
125



Clostridium_XIVa

Ascusb_1166
126



Acinetobacter

Ascusb_1170
127



Bacteroides

Ascusb_1176
128



Erysipelothrix

Ascusb_1182
129



Coprococcus

Ascusb_1199
130



Clostridium_XIVa

Ascusb_1201
131



Bacteroides

Ascusb_1218
132



Coprococcus

Ascusb_1239
133



Anaerovorax

Ascusb_1269
134



Pseudoflavonifractor

Ascusb_1296
135



Pseudoflavonifractor

Ascusb_1296
136



Prevotella

Ascusb_1298
137


Lachnospiracea_incertae_sedis
Ascusb_1304
138



Roseburia

Ascusb_1320
139



Prevotella

Ascusb_1330
140



Coprococcus

Ascusb_955
108



Prevotella

Ascusb_958
109



Clostridium

XIVa

Ascusb_980
110



Prevotella

Ascusb_982
111



Catonella

Ascusb_990
112



Methanobrevibacter

Ascusb_993
113



Ruminococcus

Ascusb_1013
114


Lachnospiracea_incertae_sedis
Ascusb_1021
115



Coprococcus

Ascusb_1033
116



Clostridium_XIVa

Ascusb_1090
117


Lachnospiracea_incertae_sedis
Ascusb_1108
118



Prevotella

Ascusb_1113
119



Anaerovorax

Ascusb_1114
120



Asteroleplasma

Ascusb_1116
121



Clostridium_XIVa

Ascusb_1118
122



Caulobacter

Ascusb_1123
123


Lachnospiracea_incertae_sedis
Ascusb_1128
124



Roseburia

Ascusb_1152
125



Clostridium_XIVa

Ascusb_1166
126



Acinetobacter

Ascusb_1170
127



Bacteroides

Ascusb_1176
128



Erysipelothrix

Ascusb_1182
129



Coprococcus

Ascusb_1199
130



Clostridium_XIVa

Ascusb_1201
131



Bacteroides

Ascusb_1218
132



Coprococcus

Ascusb_1239
133



Anaerovorax

Ascusb_1269
134



Pseudoflavonifractor

Ascusb_1296
135



Pseudoflavonifractor

Ascusb_1296
136



Prevotella

Ascusb_1298
137


Lachnospiracea_incertae_sedis
Ascusb_1304
138



Roseburia

Ascusb_1320
139



Prevotella

Ascusb_1330
140



Ruminococcus

Ascusb_1336
141



Atopobium

Ascusb_1341
142



Eubacterium

Ascusb_1347
143



Robinsoniella

Ascusb_1355
144



Neisseria

Ascusb_1357
145



Ruminococcus

Ascusb_1362
146



Prevotella

Ascusb_1364
147



Slackia

Ascusb_1389
148



Prevotella

Ascusb_1400
149



Clostridium_XIVa

Ascusb_1410
150



Bacteroides

Ascusb_1417
151



Anaerorhabdus

Ascusb_1426
152



Bacteroides

Ascusb_1433
153



Prevotella

Ascusb_1439
154



Corynebacterium

Ascusb_1440
155



Atopobium

Ascusb_1468
156



Streptophyta

Ascusb_1473
157



Prevotella

Ascusb_1485
158



Roseburia

Ascusb_1490
159



Prevotella

Ascusb_1492
160



Prevotella

Ascusb_1528
161



Eubacterium

Ascusb_1538
162



Rhodocista

Ascusb_1543
163



Prevotella

Ascusb_1546
164



Clostridium_XIVa

Ascusb_1553
165



Prevotella

Ascusb_1554
166



Prevotella

Ascusb_1571
167



Streptophyta

Ascusb_1578
168



Ochrobactrum

Ascusb_1580
169



Mogibacterium

Ascusb_1591
170



Adlercreutzia

Ascusb_1600
171



Prevotella

Ascusb_1609
172



Riemerella

Ascusb_1627
173



Prevotella

Ascusb_1640
174



Roseburia

Ascusb_1645
175



Slackia

Ascusb_1647
176



Clostridium_IV

Ascusb_1656
177



Syntrophococcus

Ascusb_1659
178



Prevotella

Ascusb_1667
179



Treponema

Ascusb_1689
180



Prevotella

Ascusb_1708
181



Anaerovorax

Ascusb_1723
182



Prevotella

Ascusb_1727
183



Methanobrevibacter

Ascusb_1739
184



Corynebacterium

Ascusb_1773
185



Clostridium_XIVa

Ascusb_1793
186



Alkaliphilus

Ascusb_1795
187



Ruminococcus

Ascusb_1797
188



Clostridium_XIVa

Ascusb_1806
189



Eubacterium

Ascusb_1819
190



Bacteroides

Ascusb_1835
191



Roseburia

Ascusb_1886
192



Lentisphaera

Ascusb_1901
193



Eubacterium

Ascusb_1905
194



Roseburia

Ascusb_1918
195



Clostridium_IV

Ascusb_1922
196



Hahella

Ascusb_1947
197



Butyricicoccus

Ascusb_1969
198



Clostridium

IV

Ascusb_2016
199



Prevotella

Ascusb_2024
200



Clostridium_IV

Ascusb_2058
201



Desulfovibrio

Ascusb_2081
202



Sphingobacterium

Ascusb_2101
203



Roseburia

Ascusb_2105
204



Bacteroides

Ascusb_2131
205



Ruminococcus

Ascusb_2141
206



Prevotella

Ascusb_2156
207



Asteroleplasma

Ascusb_2168
208



Syntrophococcus

Ascusb_2182
209



Victivallis

Ascusb_2199
210



Lachnobacterium

Ascusb_2210
211


Lachnospiracea_incertae_sedis
Ascusb_2211
212



Clostridium_IV

Ascusb_2218
213



Anaerorhabdus

Ascusb_2221
214



Altererythrobacter

Ascusb_2236
215



Clostridium_XIVa

Ascusb_2246
216



Clostridium_XIVa

Ascusb_2263
217



Proteiniclasticum

Ascusb_2264
218



Bifidobacterium

Ascusb_2308
219



Clostridium_XIVa

Ascusb_2322
220



Clostridium_XIVa

Ascusb_2323
221



Desulfovibrio

Ascusb_2332
222



Clostridium_XIVa

Ascusb_2353
223



Nitrobacter

Ascusb_2375
224



Enterorhabdus

Ascusb_2414
225



Clostridium_sensu_stricto

Ascusb_2429
226



Oscillibacter

Ascusb_2435
227



Nautilia

Ascusb_2437
228



Corynebacterium

Ascusb_2447
229



Ruminococcus

Ascusb_2452
230



Coprococcus

Ascusb_2461
231



Eubacterium

Ascusb_2462
232



Rikenella

Ascusb_2470
233



Clostridium_XIVa

Ascusb_2482
234



Paenibacillus

Ascusb_2487
235



Ruminococcus

Ascusb_2492
236



Prevotella

Ascusb_2503
237



Haematobacter

Ascusb_2504
238



Prevotella

Ascusb_2523
239



Clostridium_XIVa

Ascusb_2537
240


Lachnospiracea_incertae_sedis
Ascusb_2538
241



Enterorhabdus

Ascusb_2565
242



Blautia

Ascusb_2591
243



Sporobacter

Ascusb_2592
244



Oscillibacter

Ascusb_2607
245



Clostridium_XIVa

Ascusb_2608
246



Atopobium

Ascusb_2613
247



Sporobacter

Ascusb_2626
248



Clostridium_XIVa

Ascusb_2629
249


Candidate Phylum OD1
Ascusb_2643
250



Oscillibacter

Ascusb_2645
251



Clostridium_XIVa

Ascusb_2647
252



Clostridium_IV

Ascusb_2649
253



Mogibacterium

Ascusb_2653
254



Roseburia

Ascusb_2663
255


Lachnospiracea_incertae_sedis
Ascusb_2671
256



Pelotomaculum

Ascusb_2696
257



Pelotomaculum

Ascusb_2712
258



Clostridium_XIVa

Ascusb_2713
259



Robinsoniella

Ascusb_2730
260



Coprococcus

Ascusb_2746
261



Wautersiella

Ascusb_2757
262


Lachnospiracea_incertae_sedis
Ascusb_2762
263



Planctomyces

Ascusb_2764
264



Treponema

Ascusb_2800
265



Coprococcus

Ascusb_2806
266



Paracoccus

Ascusb_2809
267



Ruminococcus

Ascusb_2811
268



Atopobium

Ascusb_2814
269



Prevotella

Ascusb_2825
270



Clostridium_IV

Ascusb_2832
271



Clostridium_XIVa

Ascusb_2838
272



Clostridium_XIVa

Ascusb_2843
273



Clostridium_XIVa

Ascusb_2853
274



Prevotella

Ascusb_2857
275



Dethiosulfovibrio

Ascusb_2872
276



Clostridium_XI

Ascusb_2885
277



Clostridium_IV

Ascusb_2907
278



Saccharofermentans

Ascusb_2909
279



Clostridium_sensu_stricto

Ascusb_2912
280



Roseburia

Ascusb_2914
281


Lachnospiracea_incertae_sedis
Ascusb_2930
282


Candidate phylum SR1
Ascusb_2946
283



Hydrogenoanaerobacterium

Ascusb_2948
284



Victivallis

Ascusb_2966
285



Clostridium_IV

Ascusb_2983
286



Pelotomaculum

Ascusb_2988
287



Clostridium_XIVa

Ascusb_2990
288



Saccharofermentans

Ascusb_3005
289


Lachnospiracea_incertae_sedis
Ascusb_3008
290



Coprococcus

Ascusb_3010
291



Clostridium_XIVa

Ascusb_3022
292



Clostridium_XIVb

Ascusb_3029
293



Papillibacter

Ascusb_3053
294



Bartonella

Ascusb_3056
295



Clostridium_IV

Ascusb_3058
296



Eubacterium

Ascusb_3061
297



Asaccharobacter

Ascusb_3066
298



Clostridium_IV

Ascusb_3073
299



Blautia

Ascusb_3074
300



Prevotella

Ascusb_3079
301



Ruminococcus

Ascusb_3087
302



Selenomonas

Ascusb_3120
303



Treponema

Ascusb_3142
304



Adlercreutzia

Ascusb_3147
305



Butyricicoccus

Ascusb_3161
306



Pseudoflavonifractor

Ascusb_3163
307



Corynebacterium

Ascusb_3165
308



Adlercreutzia

Ascusb_3188
309



Selenomonas

Ascusb_3197
310



Coraliomargarita

Ascusb_3213
311



Paraprevotella

Ascusb_3225
312



Oscillibacter

Ascusb_3229
313



Anaerovorax

Ascusb_3240
314



Clostridium_XIVa

Ascusb_3242
315



Saccharofermentans

Ascusb_3248
316



Erysipelothrix

Ascusb_3263
317



Agaricicola

Ascusb_3275
318



Denitrobacterium

Ascusb_3285
319



Armatimonadetes

Ascusb_3299
320



Asaccharobacter

Ascusb_3304
321



Anaeroplasma

Ascusb_3322
322



Prevotella

Ascusb_3333
323


Lachnospiracea_incertae_sedis
Ascusb_3339
324



Clostridium_IV

Ascusb_3351
325



Streptococcus

Ascusb_3376
326



Cellulosilyticum

Ascusb_3393
327



Asaccharobacter

Ascusb_3405
328



Enterorhabdus

Ascusb_3408
329



Treponema

Ascusb_3415
330



Roseburia

Ascusb_3417
331



Victivallis

Ascusb_3422
332



Prevotella

Ascusb_3424
333



Roseburia

Ascusb_3446
334



Ruminococcus

Ascusb_3451
335



Mogibacterium

Ascusb_3456
336


Lachnospiracea_incertae_sedis
Ascusb_3467
337



Prevotella

Ascusb_3479
338



Clostridium_sensu_stricto

Ascusb_3480
339



Victivallis

Ascusb_3481
340


Cyanobacteria
Ascusb_3482
341



Treponema

Ascusb_3483
342



Stenotrophomonas

Ascusb_3484
343



Ascusb_3492
344



Clostridium_XIVa

Ascusb_3494
345



Sphingobium

Ascusb_3495
346


Lachnospiracea_incertae_sedis
Ascusb_3512
347



Oscillibacter

Ascusb_3518
348



Methylobacterium

Ascusb_3523
349



Zhangella

Ascusb_3530
350


Lachnospiracea_incertae_sedis
Ascusb_3545
351



Oscillibacter

Ascusb_3546
352



Clostridium_III

Ascusb_3548
353



Coraliomargarita

Ascusb_3563
354



Eubacterium

Ascusb_3575
355



Enterorhabdus

Ascusb_3578
356



Clostridium_XIVa

Ascusb_3587
357



Saccharofermentans

Ascusb_3592
358



Clostridium_IV

Ascusb_3600
359



Clostridium_sensu_stricto

Ascusb_3602
360



Victivallis

Ascusb_3638
361



Coprococcus

Ascusb_3642
362



Pseudoflavonifractor

Ascusb_3647
363



Anaeroplasma

Ascusb_3674
364



Anaeroplasma

Ascusb_3687
365



Bacteroides

Ascusb_3700
366



Acinetobacter

Ascusb_3717
367



Victivallis

Ascusb_3724
368



Victivallis

Ascusb_3725
369



Mogibacterium

Ascusb_3728
370



Oscillibacter

Ascusb_3746
371



Butyricimonas

Ascusb_3748
372



Dethiosulfovibrio

Ascusb_3750
373



Pseudoflavonifractor

Ascusb_3751
374



Clostridium_IV

Ascusb_3762
375



Anaeroplasma

Ascusb_3763
376



Oscillibacter

Ascusb_3768
377



Herbiconiux

Ascusb_3775
378



Eubacterium

Ascusb_3779
379



Armatimonadetes

Ascusb_3789
380



Selenomonas

Ascusb_3796
381



Clostridium_IV

Ascusb_3811
382



Mogibacterium

Ascusb_3825
383



Clostridium_IV

Ascusb_3838
384



Roseburia

Ascusb_3849
385



Anaerovibrio

Ascusb_3866
386



Clostridium_III

Ascusb_3875
387



Saccharofermentans

Ascusb_3903
388



Saccharofermentans

Ascusb_3911
389



Prevotella

Ascusb_3914
390



Clostridium_XIVa

Ascusb_3919
391



Robinsoniella

Ascusb_3950
392



Brevundimonas

Ascusb_3952
393



Anaerotruncus

Ascusb_3970
394



Victivallis

Ascusb_3982
395



Bacteroides

Ascusb_4008
396



Clostridium_XIVb

Ascusb_4019
397



Prevotella

Ascusb_4033
398



Ruminococcus

Ascusb_4034
399



Pelobacter

Ascusb_4040
400



Clostridium_XIVa

Ascusb_4063
401



Clostridium_XIVa

Ascusb_4067
402



Clostridium_XIVb

Ascusb_4083
403



Coprococcus

Ascusb_4085
404



Clostridium_IV

Ascusb_4086
405



Clostridium_IV

Ascusb_4095
406



Coprococcus

Ascusb_4114
407



Victivallis

Ascusb_4115
408



Clostridium_III

Ascusb_4118
409



Anaerovibrio

Ascusb_4120
410



Anaerovorax

Ascusb_4124
411



Proteiniclasticum

Ascusb_4142
412



Anaerovorax

Ascusb_4143
413



Selenomonas

Ascusb_4149
414



Hydrogenoanaerobacterium

Ascusb_4155
415



Acetanaerobacterium

Ascusb_4156
416



Clostridium_XIVa

Ascusb_4159
417



Asaccharobacter

Ascusb_4161
418



Clostridium_XIVa

Ascusb_4167
419


Lachnospiracea_incertae_sedis
Ascusb_4171
420



Saccharofermentans

Ascusb_4172
421



Prevotella

Ascusb_4176
422



Anaeroplasma

Ascusb_4179
423



Spirochaeta

Ascusb_4188
424



Alkaliphilus

Ascusb_4213
425



Paraprevotella

Ascusb_4215
426



Hippea

Ascusb_4217
427



Prevotella

Ascusb_4223
428



Prevotella

Ascusb_4237
429



Hydrogenoanaerobacterium

Ascusb_4241
430



Clostridium_sensu_stricto

Ascusb_4265
431



Paraeggerthella

Ascusb_4266
432



Clostridium_XIVa

Ascusb_4277
433



Clostridium_XIVa

Ascusb_4279
434



Clostridium_IV

Ascusb_4281
435



Clostridium_XIVa

Ascusb_4292
436



Adhaeribacter

Ascusb_4313
437



Syntrophococcus

Ascusb_4316
438



Clostridium_sensu_stricto

Ascusb_4317
439



Saccharofermentans

Ascusb_4326
440



Clostridium_IV

Ascusb_4332
441



Clostridium_IV

Ascusb_4345
442



Clostridium_sensu_stricto

Ascusb_4347
443



Coraliomargarita

Ascusb_4375
444



Sharpea

Ascusb_4380
445



Clostridium_IV

Ascusb_4394
446



Anaerovorax

Ascusb_4416
447



Blautia

Ascusb_4421
448



Clostridium_XIVa

Ascusb_4422
449



Clostridium_IV

Ascusb_4432
450



Anaerovorax

Ascusb_4433
451



Coraliomargarita

Ascusb_4434
452


Lachnospiracea_incertae_sedis
Ascusb_4442
453



Aquiflexum

Ascusb_4449
454



Pedobacter

Ascusb_4450
455



Robinsoniella

Ascusb_4457
456



Pelomonas

Ascusb_4468
457



Saccharofermentans

Ascusb_4469
458



Paracoccus

Ascusb_4479
459



Enterorhabdus

Ascusb_4486
460



Beijerinckia

Ascusb_4496
461



Sporobacter

Ascusb_4505
462



Clostridium_IV

Ascusb_4517
463



Bacillus

Ascusb_4522
464



Saccharofermentans

Ascusb_4537
465



Spirochaeta

Ascusb_4545
466



Prevotella

Ascusb_4548
467



Eubacterium

Ascusb_4556
468



Herbiconiux

Ascusb_4559
469



Brevundimonas

Ascusb_4560
470



Mogibacterium

Ascusb_4563
471



Anaerorhabdus

Ascusb_4566
472



Victivallis

Ascusb_4569
473



Prevotella

Ascusb_4573
474



Anaerovorax

Ascusb_4579
475



Aquiflexum

Ascusb_4606
476



Oscillibacter

Ascusb_4618
477



Altererythrobacter

Ascusb_4626
478



Hydrogenoanaerobacterium

Ascusb_4627
479



Clostridium_III

Ascusb_4634
480



Clostridium_XIVb

Ascusb_4639
481



Saccharofermentans

Ascusb_4644
482



Roseburia

Ascusb_4652
483



Anaeroplasma

Ascusb_4657
484



Planctomyces

Ascusb_4676
485



Ruminococcus

Ascusb_4679
486



Selenomonas

Ascusb_4695
487



Anaeroplasma

Ascusb_4696
488



Anaerovorax

Ascusb_4700
489



Rummeliibacillus

Ascusb_4701
490



Clostridium_XIVa

Ascusb_4716
491



Anaeroplasma

Ascusb_4731
492



Butyrivibrio

Ascusb_4737
493


Lachnospiracea_incertae_sedis
Ascusb_4738
494



Anaerotruncus

Ascusb_4758
495



Syntrophococcus

Ascusb_4763
496



Paraeggerthella

Ascusb_4795
497



Papillibacter

Ascusb_4800
498


Lachnospiracea_incertae_sedis
Ascusb_4805
499



Prevotella

Ascusb_4820
500



Papillibacter

Ascusb_4828
501



Streptococcus

Ascusb_4852
502



Methanobrevibacter

Ascusb_4859
503



Prevotella

Ascusb_4861
504



Prevotella

Ascusb_4867
505



Prevotella

Ascusb_4873
506



Coraliomargarita

Ascusb_4882
507



Prevotella

Ascusb_4886
508



Thermotalea

Ascusb_4893
509



Clostridium_XIVa

Ascusb_4897
510



Atopobium

Ascusb_4945
511



Prevotella

Ascusb_4969
512



Mogibacterium

Ascusb_4972
513



Clostridium_XIVa

Ascusb_4976
514



Clostridium_XIVa

Ascusb_4997
515



Eggerthella

Ascusb_4999
516



Blautia

Ascusb_5000
517



Vampirovibrio

Ascusb_5006
518



Papillibacter

Ascusb_5040
519



Beijerinckia

Ascusb_5058
520



Bacteroides

Ascusb_5060
521



Desulfotomaculum

Ascusb_5065
522


Acidobacteria
Ascusb_5069
523



Clostridium_XIVa

Ascusb_5081
524



Clostridium_XIVa

Ascusb_5089
525



Clostridium_XIVa

Ascusb_5095
526



Cryptanaerobacter

Ascusb_5103
527



Prevotella

Ascusb_5113
528



Syntrophomonas

Ascusb_5137
529



Erysipelothrix

Ascusb_5144
530



Selenomonas

Ascusb_5165
531



Clostridium_III

Ascusb_5171
532



Flavobacterium

Ascusb_5181
533



Thermotalea

Ascusb_5191
534


Lachnospiracea_incertae_sedis
Ascusb_5194
535



Mucilaginibacter

Ascusb_5197
536



Bacteroides

Ascusb_5198
537



Ruminococcus

Ascusb_5206
538



Clostridium_XIVa

Ascusb_5223
539



Asaccharobacter

Ascusb_5225
540



Blautia

Ascusb_5235
541



Mucilaginibacter

Ascusb_5247
542



Coprococcus

Ascusb_5252
543


Lachnospiracea_incertae_sedis
Ascusb_5253
544



Butyricimonas

Ascusb_5255
545


Lachnospiracea_incertae_sedis
Ascusb_5267
546



Treponema

Ascusb_5280
547



Clostridium_sensu_stricto

Ascusb_5281
548



Clostridium_XIVa

Ascusb_5289
549



Anaerovorax

Ascusb_5292
550



Saccharofermentans

Ascusb_5294
551



Clostridium_XIVa

Ascusb_5295
552



Clostridium_III

Ascusb_5301
553



Clostridium_IV

Ascusb_5313
554



Ruminococcus

Ascusb_5324
555



Clostridium_XIVa

Ascusb_5326
556



Clostridium_XI

Ascusb_5335
557



Clostridium_XIVa

Ascusb_5336
558



Eubacterium

Ascusb_5338
559


Lachnospiracea_incertae_sedis
Ascusb_5342
560



Clostridium_IV

Ascusb_5352
561



Ruminococcus

Ascusb_5353
562



Clostridium_IV

Ascusb_5354
563



Faecalibacterium

Ascusb_5360
564



Anaerovibrio

Ascusb_5368
565



Asaccharobacter

Ascusb_5397
566



Pelotomaculum

Ascusb_5411
567



Spirochaeta

Ascusb_5422
568



Prevotella

Ascusb_5429
569


Lachnospiracea_incertae_sedis
Ascusb_5440
570



Anaerovorax

Ascusb_5441
571



Clostridium_IV

Ascusb_5443
572



Victivallis

Ascusb_5451
573



Syntrophococcus

Ascusb_5456
574



Syntrophococcus

Ascusb_5463
575



Desulfovibrio

Ascusb_5481
576


Lachnospiracea_incertae_sedis
Ascusb_5485
577


Lachnospiracea_incertae_sedis
Ascusb_5495
578



Clostridium_IV

Ascusb_5509
579



Prevotella

Ascusb_5510
580



Victivallis

Ascusb_5512
581



Clostridium_XIVa

Ascusb_5515
582



Selenomonas

Ascusb_5517
583



Bacteroides

Ascusb_5530
584



Clostridium_XIVa

Ascusb_5536
585



Eggerthella

Ascusb_5554
586



Selenomonas

Ascusb_5584
587



Mogibacterium

Ascusb_5592
588



Armatimonadetes

Ascusb_5609
589



Clostridium_XIVa

Ascusb_5612
590



Victivallis

Ascusb_5623
591



Paraprevotella

Ascusb_5628
592



Brevundimonas

Ascusb_5647
593



Prevotella

Ascusb_5650
594



Prevotella

Ascusb_5652
595



Robinsoniella

Ascusb_5660
596



Clostridium_III

Ascusb_5686
597



Butyricimonas

Ascusb_5689
598



Spirochaeta

Ascusb_5691
599



Hydrogenoanaerobacterium

Ascusb_5694
600



Proteiniclasticum

Ascusb_5716
601



Roseburia

Ascusb_5725
602



Clostridium_XIVa

Ascusb_5738
603



Anaerofustis

Ascusb_5746
604



Succiniclasticum

Ascusb_5765
605



Anaeroplasma

Ascusb_5770
606



Oscillibacter

Ascusb_5777
607



Escherichia/Shigella

Ascusb_5789
608



Bacteroides

Ascusb_5812
609



Clostridium_XIVa

Ascusb_5830
610



Clostridium_XIVa

Ascusb_5838
611



Clostridium_IV

Ascusb_5841
612



Clostridium_III

Ascusb_5845
613



Prevotella

Ascusb_5847
614



Coprococcus

Ascusb_5849
615



Oscillibacter

Ascusb_5858
616



Parabacteroides

Ascusb_5862
617



Bacteroides

Ascusb_5868
618



Mogibacterium

Ascusb_5869
619



Solobacterium

Ascusb_5870
620



Bacteroides

Ascusb_5874
621



Clostridium_III

Ascusb_5877
622



Victivallis

Ascusb_5879
623



Saccharofermentans

Ascusb_5884
624



Saccharofermentans

Ascusb_5889
625



Olivibacter

Ascusb_5894
626



Thermotalea

Ascusb_5895
627



Proteiniclasticum

Ascusb_5913
628



Clostridium_III

Ascusb_5926
629



Anaeroplasma

Ascusb_5934
630



Treponema

Ascusb_5939
631



Clostridium_XIVa

Ascusb_5940
632



Clostridium_III

Ascusb_5950
633



Desulfotomaculum

Ascusb_5953
634



Bacillus

Ascusb_5969
635



Anaerovorax

Ascusb_5972
636



Ruminococcus

Ascusb_5973
637



Agarivorans

Ascusb_5975
638



Anaerotruncus

Ascusb_5979
639



Papillibacter

Ascusb_5984
640



Clostridium_XIVa

Ascusb_5991
641



Clostridium_III

Ascusb_5996
642



Bacteroides

Ascusb_5997
643



Clostridium_XIVa

Ascusb_5998
644



Ruminococcus

Ascusb_6003
645



Clostridium_XIVa

Ascusb_6005
646



Oscillibacter

Ascusb_6006
647



Nitrobacter

Ascusb_6022
648



Clostridium_XIVa

Ascusb_6026
649


Lachnospiracea_incertae_sedis
Ascusb_6035
650



Limibacter

Ascusb_6037
651



Desulfovibrio

Ascusb_6053
652



Coprococcus

Ascusb_6067
653



Anaerovorax

Ascusb_6070
654



Spirochaeta

Ascusb_6074
655


Cyanobacteria
Ascusb_6079
656



Saccharofermentans

Ascusb_6081
657



Anaeroplasma

Ascusb_6106
658



Clostridium_III

Ascusb_6115
659



Victivallis

Ascusb_6151
660



Enterorhabdus

Ascusb_6168
661



Clostridium_IV

Ascusb_6169
662



Erysipelothrix

Ascusb_6172
663



Clostridium_III

Ascusb_6200
664



Clostridium_sensu_stricto

Ascusb_6207
665



Gelidibacter

Ascusb_6212
666



Roseburia

Ascusb_6219
667



Neisseria

Ascusb_6270
668



Prevotella

Ascusb_6273
669


Cyanobacteria
Ascusb_6275
670



Oscillibacter

Ascusb_6282
671


Candidate phylum TM7
Ascusb_6313
672



Prevotella

Ascusb_6326
673



Saccharofermentans

Ascusb_6330
674


Erysipelotrichaceae_incertae_sedis
Ascusb_6337
675



Spirochaeta

Ascusb_6342
676



Clostridium_XIVa

Ascusb_6372
677



Clostridium_XIVb

Ascusb_6376
678



Clostridium_XIVa

Ascusb_6387
679



Adlercreutzia

Ascusb_6389
680



Clostridium_XIVa

Ascusb_6394
681


Lachnospiracea_incertae_sedis
Ascusb_6400
682



Clostridium_IV

Ascusb_6403
683



Adlercreutzia

Ascusb_6406
684



Prevotella

Ascusb_6409
685



Syntrophococcus

Ascusb_6420
686



Treponema

Ascusb_6433
687



Prevotella

Ascusb_6448
688



Clostridium_III

Ascusb_6450
689



Pseudoflavonifractor

Ascusb_6463
690



Clostridium_IV

Ascusb_6468
691



Sharpea

Ascusb_6473
692



Dongia

Ascusb_6499
693



Eubacterium

Ascusb_6505
694



Prevotella

Ascusb_6507
695



Clostridium_IV

Ascusb_6519
696



Parabacteroides

Ascusb_6525
697



Brevundimonas

Ascusb_6535
698



Clostridium_XIVa

Ascusb_6540
699



Ruminococcus

Ascusb_6541
700



Thermotalea

Ascusb_6558
701



Victivallis

Ascusb_6561
702



Anaeroplasma

Ascusb_6563
703



Oscillibacter

Ascusb_6564
704



Ruminococcus

Ascusb_6570
705



Clostridium_XIVa

Ascusb_6578
706



Clostridium_XIVa

Ascusb_6581
707



Clostridium_IV

Ascusb_6586
708



Roseburia

Ascusb_6593
709



Eggerthella

Ascusb_6612
710



Clostridium_III

Ascusb_6614
711



Clostridium_XIVa

Ascusb_6621
712



Lactobacillus

Ascusb_6630
713



Bacteroides

Ascusb_6633
714



Cellulosilyticum

Ascusb_6635
715



Brevundimonas

Ascusb_6645
716



Clostridium_IV

Ascusb_6670
717



Prevotella

Ascusb_6672
718



Helicobacter

Ascusb_6676
719



Clostridium_IV

Ascusb_6683
720



Proteiniclasticum

Ascusb_6684
721



Brevundimonas

Ascusb_6701
722



Clostridium_XIVa

Ascusb_6704
723



Prevotella

Ascusb_6706
724



Desulfovibrio

Ascusb_6708
725



Coraliomargarita

Ascusb_6709
726



Eubacterium

Ascusb_6715
727



Sphingomonas

Ascusb_6718
728



Prevotella

Ascusb_6730
729



Clostridium_IV

Ascusb_6734
730



Paraprevotella

Ascusb_6735
731



Ruminococcus

Ascusb_6746
732



Saccharofermentans

Ascusb_6756
733



Clostridium_III

Ascusb_6757
734



Clostridium_III

Ascusb_6774
735



Turicibacter

Ascusb_6792
736



Prevotella

Ascusb_6796
737



Clostridium_XIVa

Ascusb_6803
738



Fusibacter

Ascusb_6813
739



Clostridium_XIVa

Ascusb_6824
740



Clostridium_IV

Ascusb_6833
741



Rummeliibacillus

Ascusb_6848
742



Mogibacterium

Ascusb_6852
743



Bacteroides

Ascusb_6864
744



Pelospora

Ascusb_6875
745



Eggerthella

Ascusb_6880
746



Eubacterium

Ascusb_6887
747



Blautia

Ascusb_6889
748



Clostridium_XIVb

Ascusb_6901
749



Ehrlichia

Ascusb_6907
750



Eubacterium

Ascusb_6930
751



Prevotella

Ascusb_6943
752



Clostridium_XIVa

Ascusb_6952
753



Treponema

Ascusb_6954
754



Hydrogenoanaerobacterium

Ascusb_6957
755



Selenomonas

Ascusb_6964
756



Saccharofermentans

Ascusb_6966
757



Clostridium_IV

Ascusb_6971
758



Clostridium_sensu_stricto

Ascusb_6976
759



Anaerovorax

Ascusb_6979
760



Spirochaeta

Ascusb_6997
761



Brevundimonas

Ascusb_7001
762



Eubacterium

Ascusb_7017
763



Clostridium_XIVa

Ascusb_7025
764



Anaerovorax

Ascusb_7031
765



Ruminococcus

Ascusb_7039
766



Papillibacter

Ascusb_7040
767



Clostridium_IV

Ascusb_7043
768



Hydrogenoanaerobacterium

Ascusb_7046
769



Asaccharobacter

Ascusb_7048
770



Clostridium_XIVa

Ascusb_7054
771



Rhodocista

Ascusb_7078
772



Clostridium_XIVa

Ascusb_7087
773



Beijerinckia

Ascusb_7091
774



Lactobacillus

Ascusb_7101
775



Cryptanaerobacter

Ascusb_7102
776



Prevotella

Ascusb_7113
777



Anaerovibrio

Ascusb_7114
778



Anaerovorax

Ascusb_7123
779


Lachnospiracea_incertae_sedis
Ascusb_7128
780



Enterorhabdus

Ascusb_7131
781



Clostridium_XIVb

Ascusb_7141
782



Selenomonas

Ascusb_7148
783



Eubacterium

Ascusb_7149
784



Thermotalea

Ascusb_7151
785



Enterorhabdus

Ascusb_7153
786



Clostridium_III

Ascusb_7159
787



Acetanaerobacterium

Ascusb_7164
788



Treponema

Ascusb_7168
789



Clostridium_XIVa

Ascusb_7176
790



Enterorhabdus

Ascusb_7180
791



Prevotella

Ascusb_7188
792



Desulfovibrio

Ascusb_7199
793



Aminobacter

Ascusb_7213
794



Clostridium_IV

Ascusb_7224
795



Rikenella

Ascusb_7225
796



Gordonibacter

Ascusb_7240
797



Papillibacter

Ascusb_7245
798



Syntrophococcus

Ascusb_7246
799



Clostridium_sensu_stricto

Ascusb_7256
800



Hahella

Ascusb_7257
801



Vampirovibrio

Ascusb_7264
802



Coprococcus

Ascusb_7275
803



Coraliomargarita

Ascusb_7299
804



Clostridium_III

Ascusb_7300
805



Clostridium_XIVa

Ascusb_7304
806



Desulfotomaculum

Ascusb_7325
807



Helicobacter

Ascusb_7373
808



Syntrophococcus

Ascusb_7380
809


Lachnospiracea_incertae_sedis
Ascusb_7384
810



Clostridium_IV

Ascusb_7385
811



Paludibacter

Ascusb_7395
812


Lachnospiracea_incertae_sedis
Ascusb_7401
813


Lachnospiracea_incertae_sedis
Ascusb_7412
814



Adhaeribacter

Ascusb_7419
815



Clostridium_IV

Ascusb_7420
816



Cryptanaerobacter

Ascusb_7424
817



Idiomarina

Ascusb_7435
818



Clostridium_IV

Ascusb_7437
819



Selenomonas

Ascusb_7440
820



Acetanaerobacterium

Ascusb_7444
821



Bifidobacterium

Ascusb_7446
822



Clostridium_XIVb

Ascusb_7449
823



Asaccharobacter

Ascusb_7450
824



Eubacterium

Ascusb_7452
825



Anaeroplasma

Ascusb_7455
826



Saccharofermentans

Ascusb_7456
827



Ruminococcus

Ascusb_7467
828



Clostridium_III

Ascusb_7470
829



Acholeplasma

Ascusb_7472
830



Pedobacter

Ascusb_7476
831



Sphingomonas

Ascusb_7487
832



Verrucomicrobia

Ascusb_7525
833



Anaerovorax

Ascusb_7533
834



Spirochaeta

Ascusb_7534
835



Paraeggerthella

Ascusb_7539
836


Lachnospiracea_incertae_sedis
Ascusb_7542
837



Bacteroides

Ascusb_7543
838



Paenibacillus

Ascusb_7549
839



Prevotella

Ascusb_7553
840



Bacteroides

Ascusb_7555
841



Clostridium_XIVa

Ascusb_7563
842



Clostridium_XIVa

Ascusb_7568
843



Roseburia

Ascusb_7572
844



Clostridium_XIVa

Ascusb_7581
845



Clostridium_III

Ascusb_7591
846



Pedobacter

Ascusb_7599
847



Robinsoniella

Ascusb_7614
848



Anaeroplasma

Ascusb_7615
849



Clostridium_XIVa

Ascusb_7622
850



Hydrogenoanaerobacterium

Ascusb_7626
851



Turicibacter

Ascusb_7638
852



Papillibacter

Ascusb_7645
853



Clostridium_XIVa

Ascusb_7647
854



Saccharofermentans

Ascusb_7648
855



Clostridium_XIVb

Ascusb_7650
856



Sporobacter

Ascusb_7662
857



Asaccharobacter

Ascusb_7663
858



Bacteroides

Ascusb_7669
859



Anaeroplasma

Ascusb_7677
860



Sporobacter

Ascusb_7680
861



Streptomyces

Ascusb_7690
862



Arcobacter

Ascusb_7694
863



Clostridium_XIVa

Ascusb_7699
864



Barnesiella

Ascusb_7706
865



Lactobacillus

Ascusb_7723
866



Flavobacterium

Ascusb_7728
867



Victivallis

Ascusb_7733
868



Clostridium_XIVa

Ascusb_7735
869



Ureaplasma

Ascusb_7748
870



Acetanaerobacterium

Ascusb_7752
871



Slackia

Ascusb_7753
872


Lachnospiracea_incertae_sedis
Ascusb_7761
873



Oscillibacter

Ascusb_7763
874



Prevotella

Ascusb_7765
875



Proteiniphilum

Ascusb_7767
876



Spirochaeta

Ascusb_7784
877



Ruminococcus

Ascusb_7788
878



Prevotella

Ascusb_7792
879



Butyricicoccus

Ascusb_7796
880



Devosia

Ascusb_7817
881



Anaeroplasma

Ascusb_7828
882



Oscillibacter

Ascusb_7829
883



Barnesiella

Ascusb_7831
884



Atopobium

Ascusb_7837
885



Clostridium_XIVa

Ascusb_7838
886



Methanobrevibacter

Ascusb_7839
887



Butyricimonas

Ascusb_7849
888



Butyricimonas

Ascusb_7853
889



Asaccharobacter

Ascusb_7855
890



Enhydrobacter

Ascusb_7871
891



Treponema

Ascusb_7872
892



Clostridium_XIVa

Ascusb_7873
893



Adlercreutzia

Ascusb_7874
894



Prevotella

Ascusb_7890
895



Pseudoflavonifractor

Ascusb_7896
896



Syntrophococcus

Ascusb_7898
897



Clostridium_IV

Ascusb_7901
898



Demequina

Ascusb_7902
899


Lachnospiracea_incertae_sedis
Ascusb_7904
900



Saccharofermentans

Ascusb_7924
901



Sphaerisporangium

Ascusb_7925
902



Anaeroplasma

Ascusb_7939
903



Geobacillus

Ascusb_7958
904



Prevotella

Ascusb_7959
905



Clostridium_XIVa

Ascusb_7967
906



Victivallis

Ascusb_7973
907



Bacteroides

Ascusb_7989
908



Demequina

Ascusb_7990
909



Paraeggerthella

Ascusb_7994
910



Paraprevotella

Ascusb_7996
911



Pseudoflavonifractor

Ascusb_8013
912



Roseburia

Ascusb_8018
913



Gelidibacter

Ascusb_8038
914



Clostridium_IV

Ascusb_8069
915



Rhizobium

Ascusb_8076
916



Acholeplasma

Ascusb_8081
917



Clostridium_XIVa

Ascusb_8084
918



Bacteroides

Ascusb_8091
919



Bacteroides

Ascusb_8105
920



Papillibacter

Ascusb_8107
921



Fusibacter

Ascusb_8113
922



Coraliomargarita

Ascusb_8120
923



Papillibacter

Ascusb_8123
924



Clostridium_XIVa

Ascusb_8149
925



Acholeplasma

Ascusb_8167
926



Catenibacterium

Ascusb_8169
927



Clostridium_IV

Ascusb_8172
928



Clostridium_IV

Ascusb_8173
929



Clostridium_IV

Ascusb_8179
930



Nitrobacter

Ascusb_8182
931



Victivallis

Ascusb_8189
932



Selenomonas

Ascusb_8196
933



Enterorhabdus

Ascusb_8200
934



Eubacterium

Ascusb_8202
935



Roseburia

Ascusb_8206
936



Prevotella

Ascusb_8211
937



Asaccharobacter

Ascusb_8222
938



Bacteroides

Ascusb_8230
939



Clostridium_XIVa

Ascusb_8238
940



Gelidibacter

Ascusb_8245
941



Brevundimonas

Ascusb_8254
942



Clostridium_XIVa

Ascusb_8260
943



Prevotella

Ascusb_8266
944



Oscillibacter

Ascusb_8268
945



Asteroleplasma

Ascusb_8280
946



Anaeroplasma

Ascusb_8283
947



Oscillibacter

Ascusb_8311
948



Bilophila

Ascusb_8317
949



Oscillibacter

Ascusb_8318
950



Clostridium_IV

Ascusb_8320
951



Prevotella

Ascusb_8321
952



Geosporobacter

Ascusb_8329
953



Butyricimonas

Ascusb_8363
954



Pseudoflavonifractor

Ascusb_8366
955



Barnesiella

Ascusb_8367
956



Selenomonas

Ascusb_8370
957



Prevotella

Ascusb_8374
958



Enterorhabdus

Ascusb_8379
959



Oscillibacter

Ascusb_8384
960



Pelotomaculum

Ascusb_8394
961



Cellulosilyticum

Ascusb_8396
962



Clostridium_IV

Ascusb_8402
963



Parabacteroides

Ascusb_8410
964



Papillibacter

Ascusb_8413
965



Bacteroides

Ascusb_8439
966



Prevotella

Ascusb_8440
967



Hydrogenoanaerobacterium

Ascusb_8447
968



Clostridium_XIVa

Ascusb_8470
969



Prevotella

Ascusb_8480
970



Clostridium_IV

Ascusb_8484
971



Howardella

Ascusb_8487
972



Slackia

Ascusb_8498
973



Methylobacter

Ascusb_8500
974



Treponema

Ascusb_8508
975



Clostridium_XIVa

Ascusb_8514
976



Devosia

Ascusb_8518
977



Ruminococcus

Ascusb_8537
978


Lachnospiracea_incertae_sedis
Ascusb_8569
979



Clostridium_III

Ascusb_8580
980



Methanobrevibacter

Ascusb_8595
981



Paraprevotella

Ascusb_8600
982



Desulfobulbus

Ascusb_8627
983



Butyricicoccus

Ascusb_8639
984



Clostridium_XIVa

Ascusb_8657
985



Dialister

Ascusb_8669
986



Selenomonas

Ascusb_8681
987



Spirochaeta

Ascusb_8696
988



Clostridium_IV

Ascusb_8712
989



Cellulosilyticum

Ascusb_8713
990



Prevotella

Ascusb_8714
991



Pseudoflavonifractor

Ascusb_8715
992



Clostridium_III

Ascusb_8728
993



Oscillibacter

Ascusb_8733
994



Faecalibacterium

Ascusb_8746
995



Clostridium_XIVb

Ascusb_8753
996



Eubacterium

Ascusb_8758
997



Clostridium_III

Ascusb_8762
998



Prevotella

Ascusb_8769
999



Paenibacillus

Ascusb_8771
1000



Pedobacter

Ascusb_8782
1001



Butyricicoccus

Ascusb_8786
1002



Clostridium_XIVa

Ascusb_8787
1003



Roseburia

Ascusb_8799
1004



Hydrogenoanaerobacterium

Ascusb_8804
1005



Adhaeribacter

Ascusb_8807
1006



Eubacterium

Ascusb_8815
1007



Bacteroides

Ascusb_8822
1008



Victivallis

Ascusb_8835
1009



Roseburia

Ascusb_8840
1010



Treponema

Ascusb_8857
1011



Prevotella

Ascusb_8860
1012



Prevotella

Ascusb_8870
1013



Hydrogenoanaerobacterium

Ascusb_8873
1014



Clostridium_XIVa

Ascusb_8883
1015



Bacteroides

Ascusb_8884
1016



Bacteroides

Ascusb_8886
1017



Lactobacillus

Ascusb_8888
1018



Adlercreutzia

Ascusb_8892
1019



Dethiosulfovibrio

Ascusb_8916
1020



Lutispora

Ascusb_8934
1021



Turicibacter

Ascusb_8942
1022


Cyanobacteria
Ascusb_8953
1023



Clostridium_sensu_stricto

Ascusb_8956
1024


Cyanobacteria
Ascusb_8972
1025



Bulleidia

Ascusb_9004
1026



Aquiflexum

Ascusb_9015
1027


Lachnospiracea_incertae_sedis
Ascusb_9026
1028


Lachnospiracea_incertae_sedis
Ascusb_9073
1029



Clostridium_III

Ascusb_9075
1030



Roseburia

Ascusb_9081
1031



Glaciecola

Ascusb_9086
1032



Clostridium_XIVa

Ascusb_9090
1033



Hydrogenoanaerobacterium

Ascusb_9095
1034



Clostridium_IV

Ascusb_9097
1035



Sphaerobacter

Ascusb_9098
1036


Cyanobacteria
Ascusb_9105
1037



Prevotella

Ascusb_9109
1038



Turicibacter

Ascusb_9112
1039



Ruminococcus

Ascusb_9122
1040



Clostridium_IV

Ascusb_9131
1041



Clostridium_XIVa

Ascusb_9145
1042



Saccharofermentans

Ascusb_9151
1043



Clostridium_XIVb

Ascusb_9154
1044



Ruminococcus

Ascusb_9160
1045



Fibrobacter

Ascusb_9169
1046



Proteiniclasticum

Ascusb_9176
1047



Anaeroplasma

Ascusb_9178
1048


Cyanobacteria
Ascusb_9184
1049



Algoriphagus

Ascusb_9189
1050



Clostridium_XIVa

Ascusb_9196
1051



Howardella

Ascusb_9200
1052



Clostridium_XIVa

Ascusb_9201
1053



Barnesiella

Ascusb_9211
1054



Clostridium_IV

Ascusb_9234
1055



Prevotella

Ascusb_9238
1056



Clostridium_XIVa

Ascusb_9251
1057



Butyricimonas

Ascusb_9261
1058



Blautia

Ascusb_9264
1059



Prevotella

Ascusb_9274
1060



Clostridium_XIVa

Ascusb_9277
1061



Blautia

Ascusb_9282
1062



Clostridium_IV

Ascusb_9291
1063



Flavobacterium

Ascusb_9292
1064



Prevotella

Ascusb_9300
1065



Clostridium_XIVa

Ascusb_9301
1066



Clostridium_XIVa

Ascusb_9302
1067



Eubacterium

Ascusb_9313
1068



Butyricicoccus

Ascusb_9340
1069



Fluviicola

Ascusb_9343
1070



Anaerovibrio

Ascusb_9354
1071



Blautia

Ascusb_9355
1072



Verrucomicrobia

Ascusb_9367
1073



Clostridium_sensu_stricto

Ascusb_9368
1074



Spirochaeta

Ascusb_9369
1075



Clostridium_XI

Ascusb_9372
1076



Anaerovorax

Ascusb_9376
1077



Roseburia

Ascusb_9381
1078



Mucilaginibacter

Ascusb_9388
1079



Clostridium_XI

Ascusb_9389
1080


Lachnospiracea_incertae_sedis
Ascusb_9401
1081



Prevotella

Ascusb_9402
1082



Clostridium_III

Ascusb_9411
1083


Lachnospiracea_incertae_sedis
Ascusb_9415
1084



Coprococcus

Ascusb_9427
1085



Acholeplasma

Ascusb_9432
1086



Clostridium_III

Ascusb_9453
1087



Lactobacillus

Ascusb_9454
1088



Clostridium_IV

Ascusb_9455
1089



Prevotella

Ascusb_9465
1090



Bifidobacterium

Ascusb_9497
1091



Adhaeribacter

Ascusb_9507
1092



Hydrogenoanaerobacterium

Ascusb_9518
1093



Acetivibrio

Ascusb_9521
1094


Cyanobacteria
Ascusb_9532
1095



Flammeovirga

Ascusb_9535
1096



Dethiosulfovibrio

Ascusb_9543
1097



Hippea

Ascusb_9545
1098



Faecalibacterium

Ascusb_9558
1099



Spirochaeta

Ascusb_9559
1100



Brevundimonas

Ascusb_9563
1101



Mucilaginibacter

Ascusb_9564
1102



Hydrogenoanaerobacterium

Ascusb_9580
1103



Asaccharobacter

Ascusb_9587
1104



Clostridium_IV

Ascusb_9591
1105



Mogibacterium

Ascusb_9605
1106



Clostridium_IV

Ascusb_9617
1107



Oscillibacter

Ascusb_9619
1108



Clostridium_XIVa

Ascusb_9628
1109



Faecalibacterium

Ascusb_9640
1110



Altererythrobacter

Ascusb_9644
1111



Gelidibacter

Ascusb_9656
1112



Prevotella

Ascusb_9662
1113



Anaerovorax

Ascusb_9663
1114



Riemerella

Ascusb_9664
1115



Sphingobacterium

Ascusb_9666
1116



Syntrophococcus

Ascusb_9668
1117



Bacteroides

Ascusb_9669
1118



Papillibacter

Ascusb_9678
1119



Butyricicoccus

Ascusb_9679
1120



Clostridium_IV

Ascusb_9680
1121



Hydrogenoanaerobacterium

Ascusb_9684
1122



Marvinbryantia

Ascusb_9688
1123



Brevibacillus

Ascusb_9701
1124



Clostridium_IV

Ascusb_9715
1125



Prevotella

Ascusb_9719
1126



Clostridium_IV

Ascusb_9734
1127



Aminobacter

Ascusb_9759
1128



Sporotomaculum

Ascusb_9764
1129



Clostridium_IV

Ascusb_9779
1130



Pedobacter

Ascusb_9780
1131



Victivallis

Ascusb_9782
1132



Gelidibacter

Ascusb_9792
1133



Prevotella

Ascusb_9824
1134



Wautersiella

Ascusb_9839
1135



Slackia

Ascusb_9846
1136



Pyramidobacter

Ascusb_9851
1137


Lachnospiracea_incertae_sedis
Ascusb_9862
1138



Clostridium_XIVa

Ascusb_9869
1139



Prevotella

Ascusb_9876
1140



Lentisphaera

Ascusb_9886
1141



Desulfoluna

Ascusb_9895
1142



Clostridium_III

Ascusb_9897
1143



Clostridium_sensu_stricto

Ascusb_9925
1144



Prevotella

Ascusb_9929
1145



Clostridium_III

Ascusb_9934
1146



Clostridium_IV

Ascusb_9949
1147



Prevotella

Ascusb_9951
1148


Cyanobacteria
Ascusb_9954
1149



Helicobacter

Ascusb_9958
1150



Clostridium_XIVa

Ascusb_9977
1151



Coprococcus

Ascusb_9982
1152



Bradyrhizobium

Ascusb_9993
1153



Clostridium_IV

Ascusb_9996
1154



Sphingobacterium

Ascusb_10002
1155



Gelidibacter

Ascusb_10023
1156



Vasilyevaea

Ascusb_10029
1157



Eubacterium

Ascusb_10030
1158



Clostridium_XIVa

Ascusb_10034
1159



Eubacterium

Ascusb_10044
1160



Syntrophococcus

Ascusb_10045
1161



Prevotella

Ascusb_10050
1162



Treponema

Ascusb_10057
1163



Anaerovorax

Ascusb_10058
1164


Erysipelotrichaceae_incertae_sedis
Ascusb_10059
1165



Sulfurovum

Ascusb_10084
1166



Clostridium_IV

Ascusb_10085
1167



Papillibacter

Ascusb_10087
1168



Paracoccus

Ascusb_10094
1169



Hydrogenoanaerobacterium

Ascusb_10102
1170



Adhaeribacter

Ascusb_10121
1171


Lachnospiracea_incertae_sedis
Ascusb_10126
1172



Bacteroides

Ascusb_10127
1173



Hydrogenoanaerobacterium

Ascusb_10129
1174



Telmatospirillum

Ascusb_10138
1175



Clostridium_XIVa

Ascusb_10144
1176



Hydrogenoanaerobacterium

Ascusb_10147
1177



Clostridium_IV

Ascusb_10156
1178



Vasilyevaea

Ascusb_10164
1179



Anaeroplasma

Ascusb_10177
1180



Sporotomaculum

Ascusb_10193
1181



Clostridium_IV

Ascusb_10194
1182



Enterorhabdus

Ascusb_10204
1183



Bacteroides

Ascusb_10208
1184



Anaerotruncus

Ascusb_10210
1185



Rhodopirellula

Ascusb_10215
1186



Clostridium_XIVa

Ascusb_10221
1187



Gelidibacter

Ascusb_10243
1188



Anaerofustis

Ascusb_10268
1189



Butyricicoccus

Ascusb_10269
1190



Butyricicoccus

Ascusb_10278
1191



Clostridium_XIVa

Ascusb_10281
1192



Cryptanaerobacter

Ascusb_10284
1193



Clostridium_XIVa

Ascusb_10299
1194



Mogibacterium

Ascusb_10309
1195



Syntrophococcus

Ascusb_10313
1196



Bacteroides

Ascusb_10325
1197



Treponema

Ascusb_10327
1198



Coraliomargarita

Ascusb_10344
1199



Ruminococcus

Ascusb_10368
1200



Prevotella

Ascusb_10374
1201



Pseudaminobacter

Ascusb_10380
1202



Prevotella

Ascusb_10392
1203



Treponema

Ascusb_10450
1204



Syntrophococcus

Ascusb_10456
1205



Clostridium_IV

Ascusb_10457
1206



Tenacibaculum

Ascusb_10462
1207



Parabacteroides

Ascusb_10466
1208



Luteimonas

Ascusb_10469
1209



Eubacterium

Ascusb_10488
1210



Roseburia

Ascusb_10495
1211



Oscillibacter

Ascusb_10504
1212


Cyanobacteria
Ascusb_10529
1213



Prevotella

Ascusb_10547
1214



Clostridium_IV

Ascusb_10548
1215



Treponema

Ascusb_10557
1216



Clostridium_IV

Ascusb_10561
1217



Victivallis

Ascusb_10562
1218



Clostridium_XIVa

Ascusb_10576
1219



Oscillibacter

Ascusb_10586
1220



Papillibacter

Ascusb_10598
1221



Cellulosilyticum

Ascusb_10604
1222



Treponema

Ascusb_10607
1223



Ruminococcus

Ascusb_10609
1224



Coraliomargarita

Ascusb_10612
1225



Butyricicoccus

Ascusb_10613
1226



Blautia

Ascusb_10615
1227


Lachnospiracea_incertae_sedis
Ascusb_10617
1228



Prevotella

Ascusb_10622
1229



Clostridium_IV

Ascusb_10623
1230



Clostridium_IV

Ascusb_10635
1231



Clostridium_III

Ascusb_10655
1232



Neptunomonas

Ascusb_10677
1233



Clostridium_IV

Ascusb_10682
1234



Howardella

Ascusb_10685
1235



Clostridium_IV

Ascusb_10687
1236



Roseburia

Ascusb_10711
1237



Oscillibacter

Ascusb_10739
1238



Clostridium_XIVa

Ascusb_10740
1239



Clostridium_IV

Ascusb_10741
1240



Sporobacter

Ascusb_10749
1241



Clostridium_XIVa

Ascusb_10769
1242



Butyricicoccus

Ascusb_10774
1243



Clostridium_XIVa

Ascusb_10787
1244



Filomicrobium

Ascusb_10788
1245



Bacteroides

Ascusb_10790
1246



Clostridium_XIVa

Ascusb_10809
1247



Brevundimonas

Ascusb_10812
1248



Clostridium_IV

Ascusb_10817
1249



Paracoccus

Ascusb_10818
1250



Schlegelella

Ascusb_10837
1251



Clostridium_XI

Ascusb_10844
1252



Diaphorobacter

Ascusb_10847
1253



Clostridium_sensu_stricto

Ascusb_10858
1254



Saccharopolyspora

Ascusb_10863
1255



Prevotella

Ascusb_10871
1256



Eggerthella

Ascusb_10878
1257



Gelidibacter

Ascusb_10888
1258



Prevotella

Ascusb_10899
1259



Pseudomonas

Ascusb_10922
1260



Prevotella

Ascusb_10927
1261



Prevotella

Ascusb_10937
1262



Prevotella

Ascusb_10940
1263



Brevundimonas

Ascusb_10945
1264



Bacteroides

Ascusb_10982
1265



Clostridium_XIVa

Ascusb_11015
1266



Photobacterium

Ascusb_11027
1267



Clostridium_XIVa

Ascusb_11031
1268



Clostridium_XIVb

Ascusb_11032
1269



Prevotella

Ascusb_11037
1270



Clostridium_IV

Ascusb_11046
1271



Anaeroplasma

Ascusb_11051
1272



Caldilinea

Ascusb_11053
1273



Clostridium_XIVa

Ascusb_11059
1274



Victivallis

Ascusb_11061
1275



Brevundimonas

Ascusb_11063
1276


Cyanobacteria
Ascusb_11074
1277



Prevotella

Ascusb_11120
1278



Slackia

Ascusb_11124
1279



Pedobacter

Ascusb_11125
1280



Prevotella

Ascusb_11129
1281



Trueperella

Ascusb_11141
1282



Oscillibacter

Ascusb_11170
1283


Cyanobacteria
Ascusb_11185
1284



Victivallis

Ascusb_11199
1285



Bacteroides

Ascusb_11200
1286



Micrococcus

Ascusb_11207
1287



Olivibacter

Ascusb_11209
1288



Anaerophaga

Ascusb_11211
1289



Selenomonas

Ascusb_11214
1290



Megasphaera

Ascusb_11219
1291



Clostridium_XIVa

Ascusb_11221
1292



Clostridium_XIVa

Ascusb_11241
1293



Eubacterium

Ascusb_11245
1294


Cyanobacteria
Ascusb_11253
1295



Clostridium_XIVa

Ascusb_11287
1296



Treponema

Ascusb_11288
1297



Cryptanaerobacter

Ascusb_11289
1298



Xanthomonas

Ascusb_11301
1299



Asteroleplasma

Ascusb_11302
1300


Cyanobacteria
Ascusb_11315
1301



Sporotomaculum

Ascusb_11321
1302



Bacteroides

Ascusb_11324
1303



Asaccharobacter

Ascusb_11330
1304



Clostridium_IV

Ascusb_11343
1305


Cyanobacteria
Ascusb_11348
1306



Clostridium_XIVa

Ascusb_11362
1307



Treponema

Ascusb_11365
1308



Prevotella

Ascusb_11384
1309



Turicibacter

Ascusb_11388
1310



Clostridium_IV

Ascusb_11389
1311



Clostridium_IV

Ascusb_11397
1312



Clostridium_IV

Ascusb_11403
1313



Oscillibacter

Ascusb_11410
1314



Deinococcus

Ascusb_11423
1315



Pedobacter

Ascusb_11427
1316



Anaerovorax

Ascusb_11435
1317



Clostridium_IV

Ascusb_11442
1318



Bacteroides

Ascusb_11445
1319



Clostridium_IV

Ascusb_11461
1320



Rhodococcus

Ascusb_11463
1321



Treponema

Ascusb_11464
1322



Mucilaginibacter

Ascusb_11475
1323



Clostridium_XIVa

Ascusb_11503
1324



Olivibacter

Ascusb_11510
1325



Clostridium_XIVa

Ascusb_11519
1326



Barnesiella

Ascusb_11581
1327



Clostridium_XIVb

Ascusb_11584
1328



Gelidibacter

Ascusb_11600
1329



Methanobrevibacter

Ascusb_11602
1330



Anaerotruncus

Ascusb_11612
1331


Lachnospiracea_incertae_sedis
Ascusb_11653
1332


Erysipelotrichaceae_incertae_sedis
Ascusb_11656
1333



Mesorhizobium

Ascusb_11681
1334



Clostridium_XI

Ascusb_11695
1335



Planctomyces

Ascusb_11698
1336



Aerococcus

Ascusb_11713
1337



Victivallis

Ascusb_11721
1338


Cyanobacteria
Ascusb_11736
1339



Bacteroides

Ascusb_11752
1340



Clostridium_XI

Ascusb_11753
1341



Clostridium_XIVa

Ascusb_11757
1342



Ruminococcus

Ascusb_11761
1343



Saccharofermentans

Ascusb_11780
1344



Oscillibacter

Ascusb_11781
1345


Lachnospiracea_incertae_sedis
Ascusb_11783
1346



Fibrobacter

Ascusb_11793
1347



Kiloniella

Ascusb_11809
1348



Olivibacter

Ascusb_11819
1349



Clostridium_IV

Ascusb_11821
1350



Spirochaeta

Ascusb_11865
1351



Prevotella

Ascusb_11870
1352



Olivibacter

Ascusb_11881
1353



Prevotella

Ascusb_11884
1354



Parabacteroides

Ascusb_11885
1355



Prevotella

Ascusb_11892
1356



Leifsonia

Ascusb_11896
1357



Clostridium_IV

Ascusb_11901
1358



Victivallis

Ascusb_11903
1359



Treponema

Ascusb_11929
1360


Cyanobacteria
Ascusb_11952
1361



Sporotomaculum

Ascusb_11954
1362



Spirochaeta

Ascusb_11955
1363



Clostridium_III

Ascusb_11960
1364



Clostridium_XIVa

Ascusb_11962
1365



Anaerovorax

Ascusb_11963
1366



Oscillibacter

Ascusb_11964
1367



Victivallis

Ascusb_11988
1368


Lachnospiracea_incertae_sedis
Ascusb_11993
1369



Spirochaeta

Ascusb_11997
1370



Clostridium_XIVb

Ascusb_12000
1371



Oscillibacter

Ascusb_12004
1372



Prevotella

Ascusb_12013
1373



Anaeroplasma

Ascusb_12046
1374



Adlercreutzia

Ascusb_12054
1375



Clostridium_XIVa

Ascusb_12061
1376



Beijerinckia

Ascusb_12069
1377



Prevotella

Ascusb_12106
1378



Coprococcus

Ascusb_12110
1379



Lentisphaera

Ascusb_12116
1380



Clostridium_XIVa

Ascusb_12119
1381



Saccharofermentans

Ascusb_12127
1382



Porphyrobacter

Ascusb_12128
1383



Rhodobacter

Ascusb_12140
1384



Oscillibacter

Ascusb_12153
1385



Roseburia

Ascusb_12160
1386



Prevotella

Ascusb_12175
1387



Aquiflexum

Ascusb_12177
1388



Rhodopirellula

Ascusb_12187
1389



Bacteroides

Ascusb_12191
1390



Bacteroides

Ascusb_12216
1391



Clostridium_XIVa

Ascusb_12221
1392



Clostridium_IV

Ascusb_12227
1393



Prevotella

Ascusb_12243
1394



Mogibacterium

Ascusb_12248
1395



Prevotella

Ascusb_12252
1396



Clostridium_XIVa

Ascusb_12269
1397



Prevotella

Ascusb_12270
1398



Capnocytophaga

Ascusb_12276
1399



Acholeplasma

Ascusb_12282
1400



Clostridium_IV

Ascusb_12310
1401



Succinivibrio

Ascusb_12327
1402



Pseudonocardia

Ascusb_12339
1403



Clostridium_XIVa

Ascusb_12353
1404



Butyricimonas

Ascusb_12354
1405



Anaerovorax

Ascusb_12355
1406



Prevotella

Ascusb_12383
1407



Butyricimonas

Ascusb_12399
1408



Parabacteroides

Ascusb_12407
1409



Clostridium_XIVa

Ascusb_12413
1410



Clostridium_XIVb

Ascusb_12417
1411



Bacteroides

Ascusb_12428
1412


Cyanobacteria
Ascusb_12452
1413



Riemerella

Ascusb_12461
1414



Anaeroplasma

Ascusb_12487
1415



Ruminococcus

Ascusb_12489
1416



Verrucomicrobia

Ascusb_12499
1417


Lachnospiracea_incertae_sedis
Ascusb_12511
1418



Syntrophococcus

Ascusb_12512
1419



Clostridium_IV

Ascusb_12520
1420



Barnesiella

Ascusb_12534
1421



Olivibacter

Ascusb_12553
1422



Clostridium_XIVa

Ascusb_12574
1423



Cryptanaerobacter

Ascusb_12577
1424



Saccharofermentans

Ascusb_12578
1425



Clostridium_IV

Ascusb_12599
1426



Coprococcus

Ascusb_12600
1427



Barnesiella

Ascusb_12606
1428



Clostridium_sensu_stricto

Ascusb_12618
1429



Hydrogenoanaerobacterium

Ascusb_12627
1430



Clostridium_XIVb

Ascusb_12628
1431



Selenomonas

Ascusb_12661
1432



Prevotella

Ascusb_12662
1433



Hydrogenoanaerobacterium

Ascusb_12679
1434



Spirochaeta

Ascusb_12703
1435



Enterorhabdus

Ascusb_12704
1436



Thermoanaerobacter

Ascusb_12709
1437



Armatimonadetes

Ascusb_12719
1438



Syntrophococcus

Ascusb_12723
1439



Sphingobium

Ascusb_12731
1440



Clostridium_XIVa

Ascusb_12737
1441



Geosporobacter

Ascusb_12740
1442



Enterorhabdus

Ascusb_12746
1443



Verrucomicrobia

Ascusb_12747
1444



Clostridium_XIVa

Ascusb_12749
1445



Parabacteroides

Ascusb_12750
1446



Cryptanaerobacter

Ascusb_12769
1447



Anaeroplasma

Ascusb_12775
1448



Spirochaeta

Ascusb_12779
1449



Prevotella

Ascusb_12804
1450



Roseburia

Ascusb_12819
1451



Pedobacter

Ascusb_12826
1452



Pedobacter

Ascusb_12835
1453



Eggerthella

Ascusb_12838
1454



Prevotella

Ascusb_12853
1455



Rikenella

Ascusb_12873
1456



Anaerophaga

Ascusb_12894
1457



Spirochaeta

Ascusb_12901
1458



Clostridium_IV

Ascusb_12910
1459



Weissella

Ascusb_12931
1460



Butyricicoccus

Ascusb_12946
1461



Hahella

Ascusb_12953
1462



Acholeplasma

Ascusb_12960
1463



Clostridium_XIVa

Ascusb_12962
1464



Cellulosilyticum

Ascusb_12987
1465



Verrucomicrobia

Ascusb_12995
1466



Clostridium_XIVa

Ascusb_13002
1467



Pseudoflavonifractor

Ascusb_13028
1468



Calditerricola

Ascusb_13035
1469



Clostridium_IV

Ascusb_13039
1470



Clostridium_IV

Ascusb_13050
1471



Adlercreutzia

Ascusb_13054
1472



Bulleidia

Ascusb_13088
1473


Lachnospiracea_incertae_sedis
Ascusb_13089
1474



Mucilaginibacter

Ascusb_13115
1475



Victivallis

Ascusb_13128
1476



Anaerovorax

Ascusb_13130
1477



Clostridium_XIVb

Ascusb_13134
1478



Clostridium_XIVa

Ascusb_13154
1479



Prevotella

Ascusb_13155
1480



Bacteroides

Ascusb_13163
1481



Schwartzia

Ascusb_13165
1482



Pyramidobacter

Ascusb_13226
1483



Eubacterium

Ascusb_13230
1484


Lachnospiracea_incertae_sedis
Ascusb_13244
1485



Clostridium_XIVa

Ascusb_13249
1486



Roseburia

Ascusb_13254
1487



Clostridium_XIVb

Ascusb_13276
1488



Enterorhabdus

Ascusb_13284
1489



Pedobacter

Ascusb_13291
1490



Clostridium_sensu_stricto

Ascusb_13296
1491



Clostridium_XIVa

Ascusb_13328
1492



Clostridium_III

Ascusb_13343
1493



Desulfotomaculum

Ascusb_13349
1494



Clostridium_IV

Ascusb_13353
1495



Proteiniclasticum

Ascusb_13371
1496



Prevotella

Ascusb_13412
1497



Faecalibacterium

Ascusb_13417
1498



Microbacterium

Ascusb_13419
1499



Leucobacter

Ascusb_13424
1500



Prevotella

Ascusb_13426
1501



Sphingobacterium

Ascusb_13457
1502



Fusibacter

Ascusb_13458
1503



Howardella

Ascusb_13463
1504



Pedobacter

Ascusb_13488
1505



Caldilinea

Ascusb_13504
1506



Turicibacter

Ascusb_13513
1507



Clostridium_IV

Ascusb_13516
1508



Alistipes

Ascusb_13546
1509



Clostridium_XIVa

Ascusb_13547
1510



Clostridium_XIVa

Ascusb_13567
1511



Prevotella

Ascusb_13597
1512



Clostridium_XIVa

Ascusb_13611
1513



Butyricimonas

Ascusb_13648
1514



Anaerovibrio

Ascusb_13663
1515



Prevotella

Ascusb_13675
1516



Pseudoflavonifractor

Ascusb_13679
1517



Corynebacterium

Ascusb_13763
1518



Leucobacter

Ascusb_13780
1519



Kerstersia

Ascusb_13819
1520



Slackia

Ascusb_13835
1521



Lactococcus

Ascusb_13839
1522



Prevotella

Ascusb_13840
1523



Clostridium_IV

Ascusb_13845
1524



Prevotella

Ascusb_13848
1525



Bacteroides

Ascusb_13867
1526



Lactobacillus

Ascusb_13881
1527



Prevotella

Ascusb_13892
1528



Clostridium_XIVa

Ascusb_13895
1529



Clostridium_sensu_stricto

Ascusb_13903
1530



Syntrophococcus

Ascusb_13904
1531



Clostridium_XIVa

Ascusb_13921
1532



Victivallis

Ascusb_13923
1533



Bacteroides

Ascusb_13940
1534


Acidobacteria
Ascusb_13951
1535



Clostridium_XIVa

Ascusb_13953
1536



Prevotella

Ascusb_13954
1537



Verrucomicrobia

Ascusb_13955
1538



Clostridium_XIVa

Ascusb_13981
1539



Treponema

Ascusb_13982
1540



Pyramidobacter

Ascusb_13983
1541



Robinsoniella

Ascusb_13992
1542


Lachnospiracea_incertae_sedis
Ascusb_13995
1543



Clostridium_XI

Ascusb_13996
1544



Bifidobacterium

Ascusb_14005
1545



Bacteroides

Ascusb_14013
1546



Gordonibacter

Ascusb_14016
1547



Enterorhabdus

Ascusb_14055
1548



Lactobacillus

Ascusb_14059
1549



Bacteroides

Ascusb_14074
1550



Prevotella

Ascusb_14086
1551



Tannerella

Ascusb_14141
1552



Bacteroides

Ascusb_14145
1553



Prevotella

Ascusb_14151
1554



Clostridium_XIVb

Ascusb_14163
1555



Gelidibacter

Ascusb_14189
1556


Cyanobacteria
Ascusb_14213
1557



Rhodoplanes

Ascusb_14224
1558



Selenomonas

Ascusb_14226
1559



Escherichia/Shigella

Ascusb_14256
1560



Rikenella

Ascusb_14278
1561



Coprococcus

Ascusb_14285
1562



Clostridium_sensu_stricto

Ascusb_14290
1563



Hyphomicrobium

Ascusb_14304
1564


Erysipelotrichaceae_incertae_sedis
Ascusb_14320
1565



Verrucomicrobia

Ascusb_14324
1566



Staphylococcus

Ascusb_14335
1567



Verrucomicrobia

Ascusb_14358
1568



Victivallis

Ascusb_14359
1569



Selenomonas

Ascusb_14423
1570



Desulfobulbus

Ascusb_14425
1571



Clostridium_III

Ascusb_14450
1572



Spirochaeta

Ascusb_14451
1573



Kordia

Ascusb_14514
1574



Bosea

Ascusb_14521
1575



Enterococcus

Ascusb_14525
1576



Clostridium_III

Ascusb_14530
1577



Xanthobacter

Ascusb_14538
1578



Lactobacillus

Ascusb_14555
1579



Prevotella

Ascusb_14583
1580



Acidaminococcus

Ascusb_14595
1581



Eubacterium

Ascusb_14596
1582



Bacteroides

Ascusb_14611
1583



Clostridium_XIVa

Ascusb_14613
1584



Lactobacillus

Ascusb_14626
1585



Devosia

Ascusb_14628
1586



Pedobacter

Ascusb_14667
1587



Clostridium_IV

Ascusb_14747
1588



Clostridium_XIVa

Ascusb_14785
1589



Corynebacterium

Ascusb_14790
1590



Spirochaeta

Ascusb_14792
1591



Anaeroplasma

Ascusb_14828
1592



Clostridium_XIVa

Ascusb_14869
1593


Lachnospiracea_incertae_sedis
Ascusb_14888
1594



Saccharofermentans

Ascusb_14898
1595



Slackia

Ascusb_14906
1596



Limibacter

Ascusb_14951
1597



Sphingobium

Ascusb_14952
1598



Clostridium_XIVa

Ascusb_14987
1599



Riemerella

Ascusb_14990
1600



Saccharofermentans

Ascusb_15032
1601



Bacteroides

Ascusb_15048
1602



Prevotella

Ascusb_15076
1603



Selenomonas

Ascusb_15097
1604



Victivallis

Ascusb_15122
1605



Howardella

Ascusb_15128
1606



Pelospora

Ascusb_15132
1607



Clostridium_sensu_stricto

Ascusb_15151
1608



Selenomonas

Ascusb_15156
1609



Fibrobacter

Ascusb_15181
1610



Clostridium_III

Ascusb_15215
1611



Sphingomonas

Ascusb_15220
1612



Selenomonas

Ascusb_15226
1613



Eggerthella

Ascusb_15326
1614



Treponema

Ascusb_15352
1615



Mogibacterium

Ascusb_15357
1616



Adlercreutzia

Ascusb_15390
1617



Selenomonas

Ascusb_15394
1618



Methylomicrobium

Ascusb_15404
1619



Leuconostoc

Ascusb_15413
1620



Pyramidobacter

Ascusb_15427
1621



Butyrivibrio

Ascusb_15438
1622



Bacteroides

Ascusb_15454
1623



Butyricimonas

Ascusb_15455
1624



Ruminococcus

Ascusb_15461
1625



Clostridium_sensu_stricto

Ascusb_15482
1626



Butyrivibrio

Ascusb_15488
1627



Corynebacterium

Ascusb_15494
1628



Proteiniborus

Ascusb_15526
1629



Spirochaeta

Ascusb_15539
1630



Acetitomaculum

Ascusb_15549
1631



Selenomonas

Ascusb_15552
1632



Altererythrobacter

Ascusb_15556
1633



Atopobium

Ascusb_15587
1634



Clostridium_IV

Ascusb_15615
1635



Clostridium_XIVa

Ascusb_15624
1636



Clostridium_XIVa

Ascusb_15695
1637



Clostridium_IV

Ascusb_15703
1638



Clostridium_III

Ascusb_15720
1639


Candidate phylumTM7
Ascusb_15737
1640



Desulfotomaculum

Ascusb_15741
1641



Pedobacter

Ascusb_15746
1642



Bacteroides

Ascusb_15750
1643



Asaccharobacter

Ascusb_15754
1644



Microbacterium

Ascusb_15768
1645



Treponema

Ascusb_15824
1646



Dethiosulfovibrio

Ascusb_15830
1647



Oscillibacter

Ascusb_15832
1648



Selenomonas

Ascusb_15846
1649



Eubacterium

Ascusb_15864
1650



Ruminococcus

Ascusb_15877
1651



Treponema

Ascusb_15915
1652



Spirochaeta

Ascusb_15951
1653



Roseburia

Ascusb_15963
1654



Ruminococcus

Ascusb_15992
1655



Butyricimonas

Ascusb_16010
1656



Pedobacter

Ascusb_16051
1657



Spirochaeta

Ascusb_16066
1658



Parabacteroides

Ascusb_16101
1659



Methylococcus

Ascusb_16111
1660



Enterorhabdus

Ascusb_16113
1661



Clostridium_sensu_stricto

Ascusb_16124
1662



Gelidibacter

Ascusb_16149
1663



Sporobacter

Ascusb_16168
1664



Pedobacter

Ascusb_16185
1665


Cyanobacteria
Ascusb_16194
1666



Syntrophococcus

Ascusb_16198
1667



Slackia

Ascusb_16200
1668



Mogibacterium

Ascusb_16215
1669



Prevotella

Ascusb_16239
1670



Pseudoflavonifractor

Ascusb_16244
1671



Veillonella

Ascusb_16257
1672



Clostridium_XIVa

Ascusb_16278
1673



Bacillus

Ascusb_16299
1674



Pedobacter

Ascusb_16316
1675



Clostridium_IV

Ascusb_16329
1676



Fibrobacter

Ascusb_16330
1677



Paenibacillus

Ascusb_16336
1678



Brevundimonas

Ascusb_16345
1679



Desulfovibrio

Ascusb_16373
1680



Clostridium_XI

Ascusb_16374
1681



Helicobacter

Ascusb_16383
1682



Prevotella

Ascusb_16420
1683



Clostridium_XIVa

Ascusb_16423
1684



Prevotella

Ascusb_16436
1685



Herbiconiux

Ascusb_16453
1686



Clostridium_IV

Ascusb_16461
1687



Rikenella

Ascusb_16470
1688



Clostridium_XIVa

Ascusb_16473
1689



Hippea

Ascusb_16536
1690



Lactobacillus

Ascusb_16537
1691



Eubacterium

Ascusb_16541
1692



Clostridium_IV

Ascusb_16546
1693



Clostridium_III

Ascusb_16560
1694



Lactobacillus

Ascusb_16565
1695



Lactobacillus

Ascusb_16574
1696



Desulfotomaculum

Ascusb_16578
1697



Prevotella

Ascusb_16618
1698



Staphylococcus

Ascusb_16628
1699



Tenacibaculum

Ascusb_16632
1700



Parabacteroides

Ascusb_16655
1701



Clostridium_XIVa

Ascusb_16668
1702



Clostridium_IV

Ascusb_16671
1703



Clostridium_IV

Ascusb_16674
1704



Pedobacter

Ascusb_16682
1705



Helicobacter

Ascusb_16686
1706



Proteiniclasticum

Ascusb_16691
1707



Anaplasma

Ascusb_16711
1708



Bacteroides

Ascusb_16734
1709



Clostridium_IV

Ascusb_16749
1710



Mucilaginibacter

Ascusb_16803
1711



Verrucomicrobia

Ascusb_16829
1712



Selenomonas

Ascusb_16884
1713



Parabacteroides

Ascusb_16931
1714



Eubacterium

Ascusb_16933
1715



Coprococcus

Ascusb_16948
1716



Weissella

Ascusb_16968
1717



Pedobacter

Ascusb_16992
1718



Clostridium_XI

Ascusb_16995
1719



Sphingomonas

Ascusb_16998
1720



Treponema

Ascusb_17013
1721



Geobacter

Ascusb_17017
1722



Clostridium_XIVa

Ascusb_17018
1723



Filomicrobium

Ascusb_17036
1724



Prevotella

Ascusb_17038
1725



Pedobacter

Ascusb_17057
1726



Pedobacter

Ascusb_17058
1727



Clostridium_XIVa

Ascusb_17064
1728



Bifidobacterium

Ascusb_17066
1729



Saccharofermentans

Ascusb_17092
1730



Ruminococcus

Ascusb_17136
1731



Flavobacterium

Ascusb_17138
1732



Rhodopirellula

Ascusb_17161
1733



Roseburia

Ascusb_17171
1734



Prevotella

Ascusb_17177
1735



Limibacter

Ascusb_17182
1736



Saccharofermentans

Ascusb_17203
1737



Clostridium_sensu_stricto

Ascusb_17206
1738



Clostridium_III

Ascusb_17243
1739



Prevotella

Ascusb_17275
1740



Pseudoxanthomonas

Ascusb_17283
1741



Anaerorhabdus

Ascusb_17325
1742



Clostridium_III

Ascusb_17360
1743



Streptomyces

Ascusb_17372
1744



Pedobacter

Ascusb_17388
1745



Cellulomonas

Ascusb_17414
1746



Clostridium_XIVa

Ascusb_17416
1747



Olivibacter

Ascusb_17425
1748



Treponema

Ascusb_17433
1749



Gelidibacter

Ascusb_17437
1750



Ruminococcus

Ascusb_17439
1751



Clostridium_IV

Ascusb_17446
1752



Gemmatimonas

Ascusb_17450
1753



Prevotella

Ascusb_17459
1754



Ethanoligenens

Ascusb_17477
1755



Leucobacter

Ascusb_17494
1756



Clostridium_XIVa

Ascusb_17502
1757



Clostridium_XIVa

Ascusb_17507
1758



Eggerthella

Ascusb_17540
1759



Prevotella

Ascusb_17553
1760



Prevotella

Ascusb_17569
1761



Solobacterium

Ascusb_17571
1762



Xanthobacter

Ascusb_17581
1763



Verrucomicrobia

Ascusb_17649
1764



Desulfovibrio

Ascusb_17670
1765



Microbacterium

Ascusb_17717
1766



Oscillibacter

Ascusb_17718
1767



Blautia

Ascusb_17735
1768



Papillibacter

Ascusb_17736
1769



Prevotella

Ascusb_17759
1770



Lentisphaera

Ascusb_17766
1771



Ruminococcus

Ascusb_17767
1772



Bacteroides

Ascusb_17769
1773



Catonella

Ascusb_17771
1774



Clostridium_XIVa

Ascusb_17773
1775



Clostridium_IV

Ascusb_17782
1776



Verrucomicrobia

Ascusb_17802
1777



Clostridium_XI

Ascusb_17804
1778



Prevotella

Ascusb_17810
1779


Candidate phylumTM7
Ascusb_17824
1780



Mogibacterium

Ascusb_17838
1781



Clostridium_XIVa

Ascusb_17846
1782



Ruminococcus

Ascusb_17857
1783



Eubacterium

Ascusb_17866
1784



Clostridium_IV

Ascusb_17892
1785



Rhodomicrobium

Ascusb_17896
1786



Butyricicoccus

Ascusb_17957
1787



Saccharofermentans

Ascusb_17975
1788



Prevotella

Ascusb_17978
1789



Mannheimia

Ascusb_17981
1790



Lactobacillus

Ascusb_18078
1791



Clostridium_IV

Ascusb_18081
1792



Clostridium_IV

Ascusb_18091
1793



Adlercreutzia

Ascusb_18107
1794



Selenomonas

Ascusb_18110
1795



Paenibacillus

Ascusb_18123
1796



Clostridium_IV

Ascusb_18140
1797



Paenibacillus

Ascusb_18148
1798



Butyricimonas

Ascusb_18161
1799



Wandonia

Ascusb_18170
1800



Puniceicoccus

Ascusb_18179
1801



Lactonifactor

Ascusb_18183
1802



Selenomonas

Ascusb_18248
1803



Brevundimonas

Ascusb_18262
1804



Prevotella

Ascusb_18273
1805



Gelidibacter

Ascusb_18283
1806



Mogibacterium

Ascusb_18287
1807



Clostridium_XIVa

Ascusb_18303
1808



Coprococcus

Ascusb_18329
1809



Verrucomicrobia

Ascusb_18335
1810



Barnesiella

Ascusb_18339
1811



Verrucomicrobia

Ascusb_18351
1812



Clostridium_XIVa

Ascusb_18354
1813



Anaerovorax

Ascusb_18371
1814



Bacteroides

Ascusb_18389
1815



Parasporobacterium

Ascusb_18444
1816



Prevotella

Ascusb_18449
1817



Parapedobacter

Ascusb_18475
1818



Streptomyces

Ascusb_18495
1819


Candidate phylumTM7
Ascusb_18503
1820



Thermotalea

Ascusb_18516
1821



Alkaliflexus

Ascusb_18519
1822



Oscillibacter

Ascusb_18557
1823



Anaerotruncus

Ascusb_18564
1824



Spirochaeta

Ascusb_18566
1825



Clostridium_XI

Ascusb_18567
1826



Sporotomaculum

Ascusb_18585
1827



Sporacetigenium

Ascusb_18592
1828



Bulleidia

Ascusb_18608
1829



Clostridium_IV

Ascusb_18636
1830



Syntrophomonas

Ascusb_18648
1831



Desulfatiferula

Ascusb_18678
1832



Hydrogenoanaerobacterium

Ascusb_18680
1833



Clostridium_XIVa

Ascusb_18695
1834



Mogibacterium

Ascusb_18731
1835



Spirochaeta

Ascusb_18733
1836



Prevotella

Ascusb_18735
1837



Treponema

Ascusb_18738
1838



Spiroplasma

Ascusb_18764
1839



Clostridium_XIVa

Ascusb_18766
1840



Bacteroides

Ascusb_18795
1841



Treponema

Ascusb_18814
1842



Selenomonas

Ascusb_18829
1843



Butyricicoccus

Ascusb_18846
1844



Gelidibacter

Ascusb_18866
1845



Acetitomaculum

Ascusb_18876
1846



Proteiniclasticum

Ascusb_18907
1847



Papillibacter

Ascusb_18930
1848



Prevotella

Ascusb_18949
1849



Elusimicrobium

Ascusb_18970
1850


Lachnospiracea_incertae_sedis
Ascusb_18998
1851



Devosia

Ascusb_19006
1852



Roseburia

Ascusb_19052
1853



Mucilaginibacter

Ascusb_19054
1854



Mogibacterium

Ascusb_19056
1855



Saccharofermentans

Ascusb_19063
1856



Paenibacillus

Ascusb_19092
1857



Anaerotruncus

Ascusb_19101
1858



Leucobacter

Ascusb_19114
1859



Clostridium_XIVa

Ascusb_19148
1860



Eubacterium

Ascusb_19160
1861



Beijerinckia

Ascusb_19170
1862



Prevotella

Ascusb_19200
1863



Clostridium_III

Ascusb_19206
1864


Cyanobacteria
Ascusb_19219
1865



Pseudoflavonifractor

Ascusb_19237
1866



Butyrivibrio

Ascusb_19245
1867



Acholeplasma

Ascusb_19267
1868



Filomicrobium

Ascusb_19288
1869



Clostridium_III

Ascusb_19335
1870



Pseudoflavonifractor

Ascusb_19340
1871



Anaerophaga

Ascusb_19341
1872


Lachnospiracea_incertae_sedis
Ascusb_19347
1873



Asaccharobacter

Ascusb_19353
1874



Kordia

Ascusb_19371
1875



Ruminococcus

Ascusb_19376
1876



Clostridium_III

Ascusb_19379
1877



Ethanoligenens

Ascusb_19392
1878



Clostridium_XIVa

Ascusb_19412
1879



Barnesiella

Ascusb_19414
1880



Eubacterium

Ascusb_19444
1881



Prevotella

Ascusb_19457
1882



Anaerophaga

Ascusb_19496
1883



Acetitomaculum

Ascusb_19498
1884



Prevotella

Ascusb_19503
1885



Clostridium_III

Ascusb_19507
1886



Marinoscillum

Ascusb_19558
1887



Pedobacter

Ascusb_19568
1888



Prevotella

Ascusb_19579
1889



Prevotella

Ascusb_19613
1890



Anaerovorax

Ascusb_19633
1891



Clostridium_XIVa

Ascusb_19658
1892



Clostridium_IV

Ascusb_19662
1893


Lachnospiracea_incertae_sedis
Ascusb_19681
1894



Clostridium_sensu_stricto

Ascusb_19694
1895



Lishizhenia

Ascusb_19698
1896



Pedobacter

Ascusb_19700
1897



Howardella

Ascusb_19731
1898



Roseburia

Ascusb_19745
1899



Clostridium_XIVa

Ascusb_19754
1900



Anaerovorax

Ascusb_19765
1901



Lentisphaera

Ascusb_19772
1902



Prevotella

Ascusb_19778
1903



Saccharofermentans

Ascusb_19779
1904


Cyanobacteria
Ascusb_19818
1905



Proteiniphilum

Ascusb_19824
1906



Schwartzia

Ascusb_19855
1907



Anaerorhabdus

Ascusb_19884
1908



Robinsoniella

Ascusb_19885
1909



Clostridium_IV

Ascusb_19904
1910


Erysipelotrichaceae_incertae_sedis
Ascusb_19936
1911



Flavobacterium

Ascusb_19950
1912



Pedobacter

Ascusb_19955
1913



Clostridium_III

Ascusb_19982
1914



Selenomonas

Ascusb_20001
1915



Rhizobium

Ascusb_20027
1916



Victivallis

Ascusb_20044
1917



Butyricimonas

Ascusb_20062
1918



Parabacteroides

Ascusb_20064
1919



Adhaeribacter

Ascusb_20067
1920



Eubacterium

Ascusb_20086
1921


Acidobacteria
Ascusb_20100
1922



Treponema

Ascusb_20104
1923



Clostridium_XIVa

Ascusb_20108
1924



Clostridium_XIVa

Ascusb_20135
1925



Schwartzia

Ascusb_20143
1926



Prevotella

Ascusb_20162
1927



Selenomonas

Ascusb_20172
1928



Beijerinckia

Ascusb_20219
1929



Eubacterium

Ascusb_20221
1930



Adhaeribacter

Ascusb_20251
1931



Verrucomicrobia

Ascusb_20264
1932



Desulfobulbus

Ascusb_20275
1933



Bacteroides

Ascusb_20278
1934



Rummeliibacillus

Ascusb_20291
1935



Agarivorans

Ascusb_20293
1936



Clostridium_XIVa

Ascusb_20306
1937



Selenomonas

Ascusb_20312
1938



Verrucomicrobia

Ascusb_20365
1939



Prevotella

Ascusb_20368
1940



Spirochaeta

Ascusb_20392
1941



Selenomonas

Ascusb_20405
1942



Spiroplasma

Ascusb_20424
1943



Pedobacter

Ascusb_20440
1944



Clostridium_XIVa

Ascusb_20449
1945


Cyanobacteria
Ascusb_20456
1946



Lactobacillus

Ascusb_20463
1947



Clostridium_XIVa

Ascusb_20529
1948



Prevotella

Ascusb_20534
1949



Prevotella

Ascusb_20540
1950



Marinobacter

Ascusb_20569
1951



Butyricimonas

Ascusb_20576
1952



Prevotella

Ascusb_20594
1953



Dongia

Ascusb_20595
1954



Anaerovorax

Ascusb_20639
1955



Butyricimonas

Ascusb_20757
1956



Cryptanaerobacter

Ascusb_20826
1957



Papillibacter

Ascusb_20904
1958



Clostridium_sensu_stricto

Ascusb_20938
1959



Escherichia/Shigella

Ascusb_20943
1960



Butyricicoccus

Ascusb_20986
1961



Prevotella

Ascusb_21013
1962


Lachnospiracea_incertae_sedis
Ascusb_21027
1963



Thermotalea

Ascusb_21035
1964



Cohaesibacter

Ascusb_21042
1965



Clostridium_XVIII

Ascusb_21043
1966


Lachnospiracea_incertae_sedis
Ascusb_21085
1967



Spirochaeta

Ascusb_21095
1968



Clostridium_XIVa

Ascusb_21112
1969



Hydrogenoanaerobacterium

Ascusb_21147
1970



Clostridium_IV

Ascusb_21151
1971



Papillibacter

Ascusb_21160
1972



Sporosarcina

Ascusb_21190
1973



Selenomonas

Ascusb_21219
1974



Papillibacter

Ascusb_21229
1975


Lachnospiracea_incertae_sedis
Ascusb_21244
1976



Clostridium_XIVa

Ascusb_21271
1977



Saccharofermentans

Ascusb_21297
1978



Clostridium_IV

Ascusb_21309
1979


Lachnospiracea_incertae_sedis
Ascusb_21348
1980



Clostridium_IV

Ascusb_21425
1981


Lachnospiracea_incertae_sedis
Ascusb_21436
1982



Desulfotomaculum

Ascusb_21466
1983



Pedobacter

Ascusb_21484
1984



Anaeroplasma

Ascusb_21546
1985



Clostridium_IV

Ascusb_21585
1986



Treponema

Ascusb_21595
1987



Mogibacterium

Ascusb_21601
1988












BRIEF DESCRIPTION OF THE FIGURES


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



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.



FIG. 3A, FIG. 3B, and FIG. 3C depict the results of a field trial in which dairy cows were administered a composition comprising Ascusb_3138 and Ascusf_15; FIG. 3A reveals the average number of pounds of milk fat produced over time; FIG. 3B reveals the average number of pounds of milk protein produced over time; and FIG. 3C reveals the average number of pounds of energy corrected milk (ECM) produced over time. The vertical line intersecting the data points in each of FIG. 3A, FIG. 3B, and FIG. 3C marks the day at which administration of the microbial bioconsortia ceased.



FIG. 4 depicts the milk yield (kg) daily means (no fill) and covariate adjusted weekly least square means (solid fill)±SEM of cows assigned either to Control (circle) or Inoculated (trapezoid) by intervention period study days.



FIG. 5 depicts the milk crude protein yield (CP, kg) daily means (no fill) and weekly least square means (solid fill)±SEM of cows assigned either to Control (circle) or Inoculated (trapezoid) by Intervention period study days.



FIG. 6 depicts the milk fat yield (kg) daily means (no fill) and weekly least square means (solid fill)±SEM of cows assigned either to Control (circle) or Inoculated (trapezoid) by Intervention period study days.



FIG. 7 depicts the energy corrected milk yield (ECM, kg) daily means (no fill) and weekly least square means (solid fill)±SEM of cows assigned either to Control (circle) or Inoculated (trapezoid) by Intervention period study days.



FIG. 8A and FIG. 8B depict the shared percent similarity (percent identity) among the bacteria (FIG. 8A) and fungi (FIG. 8B) of Table 1. The data points represent the greatest percent similarity pairing for each strain.



FIG. 9 depicts the MIC score distribution for rumen bacteria and milk fat efficiency.



FIG. 10 depicts the MIC score distribution for rumen fungi and milk fat efficiency.



FIG. 11 depicts the MIC score distribution for rumen bacteria and dairy efficiency.



FIG. 12 depicts the MIC score distribution for rumen fungi and dairy efficiency.



FIG. 13 depicts the MIC score distribution for rumen bacteria and milk fat efficiency with four species of bacteria and their MIC scores, in which the species have been evaluated in 3rd party studies. The lower the MIC score, the less likely the species/strains are capable of positively modulating milk fat efficiency in dairy cows.



FIG. 14 depicts an undegraded carbon source (Day 0) and a degraded carbon source (Day 7), as utilized in the insoluble carbon source assays.



FIG. 15 depicts a decrease in the number of cows exhibiting greater than 200,000 somatic cell counts (SSC)/mL milk in dairy cows that were administered a microbial compostion of the present disclosure versus dairy cows that were not administered a microbial composition of the present disclosure.



FIG. 16 depicts a diagram that exemplifies how the diet influences the production of volatile fatty acids which in turn modulate milk production, body condition, growth, etc. Reproduced from Moran, 2005. Tropical dairy farming: feeding management for small holder dairy farmers in the humic tropics (Chapter 5), Landlinks Press, 312 pp.



FIG. 17 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 MIC scores discussed throughout the present disclosure.



FIG. 18 depicts the non-linearity of pounds of milk fate produced over the course of an experiment to determine rumen microbial community constituents that impact the production of milk fat in dairy cows.



FIG. 19 depicts the correlation of the absolute cell count with activity filter of target strain Ascus_713 to pounds (lbs) of milk fat produced.



FIG. 20 depicts the absolute cell count with activity filter of target strain Ascus_7 and the pounds (lbs) of milk fat produced over the course of an experiment.



FIG. 21 depicts the correlation of the relative abundance with no activity filter of target strain Ascus_3038 to pounds (lbs) of milk fat produced.





DETAILED DESCRIPTION
Definitions

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.


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.


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.


As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%.


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 or Table 3, 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.


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). 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.


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).


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).


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. 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. Pat. No. 7,427,408).


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.


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.


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.


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. 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. 2nd Ed. CRC Press. 504 pg.); E. W. Martin (1970. Remington's Pharmaceutical Sciences. 17th Ed. Mack Pub. Co.); and Blaser et al. (US Publication US20110280840A1).


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 B12 produced by microbes), incorporated herein by reference.


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.


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 mircrobiome).


As used herein, “probiotic” refers to a substantially pure microbe (i.e., a single isolate) or a mixture of desired microbes, and may also include any additional components that can be administered to a mammal for restoring microbiota. Probiotics or microbial inoculant compositions of the invention may be administered with an agent to allow the microbes to survive the environment of the gastrointestinal tract, i.e., 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.


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).


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.


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) could be amended.


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, deer, antelope, and others.


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


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 calculate ECM, as related to the present disclosure, is:





ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.2×protein pounds)


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 associated with application of a beneficial microbe, or consortia, of the disclosure can be demonstrated by comparing the milk produced by an ungulate treated by the microbes taught herein to the milk of an ungulate not treated. 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.”


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.


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.


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.


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


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 two alleles 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.


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.


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


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.


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.


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, the 16S 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).


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).


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. Desirable traits may also include other milk characteristics, including but 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 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; etc.


A trait may be inherited in a dominant or recessive manner, or in a partial or incomplete-dominant 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.


In the context of this disclosure, traits may also result from the interaction of one or more mammalian genes and one or more microorganism genes.


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.


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 (i.e., genotype) and the environment.


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.


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 naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.


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.


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 for their 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.


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 using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F. M. Ausubel et al., 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, Calif.). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.


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.


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.


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.


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 al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. 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 extracted from 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, N.Y.). 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) PCR Methods 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.


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.


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, 1M 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 et al., 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.


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.


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 compounds that are required during all stages of development. Non-limiting exemplary constitutive promoters include, CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.


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.


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.


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.


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.


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 from different 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).


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 an ungulate's yield of an economically important trait (e.g., milk, milk fat, milk proteins, etc.) 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.


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 Ser. No. 15/217,575, filed on Jul. 22, 2016 (issued as U.S. Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incorporated by reference in its entirety.


The maximal information coefficient (MIC) is then calculated between strains and metadata 3021a, and between strains 3021b; as seen in FIG. 17. 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 2023a, 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









multiplier[i] = 1









else









multiplier[i] = 0









end if



links_temp = multiplier*links



final_links = links_temp[links_temp != 0]



savetxt(output_file,final_links)



output_file.close( )










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.


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) only strains 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.


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).


Isolated Microbes

In some aspects, the present disclosure provides isolated microbes, including novel strains of microbes, presented in Table 1 and Table 3.


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


In some aspects, the disclosure provides for utilizing one or more microbes selected from Table 1 and Table 3 to increase a phenotypic trait of interest in a ruminant.


In some embodiments, the disclosure provides isolated microbial species belonging to taxonomic families of Clostridiaceae, Ruminococcaceae, Lachnospiraceae, Acidaminococcaceae, Peptococcaceae, Porphyromonadaceae, Prevotellaceae, Neocallimastigaceae, Saccharomycetaceae, Phaeosphaeriaceae, Erysipelotrichia, Anaerolinaeceae, Atopobiaceae, Botryosphaeriaceae, Eubacteriaceae, Acholeplasmataceae, Succinivibrionaceae, Lactobacillaceae, Selenomonadaceae, Burkholderiaceae, and Streptococcaceae.


In further embodiments, isolated microbial species may be selected from genera of family Clostridiaceae, including Acetanaerobacterium, Acetivibrio, Acidaminobacter, Alkahphilus, Anaerobacter, Anaerostipes, Anaerotruncus, Anoxynatronum, Bryantella, Butyricicoccus, Caldanaerocella, Caloramator, Caloranaerobacter, Caminicella, Candidatus Arthromitus, Clostridium, Coprobacillus, Dorea, Ethanologenbacterium, Faecalibacterium, Garciella, Guggenheimella, Hespellia, Linmingia, Natronincola, Oxobacter, Parasporobacterium, Sarcina, Soehngenia, Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicrobium, Thermobrachium, Thermohalobacter, and Tindallia.


In further embodiments, isolated microbial species may be selected from genera of family Ruminococcaceae, including Ruminococcus, Acetivibrio, Sporobacter, Anaerofilium, Papillibacter, Oscillospira, Gemmiger, Faecalibacterium, Fastidiosipila, Anaerotruncus, Ethanolingenens, Acetanaerobacterium, Subdoligranulum, Hydrogenoanaerobacterium, and Candidadus Soleaferrea.


In further embodiments, isolated microbial species may be selected from genera of family Lachnospiraceae, including Butyrivibrio, Roseburia, Lachnospira, Acetitomaculum, Coprococcus, Johnsonella, Catonella, Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, Parasporobacterium, Lachnobacterium, Shuttleworthia, Dorea, Anaerostipes, Hespellia, Marvinbryantia, Oribacterium, Moryella, Blautia, Robinsoniella, Cellulosilyticum, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter, Acetatifactor, and Eisenbergiella.


In further embodiments, isolated microbial species may be selected from genera of family Acidaminococcaceae, including Acidaminococcus, Phascolarctobacterium, Succiniclasticum, and Succinispira.


In further embodiments, isolated microbial species may be selected from genera of family Peptococcaceae, including Desulfotomaculum, Peptococcus, Desulfitobacterium, Syntrophobotulus, Dehalobacter, Sporotomaculum, Desulfosporosinus, Desulfonispora, Pelotomaculum, Thermincola, Cryptanaerobacter, Desulfitibacter, Candidatus Desulforudis, Desulfurispora, and Desulfitospora.


In further embodiments, isolated microbial species may be selected from genera of family Porphyromonadaceae, including Porphyromonas, Dysgonomonas, Tannerella, Odoribacter, Proteimphilum, Petrimonas, Paludibacter, Parabacteroides, Barnesiella, Candidatus Vestibaculum, Butyricimonas, Macellibacteroides, and Coprobacter.


In further embodiments, isolated microbial species may be selected from genera of family Anaerolinaeceae including Anaerolinea, Bellilinea, Leptolinea, Levilinea, Longilinea, Ornatilinea, and Pelolinea.


In further embodiments, isolated microbial species may be selected from genera of family Atopobiaceae including Atopbium and Olsenella.


In further embodiments, isolated microbial species may be selected from genera of family Eubacteriaceae including Acetobacterium, Alkalibacter, Alkalibaculum, Aminicella, Anaerofustis, Eubacterium, Gardella, and Pseudoramibacter.


In further embodiments, isolated microbial species may be selected from genera of family Acholeplasmataceae including Acholeplasma.


In further embodiments, isolated microbial species may be selected from genera of family Succinivibrionaceae including Anaerobiospirillum, Ruminobacter, Succinatimonas, Succinimonas, and Succinivibrio.


In further embodiments, isolated microbial species may be selected from genera of family Lactobacillaceae including Lactobacillus, Paralactobacillus, Pediococcus, and Sharpea.


In further embodiments, isolated microbial species may be selected from genera of family Selenomonadaceae including Anaerovibrio, Centipeda, Megamonas, Mitsuokella, Pectinatus, Propionispira, Schwartzia, Selenomonas, and Zymophilus.


In further embodiments, isolated microbial species may be selected from genera of family Burkholderiaceae including Burkholderia, Chitinimonas, Cupriavidus, Lautropia, Limnobacter, Pandoraea, Paraburkholderia, Paucimonas, Polynucleobacter, Ralstonia, Thermothrix, and Wautersia.


In further embodiments, isolated microbial species may be selected from genera of family Streptococcaceae including Lactococcus, Lactovum, and Streptococcus.


In further embodiments, isolated microbial species may be selected from genera of family Anaerolinaeceae including Aestuariimicrobium, Arachnia, Auraticoccus, Brooklawnia, Friedmanniella, Granulicoccus, Luteococcus, Mariniluteicoccus, Microlunatus, Micropruina, Naumannella, Propionibacterium, Propionicicella, Propioniciclava, Propioniferax, Propionimicrobium, and Tessaracoccus.


In further embodiments, isolated microbial species may be selected from genera of family Prevotellaceae, including Paraprevotella, Prevotella, hallella, Xylanibacter, and Alloprevotella.


In further embodiments, isolated microbial species may be selected from genera of family Neocallimastigaceae, including Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces, and Piromyces.


In further embodiments, isolated microbial species may be selected from genera of family Saccharomycetaceae, including Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania (syn. Arxiozyma), Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, and Zygotorulaspora.


In further embodiments, isolated microbial species may be selected from genera of family Erysipelotrichaceae, including Erysipelothrix, Solobacterium, Turicibacter, Faecalibaculum, Faecalicoccus, Faecalitalea, Holdemanella, Holdemania, Dielma, Eggerthia, Erysipelatoclostridium, Allobacterium, Breznakia, Bulleidia, Catenibacterium, Catenisphaera, and Coprobacillus.


In further embodiments, isolated microbial species may be selected from genera of family Phaeosphaeriaceae, including Barria, Bricookea, Carinispora, Chaetoplea, Eudarluca, Hadrospora, Isthmosporella, Katumotoa, Lautitia, Metameris, Mixtura, Neophaeosphaeria, Nodulosphaeria, Ophiosphaerella, Phaeosphaeris, Phaeosphaeriopsis, Setomelanomma, Stagonospora, Teratosphaeria, and Wilmia.


In further embodiments, isolated microbial species may be selected from genera of family Botryosphaeriaceae, including Amarenomyces, Aplosporella, Auerswaldiella, Botryosphaeria, Dichomera, Diplodia, Discochora, Dothidothia, Dothiorella, Fusicoccum, Granulodiplodia, Guignardia, Lasiodiplodia, Leptodothiorella, Leptodothiorella, Leptoguignardia, Macrophoma, Macrophomina, Nattrassia, Neodeightonia, Neofusicocum, Neoscytalidium, Otthia, Phaeobotryosphaeria, Phomatosphaeropsis, Phyllosticta, Pseudofusicoccum, Saccharata, Sivanesania, and Thyrostroma.


In some embodiments, the disclosure provides isolated microbial species belonging to genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. In further embodiments, the disclosure provides isolated microbial species belonging to the family of Lachnospiraceae, and the order of Saccharomycetales. In further embodiments, the disclosure provides isolated microbial species of Candida xylopsoci, Vrystaatia aloeicola, and Phyllosticta capitalensis.


In some embodiments, a microbe from the taxa disclosed herein are utilized to impart one or more beneficial properties or improved traits to milk in ruminants.


In some embodiments, the disclosure provides isolated microbial species, selected from the group consisting of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta.


In some embodiments, the disclosure provides novel isolated microbial strains of species, selected from the group consisting of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. Particular novel strains of these aforementioned taxonomic groups can be found in Table 1 and/or Table 3.


Furthermore, the disclosure relates to microbes having characteristics substantially similar to that of a microbe identified in Table 1 or Table 3.


The isolated microbial species, and novel strains of said species, identified in the present disclosure, are able to impart beneficial properties or traits to ruminant milk production.


For instance, the isolated microbes described in Table 1 and Table 3, or consortia of said microbes, are able to increase total milk fat in ruminant milk. The increase can be quantitatively measured, for example, by measuring the effect that said microbial application has upon the modulation of total milk fat.


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 heterologous polynucleotides. In further embodiments, the heterologous polynucleotides may be operably linked to one or more polynucleotides native to the microbes.


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), β-galactosidase, luciferase. In some embodiments, selectable markers may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, aminoglycoside adenyltransferase, dihydrofolate reductase, acetolactase synthase, bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, and chloramphenicol acetyltransferase. In some embodiments, the heterologous polynucleotide may be operably linked to one or more promoter.









TABLE 4





Taxa (largely Genera) of the present disclosure not


known to have been utilized in animal agriculture.



















Intestinimonas


Anaerolinea





Pseudobutyrivibrio


Olsenella





Eubacterium


Catenisphaera





Faecalibacterium


Solobacterium





Blautia


Ralsonia





Coprococcus


Casaltella





Anaeroplasma


Acholeplasma





Aminiphilus


Mitsuokella





Alistipes


Sharpea





Oscillibacter


Neocallimastix





Odoribacter


Pichia





Tannerella


Candida





Hydrogenoanaerobacterium


Orpinomyces





Succinivibrio


Sugiyamaella





Ruminobacter


Cyllamyces





Lachnospira


Caecomyces





Sinimarinibacterium


Tremella





Hydrogenoanaerobacterium


Turicibacter





Clostridium XIVa


Anaerolinea





Saccharofermentans


Piromyces





Butyricicoccus


Olsenella





Papillibacter


Clostridium XICa





Pelotomaculum


Erysipelotrichaceae





Lachnospiracea


Solobacterium





Anaeroplasma


Ralstonia





Clostridium


Eubacterium





Rikenella


Lachnobacterium





Tannerella


Acholeplasma





Howardella


Selenomonas





Butyricimonas


Sharpea





Succinivibrio


Phyllosticta





Ruminobacter


Candida xylopsoc





Syntrophococcus


Candida apicol





Pseudobutyrivibrio

Saccharomycetales




Ascomycota


Candida rugos











Microbial Consortia

In some aspects, the disclosure provides microbial consortia comprising a combination of at least any two microbes selected from amongst the microbes identified in Table 1 and/or Table 3.


In certain embodiments, the consortia 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.


In some embodiments, the disclosure provides consortia, comprising: at least two isolated microbial species belonging to genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. Particular novel strains of species of these aforementioned genera can be found in Table 1 and/or Table 3.


In some embodiments, the disclosure provides consortia, comprising: at least two isolated microbial species, selected from the group consisting of species of the family of Lachnospiraceae, and the order of Saccharomycetales.


In particular aspects, the disclosure provides microbial consortia, comprising species as grouped in Tables 5-11. With respect to Tables 5-11, the letters A through I represent a non-limiting selection of microbes of the present disclosure, defined as:


A=Strain designation Ascusb_7 identified in Table 1;


B=Strain designation Ascusb_3138 identified in Table 1;


C=Strain designation Ascusb_82 identified in Table 1;


D=Strain designation Ascusb_119 identified in Table 1;


E=Strain designation Ascusb_1801 identified in Table 1;


F=Strain designation Ascusf_23 identified in Table 1;


G=Strain designation Ascusf_24 identified in Table 1;


H=Strain designation Ascusf_45 identified in Table 1; and


I=Strain designation Ascusf_15 identified in Table 1.









TABLE 5





Eight and Nine Strain Consortia




















A, B, C, D, E, F, G, H
A, B, C, D, E, F, G, I
A, B, C, D, E, F, H, I
A, B, C, D, E, G, H, I
A, B, C, D, F, G, H, I
A, B, C, E, F, G, H, I


A, B, D, E, F, G, H, I
A, C, D, E, F, G, H, I
B, C, D, E, F, G, H, I
A, B, C, D, E, F, G, H, I
















TABLE 6





Seven Strain Consortia




















A, B, C, D, E, F, G
A, B, C, D, E, F, H
A, B, C, D, E, F, I
A, B, C, D, E, G, H
A, B, C, D, E, G, I
A, B, C, D, E, H, I


A, B, C, D, F, G, H
A, B, C, D, F, G, I
A, B, C, D, F, H, I
A, B, C, D, G, H, I
A, B, C, E, F, G, H
A, B, C, E, F, G, I


A, B, C, E, F, H, I
A, B, C, E, G, H, I
A, B, C, F, G, H, I
A, B, D, E, F, G, H
A, B, D, E, F, G, I
A, B, D, E, F, H, I


A, B, D, E, G, H, I
A, B, D, F, G, H, I
A, B, E, F, G, H, I
A, C, D, E, F, G, H
A, C, D, E, F, G, I
A, C, D, E, F, H, I


A, C, D, E, G, H, I
A, C, D, F, G, H, I
A, C, E, F, G, H, I
A, D, E, F, G, H, I
B, C, D, E, F, G, H
B, C, D, E, F, G, I


B, C, D, E, F, H, I
B, C, D, E, G, H, I
B, C, D, F, G, H, I
B, C, E, F, G, H, I
B, D, E, F, G, H, I
C, D, E, F, G, H, I
















TABLE 7





Six Strain Consortia





















A, B, C, D, E, F
A, B, C, D, E, G
A, B, C, D, E, H
A, B, C, D, E, I
A, B, C, D, F, G
A, B, C, D, F, H
A, B, C, D, F, I


A, B, C, D, G, H
A, B, C, D, G, I
A, B, C, D, H, I
A, B, C, E, F, G
A, B, C, E, F, H
A, B, C, E, F, I
A, B, C, E, G, H


A, B, C, E, G, I
A, B, C, E, H, I
A, B, C, F, G, H
A, B, C, F, G, I
A, B, C, F, H, I
A, B, C, G, H, I
A, B, D, E, F, G


A, B, D, E, F, H
A, B, D, E, F, I
A, B, D, E, G, H
A, B, D, E, G, I
A, B, D, E, H, I
A, B, D, F, G, H
A, B, D, F, G, I


D, E, F, G, H, I
C, E, F, G, H, I
A, B, D, F, H, I
A, B, D, G, H, I
A, B, E, F, G, H
A, B, E, F, G, I
A, B, E, F, H, I


A, B, E, G, H, I
A, B, F, G, H, I
A, C, D, E, F, G
A, C, D, E, F, H
A, C, D, E, F, I
A, C, D, E, G, H
A, C, D, E, G, I


A, C, D, E, H, I
A, C, D, F, G, H
A, C, D, F, G, I
A, C, D, F, H, I
A, C, D, G, H, I
A, C, E, F, G, H
A, C, E, F, G, I


A, C, E, F, H, I
A, C, E, G, H, I
A, C, F, G, H, I
A, D, E, F, G, H
A, D, E, F, G, I
A, D, E, F, H, I
A, D, E, G, H, I


A, D, F, G, H, I
A, E, F, G, H, I
B, C, D, E, F, G
B, C, D, E, F, H
B, C, D, E, F, I
B, C, D, E, G, H
B, C, D, E, G, I


B, C, D, E, H, I
B, C, D, F, G, H
B, C, D, F, G, I
B, C, D, F, H, I
B, C, D, G, H, I
B, C, E, F, G, H
B, C, E, F, G, I


B, C, E, F, H, I
B, C, E, G, H, I
B, C, F, G, H, I
B, D, E, F, G, H
B, D, E, F, G, I
B, D, E, F, H, I
B, D, E, G, H, I


B, D, F, G, H, I
B, E, F, G, H, I
C, D, E, F, G, H
C, D, E, F, G, I
C, D, E, F, H, I
C, D, E, G, H, I
C, D, F, G, H, I
















TABLE 8





Five Strain Consortia






















A, B, C, D, E
A, B, C, D, F
A, B, C, D, G
A, B, C, D, H
A, B, C, D, I
A, B, C, E, F
A, B, C, E, G
A, B, C, E, H


A, B, C, F, H
A, B, C, F, G
A, B, C, F, I
A, B, C, G, H
A, B, C, G, I
A, B, C, H, I
A, B, D, E, F
A, B, D, E, G


A, B, D, E, I
A, B, D, F, G
A, B, D, F, H
A, B, D, F, I
A, B, D, G, H
A, B, D, G, I
A, B, D, H, I
A, B, E, F, G


A, B, E, F, I
A, B, E, G, H
A, B, E, G, I
A, B, E, H, I
A, B, F, G, H
A, B, F, G, I
A, B, F, H, I
A, B, G, H, I


A, C, D, E, G
A, C, D, E, H
A, C, D, E, I
A, C, D, F, G
A, C, D, F, H
A, C, D, F, I
A, C, D, G, H
A, C, D, G, I


A, C, E, F, G
A, C, E, F, H
A, C, E, F, I
A, C, E, G, H
A, C, E, G, I
A, C, E, H, I
A, C, F, G, H
A, C, F, G, I


A, C, G, H, I
A, D, E, F, G
A, D, E, F, H
A, D, E, F, I
A, D, E, G, H
A, D, E, G, I
A, D, E, H, I
A, D, F, G, H


A, D, F, H, I
A, D, G, H, I
A, E, F, G, H
A, E, F, G, I
A, E, F, H, I
A, E, G, H, I
A, F, G, H, I
B, C, D, E, F


B, C, D, E, H
B, C, D, E, I
B, C, D, F, G
B, C, D, F, H
B, C, D, F, I
B, C, D, G, H
B, C, D, G, I
B, C, D, H, I


B, C, E, F, H
B, C, E, F, I
B, C, E, G, H
B, C, E, G, I
B, C, E, H, I
B, C, F, G, H
B, C, F, G, I
B, C, F, H, I


B, D, E, F, G
B, D, E, F, H
B, D, E, F, I
B, D, E, G, H
B, D, E, G, I
B, D, E, H, I
B, D, F, G, H
B, D, F, G, I


B, D, G, H, I
B, E, F, G, H
B, E, F, G, I
B, E, F, H, I
B, E, G, H, I
B, F, G, H, I
C, D, E, F, G
C, D, E, F, H


C, D, E, G, H
C, D, E, G, I
C, D, E, H, I
C, D, F, G, H
C, D, F, G, I
C, D, F, H, I
C, D, G, H, I
C, E, F, G, H


C, E, F, H, I
C, E, G, H, I
C, F, G, H, I
D, E, F, G, H
D, E, F, G, I
D, E, F, H, I
D, E, G, H, I
D, F, G, H, I


A, B, C, E, I
A, B, D, E, H
A, B, E, F, H
A, C, D, E, F
A, C, D, H, I
A, C, F, H, I
A, D, F, G, I
B, C, D, E, G


B, C, E, F, G
B, C, G, H, I
B, D, F, H, I
C, D, E, F, I
C, E, F, G, I
E, F, G, H, I
















TABLE 9





Four Strain Consortia























A, B, C, D
A, B, C, E
A, B, C, F
A, B, C, G
A, B, C, H
A, B, C, I
A, B, D, E
A, B, D, F
D, G, H, I


A, B, D, G
A, B, D, H
A, B, D, I
A, B, E, F
A, B, E, G
A, B, E, H
A, B, E, I
A, B, F, G
E, F, G, H


A, B, F, H
A, D, F, H
A, D, F, I
A, D, G, H
A, D, G, I
A, D, H, I
A, E, F, G
A, E, F, H
E, F, G, I


A, B, F, I
A, B, G, H
A, B, G, I
A, B, H, I
A, C, D, E
A, C, D, F
A, C, D, G
A, C, D, H
E, F, H, I


A, C, D, I
A, C, E, F
A, C, E, G
A, C, E, H
A, C, E, I
A, C, F, G
A, C, F, H
A, C, F, I
E, G, H, I


A, C, G, H
A, C, G, I
A, C, H, I
A, D, E, F
A, D, E, G
A, D, E, H
A, D, E, I
A, D, F, G
F, G, H, I


A, E, F, I
A, E, G, H
A, E, G, I
A, E, H, I
A, F, G, H
A, F, G, I
A, F, H, I
A, G, H, I
D, E, F, H


B, C, D, E
B, C, D, F
B, C, D, G
B, C, D, H
B, C, D, I
B, C, E, F
B, C, E, G
B, C, E, H
D, E, F, I


B, C, E, I
B, C, F, G
B, C, F, H
B, C, F, I
B, C, G, H
B, C, G, I
B, C, H, I
B, D, E, F
D, E, G, H


B, D, E, G
B, D, E, H
B, D, E, I
B, D, F, G
B, D, F, H
B, D, F, I
B, D, G, H
B, D, G, I
D, E, G, I


B, D, H, I
B, E, F, G
B, E, F, H
B, E, F, I
B, E, G, H
B, E, G, I
B, E, H, I
B, F, G, H
D, E, H, I


B, F, G, I
B, F, H, I
B, G, H, I
C, D, E, F
C, D, E, G
C, D, E, H
C, D, E, I
C, D, F, G
D, F, G, H


C, D, F, H
C, D, F, I
C, D, G, H
C, D, G, I
C, D, H, I
C, E, F, G
C, E, F, H
C, E, F, I
D, F, G, I


C, E, G, H
C, E, G, I
C, E, H, I
C, F, G, H
C, F, G, I
C, F, H, I
C, G, H, I
D, E, F, G
D, F, H, I
















TABLE 10





Three Strain Consortia

























A, B, C
A, B, D
A, B, E
A, B, F
A, B, G
A, B, H
A, B, I
A, C, D
A, C, E
G, H, I
E, F, H


A, C, F
A, C, G
A, C, H
A, C, I
A, D, E
A, D, F
A, D, G
A, D, H
A, D, I
F, H, I
E, F, G


A, E, F
A, E, G
A, E, H
A, E, I
A, F, G
A, F, H
A, F, I
A, G, H
A, G, I
F, G, I
D, H, I


A, H, I
B, C, D
B, C, E
B, C, F
B, C, G
B, C, H
B, C, I
B, D, E
B, D, F
F, G, H
D, G, I


B, D, G
B, D, H
B, D, I
B, E, F
B, E, G
B, E, H
B, E, I
B, F, G
B, F, H
E, H, I
E, F, I


B, F, I
B, G, H
B, G, I
B, H, I
C, D, E
C, D, F
C, D, G
C, D, H
C, D, I
E, G, I
D, G, H


C, E, F
C, E, G
C, E, H
C, E, I
C, F, G
C, F, H
C, F, I
C, G, H
C, G, I
E, G, H
D, F, I


C, H, I
D, E, F
D, E, G
D, E, H
D, E, I
D, F, G
D, F, H
















TABLE 11





Two Strain Consortia






























A, B
A, C
A, D
A, E
A, F
A, G
A, H
A, I
B, C
B, D
B, E
B, F
B, G
B, H
B, I
C, D


C, E
C, F
C, G
C, H
C, I
D, E
D, F
D, G
D, H
D, I
E, F
E, G
E, H
E, I
F, G
F, H


F, I
G, H
G, I
H, I









In some embodiments, the microbial consortia may be selected from any member group from Tables 5-11.


Isolated Microbes—Source Material

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


Isolated Microbes—Microbial Culture Techniques


The microbes of Table 1 and Table 3 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. PLOS 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).


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.


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.


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, Gherna, 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.


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, corn 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 of suitable 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, Mich. 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.


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.


Isolated Microbes—Microbial Strains

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. Microbial 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 ΔTm (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.


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.


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


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


In one embodiment, microbial strains of the present disclosure include those that comprise polynucleotide sequences that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs: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, 39, 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, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, and 2107. In a further embodiment, microbial strains of the present disclosure include those that comprise polynucleotide sequences that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs:1-2107.


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


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.


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.


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 describe growth 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.


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.


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.


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.


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.


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

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


In some embodiments, the microbial compositions include ruminant feed, such as cereals (barley, maize, oats, and the like); starches (tapioca and the like); oilseed cakes; and vegetable wastes. In some embodiments, the microbial compositions include vitamins, minerals, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickening agents, and the like.


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; products of vegetable origin such as cereal meals, tree bark meal, wood meal, and nutshell meal.


In some embodiments, the microbial compositions of the present disclosure are liquid. In further embodiments, the liquid comprises a solvent that may include water or an alcohol, 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.


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.


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 of the present disclosure comprise an animal-safe virucide or nematicide.


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 gumand 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 (U.S. Pat. No. 8,460,726B2).


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 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-p-hydroxy benzoate, methyl paraben, potassium acetate, potassium benzoiate, potassium bisulphite, potassium diacetate, potassium lactate, potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate, propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite, sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite, sodium salt of methyl-p-hydroxy benzoic acid, sodium salt of propyl-p-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).


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.


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.


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. fora 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.


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.


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 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 weeks.


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. fora 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.


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 30 to 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


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 to 100, 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.


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, about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12 to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28, about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12 to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34, about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14 to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16, about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16 to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20, about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18 to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22, about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20 to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22, about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22 to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34, about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26 to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28, about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30 to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34, or about 34 to 36 months.


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 308 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 to 30 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 to 34 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 to 36 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 to 36 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 to 34 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 to 30 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 to 36 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 to 34 30 to 32 32 to 36 32 to 34, or about 34 to 36.


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%


Encapsulation Compositions

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 ungulates. 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. (U.S. Pat. No. 5,104,662A), Ford (U.S. Pat. No. 5,733,568A), and Mosbach and Nilsson (U.S. Pat. No. 4,647,536A) for encapsulation compositions of microbes, and methods of encapsulating microbes.


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.


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.


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, corn 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.


Specific examples of fatty acids include linoleic acid, γ-linoleic acid, dihomo-γ-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, heptatriacontanoic acid, and octatriacontanoic acid.


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.


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


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.


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.


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, trisaccharides, 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 corn 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.


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.


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.


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, etc. 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.


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


Animal Feed

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, liquid, or semi-liquid.


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, the compositions of the present disclosure are mixed into the feed at the feed mill. In another embodiment, compositions of the present disclosure are mixed into the feed itself.


In some embodiments, feed of the present disclosure may be supplemented with water, premix or premixes, forage, fodder, 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, and mixtures thereof.


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.


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 and other medicaments; fermentation products, and other ingredients. In some embodiments, premixes are blended into the feed.


In some embodiments, the feed may include feed concentrates such as soybean hulls, sugar beet pulp, molasses, high protein soybean meal, ground corn, shelled corn, 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. Pat. No. 3,484,243) and Porter and Luhman (U.S. Pat. No. 9,179,694B2) for animal feed and animal feed supplements capable of use in the present compositions and methods.


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.


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

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.


In some embodiments, the microbial composition is administered in a dose comprise 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, 34 ml, 35 ml, 36 ml, 37 ml, 38 ml, 39 ml, 40 ml, 41 m, 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.


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 102 microbial cells.


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 102 to 1012, 103 to 1012, 104 to 1012, 105 to 1012, 106 to 1012, 107 to 1012, 108 to 1012, 109 to 1012, 1010 to 1012, 1011 to 1012, 102 to 1011, 103 to 1011, 104 to 1011, 105 to 1011, 106 to 1011, 107 to 1011, 108 to 1011, 109 to 1011, 1010 to 1011, 102 to 1010, 103 to 1010, 104 to 1010, 105 to 1010, 106 to 1010, 107 to 1010, 108 to 1010, 109 to 1010, 102 to 109, 103 to 109, 104 to 109, 105 to 109, 106 to 109, 107 to 109, 108 to 109, 102 to 108, 103 to 108, 104 to 108, 105 to 108, 106 to 108, 107 to 108, 102 to 107, 103 to 107, 104 to 107, 105 to 107, 106 to 107, 102 to 106, 103 to 106, 104 to 106, 105 to 106, 102 to 105, 103 to 105, 104 to 105, 102 to 104, 103 to 104, 102 to 103, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102 total microbial cells per gram or milliliter of the composition.


In some embodiments, the administered dose of the microbial composition comprises 102 to 1018, 103 to 1018, 104 to 1018, 105 to 1018, 106 to 1018, 107 to 1018, 108 to 1018, 109 to 1018, 1010 to 1018, 1011 to 1018, 1012 to 1018, 1011 to 1018, 1014 to 1018, 1015 to 1018, 1016 to 1018, 1017 to 1018, 102 to 1012, 103 to 1012, 104 to 1012, 105 to 1012, 106 to 1012, 107 to 1012, 108 to 1012, 109 to 1012, 1010 to 1012, 1011 to 1012, 102 to 1011, 103 to 1011, 104 to 1011, 105 to 1011, 106 to 1011, 107 to 1011, 108 to 1011, 109 to 1011, 1010 to 1011, 102 to 1010, 104 to 1010, 105 to 1010, 106 to 1010, 107 to 1010, 108 to 1010, 109 to 1010, 102 to 109, 103 to 109, 104 to 109, 105 to 109, 106 to 109, 107 to 109, 108 to 109, 102 to 108, 103 to 108, 104 to 108, 105 to 108, 106 to 108, 107 to 108, 102 to 107, 103 to 107, 104 to 107, 105 to 107, 106 to 107, 102 to 106, 103 to 106, 104 to 106, 105 to 106, 102 to 105, 103 to 105, 104 to 105, 102 to 104, 103 to 104, 102 to 103, 1018, 1017, 1016, 1015, 1014, 1013, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102 total microbial cells.


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.


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 week.


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.


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.


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 of mixing, 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.


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


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.


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.


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 Pat. 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.


In some embodiments, the microbes or microbial consortia 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 microbes or consortia 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.


In some embodiments, the microbes or microbial consortia 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/or esophagas of the animal, followed by the release/injection 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.


In some embodiments, the microbes or microbial consortia 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 microbes or microbial consortia into the buccal cavity and/or esophagas of the animal.


In some embodiments, the microbes or microbial consortia 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 microbes or microbial consortia 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.


In some embodiments, the microbes or microbial consortia 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.


In some embodiments, the microbes or microbial consortia 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 to 12, 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

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, protists, and viruses.


By way of example, the microorganisms may include species of the genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. The microorganisms may further include species belonging to the family of Lachnospiraceae, and the order of Saccharomycetales. In some embodiments, the microorganisms may include species of any genera disclosed herein.


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.


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).


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 an ungulate, or concentrated by filtering or centrifugation, diluted to an appropriate concentration and administered to the GI tract of an ungulate 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 ungulate to minimize the potential for damage to the animal.


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, cud, or other composition found in the gastrointestinal tract. In this embodiment, the source material may include one or more species of microorganisms.


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


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.


Whilst 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. Microorganisms can then be isolated from the enriched materials as disclosed above.


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, cud, feces, or growth media which includes the microorganisms identified to be of benefit to increased milk production in ungulates 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

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. The end products of primary degradation sustains a chain of microbes that ultimately produce a range of organic acids together with hydrogen and carbon dioxide. Because of the complex and interlinked nature of the microbiome, changing the diet and thus substrates for primary degradation may have a cascading effect on rumen 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.)


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


In some embodiments, the microbiome is shifted through the administration of one or more microbes to the gastrointestinal tract. In further embodiments, the one or more microbes are those selected from Table 1 or Table 3. 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 consortium.


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 fora 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, or 10 days.


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 fora 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, or 10 weeks.


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 fora 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.


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 or Table 3, and the corresponding rDNA sequences are those selected from SEQ ID NOs:1-60, SEQ ID NOs:2045-2107 and the SEQ ID NOs identified in Table 3.


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.)


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.


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. For example, in the case of a sample comprising a microbial community comprising bacteria (i.e., one microorganism type) and fungi (i.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).


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−1, 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.


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.


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 one embodiment, 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 charge-couple 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.


In another embodiment of in order to detect the presence and number of one or more microorganism types, the 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.


In another embodiment, the sample, or a portion thereof is subjected to Raman micro-spectroscopy 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 intrinsic biochemical “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.


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 density-gradient 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.


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 have been 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.


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 novo sequencing 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.


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.


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.


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).


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.


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.


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


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 β 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 L1 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 α subunit, ribosomal protein L15, ribosomal protein L25/L23, ribosomal protein L6 (rplF), ribosomal protein L11 (rplK), ribosomal protein L5 (rplE), ribosomal protein S12/S23, ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal protein S11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNA pseudouridine synthase B, ribosomal protein L18P/L5E, ribosomal protein S15P/S13e, Porphobilinogen deaminase, ribosomal protein S17, ribosomal protein L13 (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 assembly protein, 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, rplI 50S ribosomal protein L9, polyribonucleotide nucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 50S ribosomal protein L17, tRNA (guanine-N(1)-)-methyltransferase (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, rp1T 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 S18, DNA mismatch repair protein MutS, rpsT 30S ribosomal protein S20, DNA repair protein RecN, frr ribosome recycling factor (frr), recombination protein RecR, protein of unknown function UPF0054, miaA tRNA isopentenyltransferase, 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 reductoisomerase, 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, rpmI 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 505 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.


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.


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.


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.


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.


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).


Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodiment is used to determine absolute abundance 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.


Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp. 5463-5467, incorporated by reference 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.


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.


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. Pat. 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.


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 al. (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.


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, Calif.: 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) Next-generation 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 P1 adapter sequence so that the starting sequence of every fragment is both known and identical. Primers hybridize to the P1 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.


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 sensitive to 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.


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.


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 puposes). 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.


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) (Massol-Deya et al. (1995). Mol. Microb. Ecol. Manual. 3.3.2, pp. 1-18, incorporated by reference in its entirety for all puposes). rDNA fragments are generated by PCR using general primers, digested with restriction enzymes, electrophoresed in agarose or acrylamide gels, and stained with ethidium bromide or silver nitrate.


One fingerprinting technique used in detecting the presence and abundance of a unique first marker is single-stranded-conformation polymorphism (SSCP) (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 consortium, 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.


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.


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).


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 different DNA molecules influences the melting behaviour, 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.


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 is directly proportional to the abundance of the target organism. The universal high-density 16S microarray (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.


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.


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.


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).


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 B12).


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 as 13C or 15N) and added to the sample. Only microorganisms able to metabolize the substrate will incorporate it into their cells. Subsequently, 13C-DNA and 15N-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.


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 a 14C-labeled substrate, which during the course of growth becomes incorporated into microbial biomass. The 14C-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.


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-mass spectrometry (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).


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.


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.


In another aspect of the invention, 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).


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.


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 parameter within 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).


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.


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-Volterra modeling.


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.


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.


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 (i.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.


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


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.


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).


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 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-Volterra modeling.


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


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 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.


Bovine Pathogen Resistance and Clearance

In some aspects, the present disclosure is drawn to administering one or more microbial compositions described herein to cows 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 clears pathogens from the integument and the respiratory tract of cows, 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, inflammation, and/or incidence of liver disease.


In some embodiments, the microbial compositions of the present disclosure comprise one or more microbes that are present in the gastrointestinal tract of cows at a relative abundance of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%.


In some embodiments, after administration of microbial compositions of the present disclosure the one or more microbes are present in the gastrointestinal tract of the cow at a relative abundance of at least 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.


Pathogenic microbes of cows may include the following: Clostridium perfringens, Clostridium botulinum, Salmonella typi, Salmonella typhimurium, Salmonella enterica, Salmonella pullorum, Erysipelothrix insidiosa, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Listeria monocytogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, Mycoplasma sp., Citrobacter sp., Enterobacter sp., Pseudomonas aeruginosa, Pasteurella sp., Bacillus cereus, Bacillus licheniformis, Streptococcus uberis, Staphylococcus aureus, and pathogenic strains of Escherichia coli and Staphylococcus aureus. In some embodiments, the pathogenic microbes include viral pathogens. In some embodiments, the pathogenic microbes are pathogenic to both cows and humans. In some embodiments, the pathogenic microbes are pathogenic to either cows or humans.


In some embodiments, the administration of compositions of the present disclosure to cows modulate the makeup of the gastrointestinal microbiome such that the administered microbes outcompete microbial pathogens present in the gastrointestinal tract. In some embodiments, the administration of compositions of the present disclosure to cows harboring microbial pathogens outcompetes the pathogens and clears cows of the pathogens. In some embodiments, the administration of compositions of the present disclosure results in the stimulation of host immunity, and aid in clearance of the microbial pathogens. In some embodiments, the administration of compositions of the present disclosure introduce microbes that produce bacteriostatic and/or bactericidal components that decrease or clear the cows of the microbial pathogens. (U.S. Pat. No. 8,345,010).


In some embodiments, challenging cows with a microbial colonizer or microbial pathogen after administering one or more compositions of the present disclosure prevents the microbial colonizer or microbial pathogen from growing to a relative abundance of greater than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%. In further embodiments, challenging cows with a microbial colonizer or microbial pathogen after administering one or more compositions of the present disclosure prevents the microbial colonizer or microbial pathogen from colonizing cows.


In some embodiments, clearance of the microbial colonizer or microbial pathogen occurs in less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, or less than 2 days post administration of the one or more compositions of the present disclosure.


In some embodiments, clearance of the microbial colonizer or microbial pathogen occurs within 1-30 days, 1-25 days, 1-20 day, 1-15 days, 1-10 days, 1-5 days, 5-30 days, 5-25 days, 5-20 days, 5-15 days, 5-10 days, 10-30 days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 days, 15-20 days, 20-30 days, 20-25 days, or 25-30 days post administration of the one or more compositions of the present disclosure.


Improved Traits

In some aspects, the present disclosure is drawn to administering microbial compositions described herein to ruminants to improve one or more traits through the modulation of aspects of milk production, milk quantity, milk quality, ruminant digestive chemistry, and efficiency of feed utilization and digestibility.


In some embodiments, improving the quantity of milk fat produced by a ruminant is desirable, wherein milk fat includes triglycerides, triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, cholesterol, glycolipids, and free fatty acids. In further embodiments, free fatty acids include short chain fatty acids (i.e., C4:0, C6:0, and C8:0), medium chain fatty acids (i.e., C10:0, C10:1, C12:0, C14:0, C14:1, and C15:0), and long chain fatty acids (i.e., C16:0, C16:1, C17:0, C17:1, C18:0, C18:1, C18:2, C18:3, and C20:0). In further embodiments, it is desirable to achieve an increase in milk fat efficiency, which is measured by the total weight of milk fat produced, divided by the weight of feed ingested. The weight of milk fat produced is calculated from the measured fat percentage multiplied by the weight of milk produced.


In some embodiments, improving the quantity of carbohydrates in milk produced by a ruminant is desirable, wherein carbohydrates include lactose, glucose, galactose, and oligosaccharides. Tao et al. (2009. J. Dairy Sci. 92:2991-3001) disclose numerous oligosaccharides that may be found in bovine milk.


In some embodiments, improving the quantity of proteins in milk produced by a ruminant, wherein proteins include caseins and whey. In some embodiments, proteins of interest are only those proteins produced in milk. In other embodiments, proteins of interest are not required to be produced only in milk. Whey proteins include immunoglobulins, serum albumin, beta-lactoglobulin, and alpha-lactoglobulin.


In some embodiments, improving the quantity of vitamins in milk produced by a ruminant is desirable. Vitamins found in milk include the fat-soluble vitamins of A, D, E, and K; as well as the B vitamins found in the aqueous phase of the milk.


In some embodiments, improving the quantity of minerals in milk produced by a ruminant is desirable. Minerals found in milk include iron, zinc, copper, cobalt, magnesium, manganese, molybdenum, calcium, phosphorous, potassium, sodium, chlorine, and citric acid. Trace amounts of the following may be found in milk: aluminum, arsenic, boron, bromine, cadmium, chromium, fluorine, iodine, lead, nickel, selenium, silicon, silver, strontium, and vanadium.


In some embodiments, improving the milk yield and milk volume produced by a ruminant is desirable. In some embodiments, it is further desirable if the increase in milk yield and volume is not accompanied by simply an increase in solute volume.


In some embodiments improving energy-corrected milk (ECM) is desirable. In further embodiments, improving ECM amounts to increasing the calculated ECM output. In some embodiments, the ECM is calculated as follows: ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.2×protein pounds).


In some embodiments, improving the efficiency and digestibility of animal feed is desirable. In some embodiments, increasing the degradation of lignocellulosic components from animal feed is desirable. Lignocellulosic components include lignin, cellulose, and hemicellulose.


In some embodiments, increasing the concentration of fatty acids in the rumen of ruminants is desirable. Fatty acids include acetic acid, propionic acid, and butyric acid. In some embodiments, maintaining the pH balance in the rumen to prevent lysis of beneficial microbial consortia is desirable. In some embodiments, maintaining the pH balance in the rumen to prevent a reduction of beneficial microbial consortia is desirable.


In some embodiments, decreasing the amount of methane and manure produced by ruminants is desirable.


In some embodiments, improving the dry matter intake is desirable. In some embodiments, improving the efficiency of nitrogen utilization of the feed and dry matter ingested by ruminants is desirable.


In some embodiments, the improved traits of the present disclosure are the result of the administration of the presently described microbial compositions. It is thought that the microbial compositions modulate the microbiome of the ruminants such that the biochemistry of the rumen is changed in such a way that the ruminal liquid and solid substratum are more efficiently and more completely degraded into subcomponents and metabolites than the rumens of ruminants not having been administered microbial compositions of the present disclosure.


In some embodiments, the increase in efficiency and the increase of degradation of the ruminal substratum result in an increase in improved traits of the present disclosure.


Mode of Action: Digestibility Improvement in Ruminants

The rumen is a specialized stomach dedicated to the digestion of feed components in ruminants. A diverse microbial population inhabits the rumen, where their primary function revolves around converting the fibrous and non-fibrous carbohydrate components into useable sources of energy and protein (FIG. 16). Cellulose, in particular, forms up to 40% of plant biomass and is considered indigestible by mammals. It also is tightly associated with other structural carbohydrates, including hemicellulose, pectin, and lignin. The cellulolytic microbes in the rumen leverage extensive enzymatic activity in order break these molecules down into simple sugars and volatile fatty acids. This enzymatic activity is critical to the extraction of energy from feed, and more efficient degradation ultimately provides more energy to the animal. The soluble sugars found in the non-fibrous portion of the feed are also fermented into gases and volatile fatty acids such as butyrate, propionate, and acetate. Volatile fatty acids arising from the digestion of both the fibrous and non-fibrous components of feed are ultimately the main source of energy of the ruminant.


Individual fatty acids have been tested in ruminants in order to identify their impacts on varying aspects of production.


Acetate: Structural carbohydrates produce large amounts of acetate when degraded. An infusion of acetate directly into the rumen was shown to improve the yield of milk, as well as the amount of milk fat produced. Acetate represents at least 90% of acids in the peripheral blood—it is possible that acetate can be directly utilized by mammary tissue as a source of energy. See Rook and Balch. 1961. Brit. J. Nutr. 15:361-369.


Propionate: Propionate has been shown to increase milk protein production, but decrease milk yield. See Rook and Balch. 1961. Brit. J. Nutr. 15:361-369.


Butyrate: An infusion of butyrate directly into the rumen of dairy cows increases milk fat production without changing milk yield. See Huhtanen et al. 1993. J. Dairy Sci. 76:1114-1124.


Network Analysis

A 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 mutual information, maximal information coefficient (MIC) calculations, or other nonparametric methods between variables to establish connectivity. In another embodiment, network analysis comprises differential equation based modeling of populations. In yet another embodiment, network analysis comprises Lotka-Volterra modeling.


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 (i.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. In some embodiments, an environmental parameter is referred to as a metadata parameter.


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.


EXAMPLES
Example I
Increase Total Milk Fat, Milk Protein, and Energy-Corrected Milk (ECM) in Cows

The methods of Example I aim to increase the total amount of milk fat and milk protein produced by a lactating ruminant, and the calculated ECM.


The methodologies presented herein—based upon utilizing the disclosed isolated microbes, consortia, and compositions comprising the same—demonstrate an increase in the total amount of milk fat and milk protein produced by a lactating ruminant. These increases were realized without the need for further addition of hormones.


In this example, a microbial consortium comprising two isolated microbes, Ascusb_3138 (SEQ ID NO:28; deposited as NRRL Y-67248) and Ascusf_15 (SEQ ID NO:32; deposited as NRRL Y67249), was administered to Holstein cows in mid-stage lactation over a period of five weeks.


The cows were randomly assigned into 2 groups of 8, wherein one of the groups was a control group that received a buffer lacking a microbial consortium. The second group, the experimental group, was administered a microbial consortium comprising Ascusb_3138 (SEQ ID NO:28) and Ascusf_15 (SEQ ID NO:32) once per day for five weeks. Each of the cows were housed in individual pens and were given free access to feed and water. The diet was a high milk yield diet. Cows were fed ad libitum and the feed was weighed at the end of the day, and prior day refusals were weighed and discarded. Weighing was performed with a PS-2000 scale from Salter Brecknell (Fairmont, Minn.).


Cows were cannulated such that a cannula extended into the rumen of the cows. Cows were further provided at least 10 days of recovery post cannulation prior to administering control dosages or experimental dosages.


Each administration consisted of 20 ml of a neutral buffered saline, and each administration consisted of approximately 109 cells suspended in the saline. The control group received 20 ml of the saline once per day, while the experimental group received 20 ml of the saline further comprising 109 microbial cells of the described microbial consortium.


The rumen of every cow was sampled on days 0, 7, 14, 21, and 35, wherein day 0 was the day prior to microbial administration. Note that the experimental and control administrations were performed after the rumen was sampled on that day. Daily sampling of the rumen, beginning on day 0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) was inserted into the collected rumen fluid for recordings. Rumen sampling included both particulate and fluid sampling from the center, dorsal, ventral, anterior, and posterior regions of the rumen through the cannula, and all five samples were pooled into 15 ml conical vials containing 1.5 ml of stop solution (95% ethanol, 5% phenol). A fecal sample was also collected on each sampling day, wherein feces were collected from the rectum with the use of a palpation sleeve. Cows were weighed at the time of each sampling.


Fecal samples were placed in a 2 ounce vial, stored frozen, and analyzed to determine values for apparent neutral detergent fibers (NDF) digestibility, apparent starch digestibility, and apparent protein digestibility. Rumen sampling consisted of sampling both fluid and particulate portions of the rumen, each of which was stored in a 15 ml conical tube. Cells were fixed with a 10% stop solution (5% phenol/95% ethanol mixture) and kept at 4° C. and shipped to Ascus Biosciences (Vista, Calif.) on ice.


The milk yield was measured twice per day, once in the morning and once at night. Milk composition (% fats and % proteins, etc.) was measured twice per day, once in the morning and once at night. Milk samples were further analyzed with near-infrared spectroscopy for protein fats, solids, analysis for milk urea nitrogen (MUN), and somatic cell counts (SCC) at the Tulare Dairy Herd Improvement Association (DHIA) (Tulare, Calif.). Feed intake of individual cows and rumen pH were determined once per day.


A sample of the total mixed ration (TMR) was collected the final day of the adaptation period, and then successively collected once per week. Sampling was performed with the quartering method, wherein the samples were stored in vacuum sealed bags which were shipped to Cumberland Valley Analytical Services (Hagerstown, Md.) and analyzed with the NIR1 package.


The final day of administration of buffer and/or microbial bioconsortia was on day 35, however all other measurements and samplings continued as described until day 46.









TABLE 12







Dry matter intake, milk production and composition, body


weight (BW) gain, and rumen pH least square means (±SEM)


of cows assigned to Control and Inoculated groups.










Treatment











Control
Inoculated















Outcome





Dry matter intake, kg
26.2 ± 2.8 
30.2 ± 1.2 



Milk yield, kg
25.7 ± 1.9 
30.6 ± 1.9 



FCM, kg
27.7 ± 2.5 
32.5 ± 2.5 



ECM, kg
27.2 ± 2.4 
32.1 ± 2.4 



Milk components, %



Crude Protein
3.08 ± 0.06
3.27 ± 0.11



Fat
3.87 ± 0.08
4.06 ± 0.08



Lactose
4.64 ± 0.10
4.73 ± 0.03



Milk components yield, kg



Crude Protein
0.80 ± 0.07
0.97 ± 0.07



Fat
1.01 ± 0.10
1.20 ± 0.10



MUN, mg/dL
6.17 ± 0.60
7.41 ± 0.45



FCM/DMI
1.22 ± 0.07
1.10 ± 0.07



Body weight gain, kg/day
0.78 ± 0.44
1.46 ± 0.43



Rumen pH
6.24 ± 0.09
6.05 ± 0.09










Table 12 reveals the effects of daily administration of an Ascus microbial consortium on the performance of multiparous Holstein cows (between 60 and 120 days in milk). Marked differences between the control and inoculated treatments were observed. The inoculated group experienced increases in all parameters except FCM/DMI and rumen pH. The weekly values at the beginning of the intervention period when cows were still adapting to the treatment are included in the calculations.



FIGS. 4-7 demonstrate the significant effects of the microbial consortia on dairy cows for daily milk yield, daily milk crude protein yield, daily milk fat yield, and daily energy corrected milk yield over time. After an initial adaptation period, during which the microbes were observed to colonize the rumen, the production characteristics of the inoculated treatment group increased and diverged from the control group.



FIG. 3A demonstrates that cows that were administered the microbial consortia exhibited a 20.9% increase in the average production of milk fat versus cows that were administered the buffered solution alone. FIG. 3B demonstrates that cows that were administered the microbial consortia exhibited a 20.7% increase in the average production of milk protein versus cows that were administered the buffered solution alone. FIG. 3C demonstrates that cows that were administered the microbial consortia exhibited a 19.4% increase in the average production of energy corrected milk. The increases seen in FIG. 3A-C became less pronounced after the administration of the consortia ceased, as depicted by the vertical line intersecting the data points.



FIG. 14 clearly identifies the effect of microbial consortia on the somatic cell count in the milk. The experimental group of cows receiving the microbial consortia exhibited a decrease in the number of cows with greater than 200,000 somatic cells/ml of milk. In the field of dairy farming, the SCC is a strong indicator of milk quality. The majority of somatic cells found in milk are leukocytes, immune cells that accumulate in a particular tissue/fluid in increasing numbers usually due to an immune response to a pathogen. Generally, the lower the SCC the higher the quality of milk. Dosogne et al. 2011. J. Dairy Sci. 86(3):828-834.


Example II
Increase Total Milk Fat and Milk Protein in Cows

In certain embodiments of the disclosure, the present methods aim to increase the total amount of milk fat and milk protein produced by a lactating ruminant.


The methodologies presented herein—based upon utilizing the disclosed isolated microbes, consortia, and compositions comprising the same—have the potential to increase the total amount of milk fat and milk protein produced by a lactating ruminant. These increases can be realized without the need for further addition of hormones.


In this example, seven microbial consortia comprising isolated microbes from Table 1 are administered to Holstein cows in mid-stage lactation over a period of six weeks. The consortia are as follows:


Consortium 1—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_24;


Consortium 2—Ascusb_7, Ascusb_1801, Ascusf_45, and Ascusf_24;


Consortium 3—Ascusb_7, Ascusb_268, Ascusf_45, and Ascusf_24;


Consortium 4—Ascusb_7, Ascusb_232, Ascusf_45, and Ascusf_24;


Consortium 5—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_249;


Consortium 6—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_353; and


Consortium 7—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_23.


Consortium 8—Ascusb_3138, Ascusb_1801, Ascusf_45, and Ascusf_15.


Consortium 9—Ascusb_3138, Ascusb_268, Ascusf_45, and Ascusf_15.


Consortium 10—Ascusb_3138, Ascusb_232, Ascusf_23, and Ascusf_15.


Consortium 11—Ascusb_7, Ascusb_3138, Ascusf_15, and Ascusf_249.


Consortium 12—Ascusb_7, Ascusb_3138, Ascusf_45, and Ascusf_15.


Consortium 13—Ascusb_3138, Ascusb_32, Ascusf_15, and Ascusf_23.


Consortium 14—Ascusb_3138 and Ascusf_15.


The cows are randomly assigned into 15 groups of 8, wherein one of the groups is a control group that receives a buffer lacking a microbial consortium. The remaining seven groups are experimental groups and will each be administered one of the thirteen microbial bioconsortia once per day for six weeks. Each of the cows are held in individual pens to mitigate cross-contamination and are given free access to feed and water. The diet is a high milk yield diet. Cows are fed twice per day and the feed will be weighed at each feeding, and prior day refusals will be weighed and discarded. Weighing is performed with a PS-2000 scale from Salter Brecknell (Fairmont, Minn.).


Cows are cannulated such that a cannula extends into the rumen of the cows. Cows are further provided at least 10 days of recovery post cannulation prior to administering control dosages or experimental dosages.


Each administration consists of 5 ml of a neutral buffered saline, and each administration consists of approximately 109 cells suspended in the saline. The control group receives 5 ml of the saline once per day, while the experimental groups receive 5 ml of the saline further comprising 109 microbial cells of the described consortia.


The rumen of every cow is sampled on days 0, 7, 14, 21, and 35, wherein day 0 is the day prior to microbial administration. Note that the experimental and control administrations are performed after the rumen has been sampled on that day. Daily sampling of the rumen, beginning on day 0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) is inserted into the collected rumen fluid for recordings. Rumen sampling included both particulate and fluid sampling from the center, dorsal, ventral, anterior, and posterior regions of the rumen through the cannula, and all five samples were pooled into 15 ml conical vials containing 1.5 ml of stop solution (95% ethanol, 5% phenol). A fecal sample is also collected on each sampling day, wherein feces are collected from the rectum with the use of a palpation sleeve. Cows are weighed at the time of each sampling.


Fecal samples are placed in a 2 ounce vial, stored frozen, and analyzed to determine values for apparent NDF digestibility, apparent starch digestibility, and apparent protein digestibility. Rumen sampling consists of sampling both fluid and particulate portions of the rumen, each of which is stored in a 15 ml conical tube. Cells are fixed with a 10% stop solution (5% phenol/95% ethanol mixture) and kept at 4° C. and shipped to Ascus Biosciences (Vista, Calif.) on ice.


The milk yield is measured twice per day, once in the morning and once at night. Milk composition (% fats and % proteins, etc.) is measured twice per day, once in the morning and once at night. Milk samples are further analyzed with near-infrared spectroscopy for protein fats, solids, analysis for milk urea nitrogen (MUN), and somatic cell counts (SCC) at the Tulare Dairy Herd Improvement Association (DHIA) (Tulare, Calif.). Feed intake of individual cows and rumen pH are determined once per day.


A sample of the total mixed ration (TMR) is collected the final day of the adaptation period, and then successively collected once per week. Sampling is performed with the quartering method, wherein the samples are stored in vacuum sealed bags which are shipped to Cumberland Valley Analytical Services (Hagerstown, Md.) and analyzed with the NIR1 package.


In some embodiments, the percent fats and percent protein of milk in each of the experimental cow groups is expected to demonstrate a statistically significant increase over the percent fats and percent protein of milk in the control cow group. In other embodiments, the increase is not expected to be statistically significant, but it is expected to be still quantifiable.


Example III
Shifting the Microbiome of Ruminants

In certain embodiments of the disclosure, the present methods aim to modulate the microbiome of ruminants through the administration of one or more microbes to the gastrointestinal tract of ruminants.


The methodologies presented herein—based upon utilizing the disclosed isolated microbes, consortia, and compositions comprising the same—have the potential to modulate the microbiome of ruminants. The modulation of a ruminant's gastrointestinal microbiome may lead to an increase of desirable traits of the present disclosure.


In this example, the microbial consortia of Table 5 are administered to Holstein cows over a period of six weeks.


The cows are randomly assigned into 37 groups of 8, wherein one of the groups is a control group that receives a buffer lacking a microbial consortium. The remaining thirty-six groups are experimental groups and will each be administered one of the thirty-six microbial consortia once per day for six weeks. Each of the cows are held in individual pens to mitigate cross-contamination and are given free access to feed and water. The diet is a high milk yield diet. Cows are fed twice per day and the feed will be weighed at each feeding, and prior day refusals will be weighed and discarded. Weighing is performed with a PS-2000 scale from Salter Brecknell (Fairmont, Minn.).


Cows are cannulated such that a cannula extends into the rumen of the cows. Cows are further provided at least 10 days of recovery post cannulation prior to administering control dosages or experimental dosages.


Each administration consists of 5 ml of a neutral buffered saline, and each administration consists of approximately 109 cells suspended in the saline. The control group receives 5 ml of the saline once per day, while the experimental groups receive 5 ml of the saline further comprising 109 microbial cells of the described consortia.


The rumen of every cow is sampled on days 0, 7, 14, 21, and 35, wherein day 0 is the day prior to administration. Note that the experimental and control administrations are performed after the rumen has been sampled on that day. Daily sampling of the rumen, beginning on day 0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) is inserted into the collected rumen fluid for recordings. Rumen sampling included both particulate and fluid sampling from the center, dorsal, ventral, anterior, and posterior regions of the rumen through the cannula, and all five samples were pooled into 15 ml conical vials containing 1.5 ml of stop solution (95% ethanol, 5% phenol). A fecal sample is also collected on each sampling day, wherein feces are collected from the rectum with the use of a palpation sleeve. Cows are weighed at the time of each sampling.


Fecal samples are placed in a 2 ounce vial, stored frozen, and analyzed to determine values for apparent NDF digestibility, apparent starch digestibility, and apparent protein digestibility. Rumen sampling consists of sampling both fluid and particulate portions of the rumen, each of which is stored in a 15 ml conical tube. Cells are fixed with a 10% stop solution (5% phenol/95% ethanol mixture) and kept at 4° C. and shipped to Ascus Biosciences (Vista, Calif.) on ice.


The samples of fluid and particulate portions of the rumen, as well as the fecal samples are each evaluated for microbiome fingerprinting utilizing the T-RFLP method combined with nMDS ordination and PERMANOVA statistics.


In some embodiments, the ruminal and fecal microbiome in each of the experimental cow groups is expected to demonstrate a statistically significant change in the microbiomes over the microbiomes in the control cow group as well as the 0 day microbiome samples, wherein the change is a significant increase in the proportion of microbes administered in the experimental administrations. In other embodiments, the increase is not expected to be statistically significant, but it is expected to be still quantifiable.


Example IV
Milk Fat Produced Versus Absolute Abundance of Microbes

Determine rumen microbial community constituents that impact the production of milk fat in dairy cows.


Eight lactating, ruminally cannulated, Holstein cows were housed in individual tie-stalls for use in the experiment. Cows were fed twice daily, milked twice a day, and had continuous access to fresh water. One cow (cow 4201) was removed from the study after the first dietary Milk Fat Depression due to complications arising from an abortion prior to the experiment.


Experimental Design and Treatment: The experiment used a crossover design with 2 groups and 1 experimental period. The experimental period lasted 38 days: 10 days for the covariate/wash-out period and 28 days for data collection and sampling. The data collection period consisted of 10 days of dietary Milk Fat Depression (MFD) and 18 days of recovery. After the first experimental period, all cows underwent a 10-day wash out period prior to the beginning of period 2.


Dietary MFD was induced with a total mixed ration (TMR) low in fiber (29% NDF) with high starch degradability (70% degradable) and high polyunsaturated fatty acid levels (PUFA, 3.7%). The Recovery phase included two diets variable in starch degradability. Four cows were randomly assigned to the recovery diet high in fiber (37% NDF), low in PUFA (2.6%), and high in starch degradability (70% degradable). The remaining four cows were fed a recovery diet high in fiber (37% NDF), low in PUFA (2.6%), but low in starch degradability (35%).


During the 10-day covariate and 10-day wash out periods, cows were fed the high fiber, low PUFA, and low starch degradability diet.


Samples and Measurements: Milk yield, dry matter intake, and feed efficiency were measured daily for each animal throughout the covariate, wash out, and sample collection periods. TMR samples were measured for nutrient composition. During the collection period, milk samples were collected and analyzed every 3 days. Samples were analyzed for milk component concentrations (milk fat, milk protein, lactose, milk urea nitrogen, somatic cell counts, and solids) and fatty acid compositions.


Rumen samples were collected and analyzed for microbial community composition and activity every 3 days during the collection period. The rumen was intensively sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding during day 0, day 7, and day 10 of the dietary MFD. Similarly, the rumen was intensively sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding on day 16 and day 28 of the sample collection period. Rumen contents were analyzed for pH, acetate concentration, butyrate concentration, propionate concentration, isoacid concentration, and long chain and CLA isomer concentrations. Rumen sampling included both particulate and fluid sampling from the center, dorsal, ventral, anterior, and posterior regions of the rumen through the cannula, and all five samples were pooled into 15 ml conical vials.


Rumen Sample Preparation and Sequencing: After collection, rumen samples were centrifuged at 4,000 rpm in a swing bucket centrifuge for 20 minutes at 4° C. The supernatant was decanted, and an aliquot of each rumen content sample (1-2 mg) was added to a sterile 1.7 mL tube prefilled with 0.1 mm glass beads. A second aliquot was collected and stored in an empty, sterile 1.7 mL tube for cell counting.


Rumen samples in empty tubes were stained and put through a flow cytometer to quantify the number of cells of each microorganism type in each sample. Rumen samples with glass beads were homogenized with bead beating to lyse microorganisms. DNA and RNA was extracted and purified from each sample and prepared for sequencing on an Illumina Miseq. Samples were sequenced using paired-end chemistry, with 300 base pairs sequenced on each end of the library.


Sequencing Read Processing and Data Analysis: Sequencing reads were quality trimmed and processed to identify bacterial species present in the rumen based on a marker gene, 16S rDNA, or ITS1 and/or ITS2. Count datasets and activity datasets were integrated with the sequencing reads to determine the absolute cell numbers of active microbial species within the rumen microbial community. Production characteristics of the cow over time, including pounds of milk produced, were linked to the distribution of active microorganisms within each sample over the course of the experiment using mutual information.


Tests cases to determine the impact of count data, activity data, and count and activity on the final output were run by omitting the appropriate datasets from the sequencing analysis. To assess the impact of using a linear correlation rather than the MIC on target selection, Pearson's coefficients were also calculated for pounds of milk fat produced as compared to the relative abundance of all microorganisms and the absolute abundance of active microorganisms.


Results

One component of the Ascus Biosciences technology utilized in this application leverages mutual information to rank the importantce of native microbial strains residing in the gastrointestinal tract of the animal to specific animal traits. The maximal information coefficient (MIC) scores are calculated for all microorganisms and the desired animal trait. Relationships were scored on a scale of 0 to 1, with 1 represending a strong relationship between the microbial strain and the animal trait, and 0 representing no relationship. A cut-off based on this score is used to define useful and non-useful microorganisms with respect to the improvement of specific traits. FIG. 9 and FIG. 10 depict the MIC score distribution for rumen microbial strains that share a relationship with milk fat efficiency in dairy cows. The point where the curve shifts from exponential to linear (˜0.45-0.5 for bacteria and ˜0.3 for fungi) represents the cutoff between useful and non-useful microorganism strains pertaining to milk fat efficiency. FIG. 11 and FIG. 12 depict the MIC score distributions for rumen microbial strains that share a relationship with dairy efficiency. The point where the curve shifts from exponential to linear (˜0.45-0.5 for bacteria and ˜0.25 for fungi) represents the cutoff between useful and non-useful microorganism strains.


The MICs were calculated between pounds of milk fat produced and the absolute abundance of each active microorganism. Microorganisms were ranked by MIC score, and microorganisms with the highest MIC scores were selected as the target species most relevant to pounds of milk produced. MIC scores of the microbes of the present disclosure are recited in Table 1. The greater the MIC score, the greater the ability of the microbe to confer an increase in the weight of milk fat produced by a cow


Example V
Comparative Analysis of MIC Scores From Published Work of Other
Groups

Utilizing Ascus Biosciences' technology, the performance of currently available microbial feed additive products was predicted.


Direct-fed microbial products that claim to enhance dairy performance are openly available on the market. Some of these products contain microorganism strains that are native rumen microorganisms (Megasphaera elsdenii), or are within 97% sequence similarity of native rumen microorganisms. We have identified the species of microbes utilized in these products, and calculated their MIC score with respect to milk fat efficiency (FIG. 13). As evidenced by the curve in FIG. 13, all of the assayed strains that were available fell below the threshold used to define useful and non-useful strains, as describe above. The species/strain closest to the cutoff, Prevotella bryantii, has shown a positive effect in one study.



Lactobacillus plantarum: MIC 0.28402


The calculated MIC predicts that Lactobacillus plantarum is poorly associated with milk fat efficiency, and the art discloses that an inoculation of L. plantarum yields no increase in milk fat product, and at least one study discloses that some strains of L. plantarum create molecules that cause milk fat depression. See Lee et al. 2007. J. Appl. Microbiol. 103(4):1140-1146 and Mohammed et al. 2012. J. Dairy Sci. 95(1):328-339.



Lactobacillus acidophilus: MIC 0.30048


The calculated MIC predicts that Lactobacillus acidophilus is poorly associated with milk fat efficiency, and the art discloses that the administration of L. acidophilus to dairy cows/calves had no effect of various aspects of milk yield/milk component yield. See Higginbotham and Bath. 1993. J. Dairy Sci. 76(2):615-620; Abu-Tarboush et al. 1996. Animal Feed Sci. Tech. 57(1-2):39-49; McGilliard and Stallings. 1998. J. Dairy Sci. 81(5):1353-1357; and Raeth-Knight et al. 2007. J. Dairy Sci. 90(4):1802-1809; But see Boyd et al. 2011. 94(9):4616-4622 (discloses an increase in milk yield and milk protein yield).While Boyd et al. does disclose an increase in milk and milk protein yield, the controls of this single study do not appear to adequately isolate the the presence of L. acidophilus as the cause of the increase. The body of prior art contradicts the finding of Boyd et al.



Megasphaera elsdenii: MIC 0.32548


The calculated MIC predicts that Megasphaera elsdenii is poorly associated with milk fat efficiency, and the art provides substantial evidence to suggest that Megasphaera elsdenii has no positive effect upon milk fat efficiency, but multiple references provide evidence to suggest that it has a negative effect on milk fat efficiency. See Kim et al. 2002. J. Appl. Micro. 92(5):976-982; Hagg. 2008. Dissertation, University of Pretoria. 1-72; Hagg et al. 2010. S. African. J. Animal Sci. 40(2):101-112; Zebeli et al. 2011. J. Dairy Res. 79(1):16-25; Aikman et al. 2011. J. Dairy Sci. 94(6):2840-2849; Mohammed et al. 2012. J. Dairy Sci. 95(1):328-339; and Cacite and Weimer. 2016. J. Animal Sci. Poster Abstract. 94(sup. 5):784.



Prevotella bryantii: MIC 0.40161


The calculated MIC predicts that Prevotella bryantii is not highly associated with milk fat efficiency, and the art provides evidence that P. bryantii administered during subacute acidosis challenge in midlactation dairy cows has no apparent effect on milk yield, whereas administration of the microbe to dairy cows in early lactation yields improved milk fat concentrations. See Chiquette et al. 2012. J. Dairy Sci. 95(10):5985-5995, but see Chiquette et al. 2008. 91(9):3536-3543; respectively.


Example VI
Shift in Rumen Microbial Composition after Administration of a Microbial Composition

The methods of the instant example aim to increase the total amount of milk fat and milk protein produced by a lactating ruminant, and the calculated energy corrected milk (ECM).


The methodologies presented herein—based upon utilizing the disclosed isolated microbes, consortia, and compositions comprising the same—demonstrate an increase in the total amount of milk fat and milk protein produced by a lactating ruminant. These increases were realized without the need for further addition of hormones.


In this example, a microbial consortium comprising two isolated microbes, Ascusb_3138 (SEQ ID NO:28) and Ascusf_15 (SEQ ID NO:32), was administered to Holstein cows in mid-stage lactation over a period of five weeks.


The cows were randomly assigned into 2 groups of 8, in which one of the groups was a control group that received a buffer lacking a microbial consortium. The second group, the experimental group, was administered a microbial consortium comprising Ascusb_3138 (SEQ ID NO:28) and Ascusf_15 (SEQ ID NO:32) once per day for five weeks. Each cow was housed in an individual pen and was given free access to feed and water. The diet was a high milk yield diet. Cows were fed ad libitum and the feed was weighed at the end of each day, and prior day refusals were weighed and discarded. Weighing was performed with a PS-2000 scale from Salter Brecknell (Fairmont, Minn.).


Cows were cannulated such that a cannula extended into the rumen of the cows. Cows were further provided at least 10 days of recovery post cannulation prior to administering control dosages or experimental dosages.


Each administration consisted of 20 ml of a neutral buffered saline, and each administration consisted of approximately 109 cells suspended in the saline. The control group received 20 ml of the saline once per day, while the experimental group received 20 ml of the saline further comprising 109 microbial cells of the described microbial consortium.


The rumen of every cow was sampled on days 0, 7, 14, 21, and 35, wherein day 0 was the day prior to microbial administration. Note that the experimental and control administrations were performed after the rumen was sampled on that day. Daily sampling of the rumen, beginning on day 0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) was inserted into the collected rumen fluid for recordings. Rumen sampling included both particulate and fluid sampling from the center, dorsal, ventral, anterior, and posterior regions of the rumen through the cannula, and all five samples were pooled into 15 ml conical vials containing 1.5 ml of stop solution (95% ethanol, 5% phenol) and stored at 4° C. and shipped to Ascus Biosciences (Vista, Calif.) on ice.


A portion of each rumen sample was stained and put through a flow cytometer to quantify the number of cells of each microorganism type in each sample. A separate portion of the same rumen sample was homogenized with bead beating to lyse microorganisms. DNA and RNA was extracted and purified from each sample and prepared for sequencing on an Illumina Miseq. Samples were sequenced using paired-end chemistry, with 300 base pairs sequenced on each end of the library. The sequencing reads were used to quantify the number of cells of each active, microbial member present in each animal rumen in the control and experimental groups over the course of the experiment.


Ascusb_3138 and Ascusf_15 both colonized, and were active in the rumen after ˜3-5 days of daily administration, depending on the animal. This colonization was observed in the experimental group, but not in the control group. The rumen is a dynamic environment, where the chemistry of the cumulative rumen microbial population is highly intertwined. The artificial addition of Ascusb_3138 and Ascuf_15 could have effects on the overall structure of the community. To assess this potential impact, the entire microbial community was analyzed over the course of the experiment to identify higher level taxonomic shifts in microbial community population.


Distinct trends were not observed in the fungal populations over time, aside from the higher cell numbers of Ascusf_15 in the experimental animals. The bacterial populations, however, did change more predictably. To assess high level trends across individual animals over time, percent compositions of the microbial populations were calculated and compared. See


Table 13. Only genera composing greater than 1% of the community were analyzed. The percent composition of genera containing known fiber-degrading bacteria, including Ruminococcus, was found to increase in experimental animals as compared to control animals. Volatile fatty acid-producing genera, including Clostridial cluster XIVa, Clostridium, Pseudobutyrivibrio, Butyricimonas, and Lachnospira were also found at higher abundances in the experimental animals. The greatest shift was observed in the genera Prevotella. Members of this genus have been shown to be involved in the digestion of cellobiose, pectin, and various other structural carbohydrates within the rumen. Prevotella sp. have further been implicated in the conversion of plant lignins into beneficial antioxidants (Schogor et al. PLOS One. 9(4):e87949 (10 p.)).


To more directly measure quantitative changes in the rumen over time, cell count data was integrated with sequencing data to identify bulk changes in the population at the cell level. Fold changes in cell numbers were determined by dividing the average number of cells of each genera in the experimental group by the average number of cells of each genera in the control group. See Table 13. The cell count analysis captured many genera that fell under the threshold in the previous analysis Promicromonospora, Rhodopirellula, Olivibacter, Victivallis, Nocardia, Lentisphaera, Eubacteiru, Pedobacter, Butyricimonas, Mogibacterium, and Desulfovibrio were all found to be at least 10 fold higher on average in the experimental animals. Prevotella, Lachnospira, Butyricicoccus, Clostridium XIVa, Roseburia, Clostridium_sensu_stricto, and Pseudobutyrivibrio were found to be ˜1.5 times higher in the experimental animals.









TABLE 13







Family and Genus Level Analysis of Shifts in Bacterial Populations














Experi-




Control
Varia-
mental
Varia-


Taxonomy
(%)
tion
(%)
tion










Family Level Analysis











Prevotellaceae
15.27
6.43
18.62
5.63


Ruminococcaceae
16.40
5.14
17.84
6.44


Lachnospiraceae
23.85
7.63
24.58
6.96







Genus Level Analysis












Prevotella

16.14
5.98
19.14
5.27


Clostridium_XIVa
12.41
5.35
12.83
4.81


Lachnospiracea_incertae_sedis
3.68
1.68
3.93
1.33



Ruminococcus

3.70
2.21
3.82
1.82


Clostridium_IV
3.02
1.87
3.51
1.74



Butyricimonas

1.68
1.35
1.83
2.38


Clostridium_sensu_stricto
1.52
0.65
1.81
0.53



Pseudobutyrivibrio

1.00
0.64
1.42
1.03



Citrobacter

0.71
1.86
1.95
3.00



Selenomonas

1.04
0.83
1.34
0.86



Hydrogeno-

1.03
1.08
1.11
0.78



anaerobacterium

















TABLE 14







Analysis of Fold Changes in Bacterial cells








Genus
Fold change (experimental/control)












Promicromonospora

22619.50



Rhodopirellula

643.31



Olivibacter

394.01



Victivallis

83.97



Nocardia

73.81



Lentisphaera

57.70



Eubacterium

50.19



Pedobacter

26.15



Butyricimonas

15.47



Mogibacterium

15.23



Desulfovibrio

13.55



Anaeroplasma

8.84



Sharpea

8.78


Erysipelotrichaceae_incertae_sedis
5.71



Saccharofermentans

5.09



Parabacteroides

4.16



Papillibacter

3.63



Citrobacter

2.95


Lachnospiracea_incertae_sedis
2.27



Prevotella

1.60



Butyricicoccus

1.95


Clostridium_XIVa
1.47



Roseburia

1.44



Pseudobutyrivibrio

1.43


Clostridium_sensu_stricto
1.29



Selenomonas

1.25



Olsenella

1.04









Example VII
Analysis of Rumen Microbes for Volatile Fatty Acid Production and Carbon Source Use
A. Volatile Fatty Acid (VFA) Production

To assess the ability of the strains to produce volatile fatty acids, High Performance Liquid Chromatography (HPLC) was utilized to measure the conventration sof acetate, butyrate, and propionate in spent media. M2GSC media was used in an assay mimicking rumen conditions as closely as possibile.


For pure cultures, a single colony from each of the desired strains (from anaerobic agar plates) was inoculated into M2GSC media. A medium blank (control) was also prepared. Cultures and the medium blank were incubated at 37° C. until significant growth was visible. An optical density (OD600) was determined for each culture, and the strain ID was confirmed with Illumina sequencing. An aliquot of culture was subjected to sterile filtration into a washed glass 15 ml sample vial and analyzed by HPLC; HPLC assays were performed at Michigan State University. Enrichments that exhibited growth were also stained and cell counted to confirm that the individual strains within each enrichment grew. Strains often appeared in multiple enrichments, so the enrichment with the highest amount of growth for the strain (i.e. the highest increase in cell number of that strain) is reported in Table 15.


Due to the vast complexity of metabolisms and microbial lifestyles present in the rumen, many rumen microorganisms are incapable of axenic growth. In order to assay these organisms for desirable characteristics, enrichments cultures were established under a variety of conditions that mimicked particular features of the rumen environment. Diluted rumen fluid ( 1/100 dilution) was inoculated into M2GSC or M2 media supplemented with a variety of carbon sources including xylose (4 g/L), mannitol (4 g/L), glycerol (4 g/L), xylan (2 g/L), cellobiose (2 g/L), arabinose (4 g/L), mannose (4 g/L), rhaminose (2 g/L), maltose (2 g/L), maltose (2 g/L), and molasses. Rumen fluid was also sometimes omitted from the recipe. Additions including amino acids, volatile fatty acids, and antibiotics, were also varied across the enrichments. A medium blank (control) was also prepared. Cultures and the medium blank were incubated at 37° C. until significant growth was visible. An optical density (OD600) was determined for each culture, and the strain IDs were confirmed with Illumina sequencing. An aliquot of culture was subjected to sterile filtration into a washed glass 15 ml sample vial and analyzed by HPLC; HPLC assays were performed at Michigan State University. Enrichments that exhibited growth were also stained and cell counted to confirm that the individual strains within each enrichment grew. Strains often appeared in multiple enrichments, so the enrichment with the highest amount of growth for the strain (i.e, the highest increase in cell number of that strain) is reported in Table 15.


Concentrations of acetate, butyrate, and propionate were quantified for the medium blanks as well as the sterile filtered culture samples for both pure strain and enrichment experiments. HPLC parameters were as follows: Biorad Aminex HPX-87H column, 60° C., 0.5 ml/minute mobile phase 0.00325 N H2SO4, 500 psi, 35C RI detector, 45 minute run time, and 5 μL injection volume. Concentrations of acetate, butyrate, and propionate for both pure cultures and enrichments are reported in Table 15.









TABLE 15







Volatile Fatty Acid Production of Microbial Strains


as Analyzed with HPLC, Normalized to 1 OD










Sample ID
Acetate (g/L)
Propionate (g/L)
Butyrate (g/L)













Ascusb_5
3.59
0.00
0.00


Ascusb_7
1.54
4.08
0.03


Ascusb_11
−6.88
−0.28
−0.04


Ascusb_26
6.10
7.57
1.38


Ascusb_27
0.59
1.48
4.98


Ascusb_32
6.10
7.57
1.38


Ascusb_36
4.30
0.68
0.00


Ascusb_79
2.00
0.00
0.00


Ascusb_82
6.10
7.57
1.38


Ascusb_89
1.69
4.20
0.27


Ascusb_101
1.45
−0.21
0.00


Ascusb_102
2.00
0.00
0.00


Ascusb_104
27.13
34.55
3.31


Ascusb_111
1.69
4.20
0.27


Ascusb_119
1.54
4.08
0.03


Ascusb_125
10.97
5.68
4.69


Ascusb_145
1.69
4.20
0.27


Ascusb_149
0.00
0.00
0.47


Ascusb_159
7.05
4.52
1.42


Ascusb_183
0.00
0.00
2.03


Ascusb_187
10.97
5.68
4.69


Ascusb_190
7.40
7.36
7.91


Ascusb_199
11.36
1.17
7.65


Ascusb_205
6.10
7.57
1.38


Ascusb_232
7.83
1.15
3.19


Ascusb_268
2.00
0.00
0.00


Ascusb_278
7.05
4.52
1.42


Ascusb_329
7.83
1.15
3.19


Ascusb_368
1.69
4.20
0.27


Ascusb_374
7.83
1.15
3.19


Ascusb_411
1.69
4.20
0.27


Ascusb_546
4.30
0.68
0.00


Ascusb_728
2.36
0.00
0.00


Ascusb_765
−11.63
0.00
0.00


Ascusb_810
1.54
4.08
0.03


Ascusb_812
2.00
0.00
0.00


Ascusb_817
1.16
0.00
0.09


Ascusb_826
0.42
0.00
0.51


Ascusb_880
−0.12
0.00
0.00


Ascusb_913
10.97
5.68
4.69


Ascusb_974
4.30
0.68
0.00


Ascusb_1069
0.00
0.00
2.32


Ascusb_1074
7.05
4.52
1.42


Ascusb_1295
1.54
4.08
0.03


Ascusb_1367
7.40
7.36
7.91


Ascusb_1632
1.54
4.08
0.03


Ascusb_1674
0.68
0.30
0.00


Ascusb_1763
1.69
4.20
0.27


Ascusb_1780
1.32
0.00
0.21


Ascusb_1786
1.69
4.20
0.27


Ascusb_1801
5.47
26.95
−0.60


Ascusb_1812
1.54
4.08
0.03


Ascusb_1833
7.83
1.15
3.19


Ascusb_1850
1.32
0.00
0.21


Ascusb_2090
1.54
4.08
0.03


Ascusb_2124
1.69
4.20
0.27


Ascusb_2511
0.00
0.00
0.11


Ascusb_2530
11.36
1.17
7.65


Ascusb_2597
4.30
0.68
0.00


Ascusb_2624
0.00
0.00
0.00


Ascusb_2667
3.16
1.46
1.02


Ascusb_2836
1.32
0.00
0.21


Ascusb_3003
0.00
0.00
0.11


Ascusb_3138
0.00
0.00
2.50


Ascusb_3504
1.69
4.20
0.27


Ascusb_3881
7.05
4.52
1.42


Ascusb_6589
5.47
26.95
−0.60


Ascusb_12103
0.94
0.00
0.00


Ascusb_14245
1.76
0.00
0.00


Ascusb_20083
27.13
34.55
3.31


Ascusb_20187
7.40
7.36
7.91









B. Soluble Carbon Source Assay

To assess the ability of the strains to degrade various carbon sources, an optical density (OD600) was used to measure growth of strains on multiple carbon sources over time.


For pure isolates, a single colony from each of the desired strains (from anaerobic agar plates) was inoculated into M2GSC media. A medium blank (control) was also prepared. Strains were inoculated into a carbon source assay anaerobically, wherein the assay was set up in a 2 mL sterile 96-well plate, with each well containing RAMM salts, vitamins, minerals, cysteine, and a single carbon source. Carbon sources included glucose, xylan, lactate, xylose, mannose, glycerol, pectin, molasses, and cellobiose. Cells were inoculated such that each well started at an OD600 of 0.01. Optican densities were read at 600 nm with the Synergy H4 hybrid plate reader. The strain IDs were confirmed with Illumina sequencing after all wells were in stationary phase.


As in the volatile fatty acid assay above, enrichments were also used to assay carbon source degradation. Diluted rumen fluid ( 1/100 dilution) was inoculated into M2GSC or M2 media supplemented with a variety of carbon sources including xylose (4 g/L), mannitol (4 g/L), glycerol (4 g/L), xylan (2 g/L), cellobiose (2 g/L), arabinose (4 g/L), mannose (4 g/L), rhaminose (2 g/L), maltose (2 g/L), maltose (2 g/L), and molasses. Rumen fluid was also sometimes omitted from the recipe. Additions including amino acids, volatile fatty acids, and antibiotics, were also varied across the enrichments. A medium blank (control) was also prepared. Cultures and the medium blank were incubated at 37° C. until significant growth was visible. An optical density (OD600) was determined for each culture, and the strain IDs were confirmed with Illumina sequencing. Enrichments that exhibited growth were also stained and cell counted to confirm that the individual strains within each enrichment grew.


C. Insoluble Carbon Source Assay

To assess the ability of the strains to degrade insoluble carbon sources, visual inspection was leveraged to qualitatively determine a strain's degradation capabilities.


For pure cultures, a single colony from each of the desired strains (from anaerobic agar plates) was inoculated into anaerobic Hungate tubes containing Lowe's semi defined media with cellulose paper, starch, or grass as the sole carbon source. (Lowe et al. 1985. J. Gen. Microbiol. 131:2225-2229). Enrichment cultures using a 1/100 dilutiopn of rumen fluid were also set up using the same medium conditions. Cultures were checked visually for degradation of insoluble carbon sources (See FIG. 14). Strain ID was confirmed using Illumina sequencing. Enrichments that exhibited growth were also stained and cell counted to confirm that the individual strains within each enrichment grew.









TABLE 16







Analysis of Degradation of Various Soluable and Non-Soluable Carbon Sources by Strains of the Present Disclosure


















Strain ID
D-Glucose
Xylan
Lactate
D-Xylose
D-Mannose
Glycerol
Pectin
Molasses
Cellobiose
Cellulose
Starch





Ascusb_5
+
+

+
+
+
+

+
Unknown
Unknown


Ascusb_7
+

+


+


+
Unknown
Unknown


Ascusb_11



+

+
+
+
+
Unknown
Unknown


Ascusb_26
+

+


+


+
Unknown
Unknown


Ascusb_27
+








Unknown
Unknown


Ascusb_32
+

+

+
+


+
Unknown
Unknown


Ascusb_36

+



+



Unknown
Unknown


Ascusb_79
+




+


+
Unknown
Unknown


Ascusb_82
+
+
+
+

+


+
Unknown
Unknown


Ascusb_89
+




+



Unknown
Unknown


Ascusb_101


+


+



Unknown
Unknown


Ascusb_102
+
+
+


+



Unknown
Unknown


Ascusb_104


+






Unknown
Unknown


Ascusb_111

+
+


+


+
Unknown
Unknown


Ascusb_119



+

+



Unknown
Unknown


Ascusb_125


+
+

+

+

Unknown
Unknown


Ascusb_145
+




+



Unknown
Unknown


Ascusb_149
+


+
+
+

+

Unknown
Unknown


Ascusb_159
+

+
+

+

+

Unknown
Unknown


Ascusb_183
+


+

+

+
+
Unknown
Unknown


Ascusb_187
+
+

+

+

+

Unknown
Unknown


Ascusb_190
+

+


+

+

Unknown
Unknown


Ascusb_199



+

+



Unknown
Unknown


Ascusb_205


+


+



Unknown
Unknown


Ascusb_232



+

+



Unknown
Unknown


Ascusb_268





+



Unknown
Unknown


Ascusb_278





+

+
+
Unknown
Unknown


Ascusb_329



+





Unknown
Unknown


Ascusb_368





+



Unknown
Unknown


Ascusb_374
+


+
+
+


+
Unknown
Unknown


Ascusb_411

+







Unknown
Unknown


Ascusb_546

+



+



Unknown
Unknown


Ascusb_728
+


+
+
+


+
Unknown
Unknown


Ascusb_765





+


+
Unknown
Unknown


Ascusb_810
+




+



Unknown
Unknown


Ascusb_812


+






Unknown
Unknown


Ascusb_817


+

+
+


+
Unknown
Unknown


Ascusb_826
+


+

+


+
Unknown
Unknown


Ascusb_880
+


+

+

+
+
Unknown
Unknown


Ascusb_913
+
+

+

+

+

Unknown
Unknown


Ascusb_974

+



+



Unknown
Unknown


Ascusb_1069








+
Unknown
Unknown


Ascusb_1074

+
+


+



Unknown
Unknown


Ascusb_1295
+



+
+


+
Unknown
Unknown


Ascusb_1367
+
+



+

+
+
Unknown
Unknown


Ascusb_1632





+



Unknown
Unknown


Ascusb_1674
+


+
+

+

+
Unknown
Unknown


Ascusb_1763
+




+



Unknown
Unknown


Ascusb_1780




+
+


+
Unknown
Unknown


Ascusb_1786
+








Unknown
Unknown


Ascusb_1801


+


+



Unknown
Unknown


Ascusb_1812
+








Unknown
Unknown


Ascusb_1833

+
+
+
+
+


+
Unknown
Unknown


Ascusb_1850




+
+


+
Unknown
Unknown


Ascusb_2090
+







+
Unknown
Unknown


Ascusb_2124
+








Unknown
Unknown


Ascusb_2511

+

+

+


+
Unknown
Unknown


Ascusb_2530
+




+



Unknown
Unknown


Ascusb_2597

+



+



Unknown
Unknown


Ascusb_2624





+



Unknown
Unknown


Ascusb_2667
+




+


+
Unknown
Unknown


Ascusb_2836




+
+


+
Unknown
Unknown


Ascusb_3003
+


+




+
Unknown
Unknown


Ascusb_3138
+

+


+

+
+
Unknown
Unknown


Ascusb_3504
+




+



Unknown
Unknown


Ascusb_3881

+



+



Unknown
Unknown


Ascusb_6589


+






Unknown
Unknown


Ascusb_12103
+







+
Unknown
Unknown


Ascusb_14245
+




+


+
Unknown
Unknown


Ascusb_20083


+






Unknown
Unknown


Ascusb_20187
+




+



Unknown
Unknown


Ascusf_15
+
+
Unknown
+
+
Unknown
+
+
+
+
+


Ascusf_22


Unknown


Unknown

Unknown

+



Ascusf_23
+

Unknown


Unknown

Unknown
+
+



Ascusf_24


Unknown


Unknown

Unknown

+



Ascusf_25
+

Unknown


Unknown

Unknown
+




Ascusf_38


Unknown


Unknown

Unknown

+



Ascusf_45
+

Unknown


Unknown

Unknown
+
+
+


Ascusf_77
+

Unknown
+

Unknown

Unknown
+
+
+


Ascusf_94
+
+
Unknown
+

Unknown

Unknown
+
+
+


Ascusf_108
+

Unknown


Unknown

Unknown
+




Ascusf_206


Unknown


Unknown

Unknown

+



Ascusf_208


Unknown


Unknown

Unknown

+



Ascusf_307
+

Unknown


Unknown

Unknown
+
+
+


Ascusf_334
+
+
Unknown
+
+
Unknown

Unknown
+
+
+


Ascusf_353
+

Unknown
+

Unknown

Unknown
+




Ascusf_1012


Unknown


Unknown

Unknown

+

















TABLE 17







M2GSC and M2 Media Recipes








M2GSC
M2










Component
Amount
Component
Amount















Beef Extract
5
g
NaHCO3
4
g


Yeast Extract
1.25
g
HCl-L-cysteine
0.3
g


NaHCO3
2
g
(NH4)2SO4
0.10
g


Cellobiose
1
g
MgSO47H2O
0.005
g


Starch
1
g
K2HPO4
0.05
g


Glucose
1
g
KH2PO4
0.05
g











(NH4)2SO4 (1M)
2.55
mL
DI H2O
Up to 1000 mL












MgSO47H2O (0.25M)
0.288
mL





K2HPO4 (1M)
1
mL


KH2PO4 (1M)
1.275
mL


Clarified Rumen Fluid
50
mL


HCl-L-cysteine
0.3
g











DI H2O
Up to 500 mL
















TABLE 18







Modified Wolfe's Media Recipes









250X Modified Wolfe's
Modified Wolfe's



Vitamin Mix
Mineral Solution










Component
g/200 mL
Component
g/L













Pyridoxine-HCl
0.5
MgSO4 7H2O
140


p-Aminobenzoic
0.25
Nitrilotriacetic acid
10.96


Lipoic Acid
0.216
NaCl
50.06


Nicotinic Acid
0.252
MnSO4 H2O
24.99


Riboflavin
0.013
CaCl2
5


Thiamine HCL
0.25
CoCl2 6H2O
4.997


Calcium - DL -
0.1
FeSO2 7H2O
4.997


Pantothenate


Biotin
0.044
ZnSO2 7H2O
5.003


Folic Acid
0.004
AlK(SO4)2 12 H2O
0.5


Vitamin B12
0.007
CuSO4 5H2O
0.499




H3BO3
0.498




NaMoO4 2H2O
0.503




DI H2O
1 L









All media was prepared with anaerobic water (boiled DI H2O for 15 minutes then cooled to room temperature in a water bath while sparging with N2. All media was adjusted to a pH of 6.8 with 2M HCl. 10 mL of media was then aliquoted into 15 mL hungate tubs, and the tubes were then sparged with 80% N2 20% CO2 for 3 minutes.









TABLE 19







RAMM Salts Media Recipe










Component
g/500 mL














KH2PO4
0.11



K2HPO4
0.08



NH4Cl
0.265



NaHCO3
0.6



DI H2O
500 mL










After sterilization (autoclave) added: 2 mL of 250× modified Wolfe's vitamin mix, 10 mL of 50× modified Wolfe's mineral mix, 5 mL of 100 mM cysteine.


Example VIII
Determination of Maximal Information Coefficient (MIC) Scores for Microbe Strains Relevant to Pounds of Milk Produced
Experimental Design and Materials and Methods

Objective: Determine rumen microbial community constituents that impact the production of milk fat in dairy cows.


Animals: Eight lactating, ruminally cannulated, Holstein cows were housed in individual tie-stalls for use in the experiment. Cows were fed twice daily, milked twice a day, and had continuous access to fresh water. One cow (cow 1) was removed from the study after the first dietary Milk Fat Depression due to complications arising from an abortion prior to the experiment.


Experimental Design and Treatment: The experiment used a crossover design with 2 groups and 1 experimental period. The experimental period lasted 38 days: 10 days for the covariate/wash-out period and 28 days for data collection and sampling. The data collection period consisted of 10 days of dietary Milk Fat Depression (MFD) and 18 days of recovery. After the first experimental period, all cows underwent a 10-day wash out period prior to the beginning of period 2.


Dietary MFD was induced with a total mixed ration (TMR) low in fiber (29% NDF) with high starch degradability (70% degradable) and high polyunsaturated fatty acid levels (PUFA, 3.7%). The Recovery phase included two diets variable in starch degradability. Four cows were randomly assigned to the recovery diet high in fiber (37% NDF), low in PUFA (2.6%), and high in starch degradability (70% degradable). The remaining four cows were fed a recovery diet high in fiber (37% NDF), low in PUFA (2.6%), but low in starch degradability (35%).


During the 10-day covariate and 10-day wash out periods, cows were fed the high fiber, low PUFA, and low starch degradability diet.


Samples and Measurements: Milk yield, dry matter intake, and feed efficiency were measured daily for each animal throughout the covariate, wash out, and sample collection periods. TMR samples were measured for nutrient composition. During the collection period, milk samples were collected and analyzed every 3 days. Samples were analyzed for milk component concentrations (milk fat, milk protein, lactose, milk urea nitrogen, somatic cell counts, and solids) and fatty acid compositions.


Rumen samples were collected and analyzed for microbial community composition and activity every 3 days during the collection period. The rumen was intensively sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding during day 0, day 7, and day 10 of the dietary MFD. Similarly, the rumen was intensively sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding on day 16 and day 28 during the recovery period. Rumen contents were analyzed for pH, acetate concentration, butyrate concentration, propionate concentration, isoacid concentration, and long chain and CLA isomer concentrations.


Rumen Sample Preparation and Sequencing: After collection, rumen samples were centrifuged at 4,000 rpm in a swing bucket centrifuge for 20 minutes at 4° C. The supernatant was decanted, and an aliquot of each rumen content sample (1-2 mg) was added to a sterile 1.7 mL tube prefilled with 0.1 mm glass beads. A second aliquot was collected and stored in an empty, sterile 1.7 mL tube for cell counting.


Rumen samples with glass beads (1st aliquot) were homogenized with bead beating to lyse microorganisms. DNA and RNA was extracted and purified from each sample and prepared for sequencing on an Illumina Miseq. Samples were sequenced using paired-end chemistry, with 300 base pairs sequenced on each end of the library. Rumen samples in empty tubes (2nd aliquot) were stained and put through a flow cytometer to quantify the number of cells of each microorganism type in each sample.


Sequencing Read Processing and Data Analysis: Sequencing reads were quality trimmed and processed to identify bacterial species present in the rumen based on a marker gene. Count datasets and activity datasets were integrated with the sequencing reads to determine the absolute cell numbers of active microbial species within the rumen microbial community. Production characteristics of the cow over time, including pounds of milk produced, were linked to the distribution of active microorganisms within each sample over the course of the experiment using mutual information. Maximal information coefficient (MIC) scores were calculated between pounds of milk fat produced and the absolute cell count of each active microorganism. Microorganisms were ranked by MIC score, and microorganisms with the highest MIC scores were selected as the target species most relevant to pounds of milk produced.


Tests cases to determine the impact of count data, activity data, and count and activity on the final output were run by omitting the appropriate datasets from the sequencing analysis. To assess the impact of using a linear correlation rather than the MIC on target selection, Pearson's coefficients were also calculated for pounds of milk fat produced as compared to the relative abundance of all microorganisms and the absolute cell count of active microorganisms.


Results and Discussion

Relative Abundances vs. Absolute Cell Counts


The top 15 target species were identified for the dataset that included cell count data (absolute cell count, Table 21) and for the dataset that did not include cell count data (relative abundance, Table 20) based on MIC scores. Activity data was not used in this analysis in order to isolate the effect of cell count data on final target selection. Ultimately, the top 8 targets were the same between the two datasets. Of the remaining 7, 5 strains were present on both lists in varying order. Despite the differences in rank for these 5 strains, the calculated MIC score for each strain was the identical between the two lists. The two strains present on the absolute cell count list but not the relative abundance list, ascus_111 and ascus_288, were rank 91 and rank 16, respectively, on the relative abundance list. The two strains present on the relative abundance list but not the absolute cell count list, ascus_102 and ascus_252, were rank 50 and rank 19, respectively, on the absolute cell count list. These 4 strains did have different MIC scores on each list, thus explaining their shift in rank and subsequent impact on the other strains in the list.









TABLE 20







Top 15 Target Strains using Relative Abundance with no Activity Filter









Target




Strain
MIC
Nearest Taxonomy












ascus_7
0.97384
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),




o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605)


ascus_82
0.97173
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),




o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073)


ascus_209
0.95251
d: Bacteria(1.0000), p: TM7(0.9991), g: TM7_genera_incertae_sedis(0.8645)


ascus_126
0.91477
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),




o: Clostridiales(0.2714), f: Ruminococcaceae(0.1242), g: Saccharofermentans(0.0073)


ascus_1366
0.89713
d: Bacteria(1.0000), p: TM7(0.9445), g: TM7_genera_incertae_sedis(0.0986)


ascus_1780
0.89466
d: Bacteria(0.9401), p: Bacteroidetes(0.4304), c: Bacteroidia(0.0551),




o: Bacteroidales(0.0198), f: Prevotellaceae(0.0067), g: Prevotella(0.0052)


ascus_64
0.89453
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823),




o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605)


ascus_299
0.88979
d: Bacteria(1.0000), p: TM7(0.9963), g: TM7_genera_incertae_sedis(0.5795)


ascus_102
0.87095
d: Bacteria(1.0000), p: Firmicutes(0.9628), c: Clostridia(0.8317),




o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g: Saccharofermentans(0.0283)


ascus_1801
0.87038
d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365),




o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047)


ascus_295
0.86724
d: Bacteria(1.0000), p: SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793)


ascus_1139
0.8598
d: Bacteria(1.0000), p: TM7(0.9951), g: TM7_genera_incertae_sedis(0.4747)


ascus_127
0.84082
d: Bacteria(1.0000), p: TM7(0.9992), g: TM7_genera_incertae_sedis(0.8035)


ascus_341
0.8348
d: Bacteria(1.0000), p: TM7(0.9992), g: TM7_genera_incertae_sedis(0.8035)


ascus_252
0.82891
d: Bacteria(1.0000), p: Firmicutes(0.9986), c: Clostridia(0.9022),




o: Clostridiales(0.7491), f: Lachnospiraceae(0.3642), g: Lachnospiracea_incertae_sedis(0.0859)
















TABLE 21







Top 15 Target Strains using Absolute cell count with no Activity Filter









Target




Strain
MIC
Nearest Taxonomy












ascus_7
0.97384
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),




o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605)


ascus_82
0.97173
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),




o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073)


ascus_209
0.95251
d: Bacteria(1.0000), p: TM7(0.9991), g: TM7_genera_incertae_sedis(0.8645)


ascus_126
0.91701
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),




o: Clostridiales(0.2714), f: Ruminococcaceae(0.1242), g: Saccharofermentans(0.0073)


ascus_1366
0.89713
d: Bacteria(1.0000), p: TM7(0.9445), g: TM7_genera_incertae_sedis(0.0986)


ascus_1780
0.89466
d: Bacteria(0.9401), p: Bacteroidetes(0.4304), c: Bacteroidia(0.0551),




o: Bacteroidales(0.0198), f: Prevotellaceae(0.0067), g: Prevotella(0.0052)


ascus_64
0.89453
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823),




o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605)


ascus_299
0.88979
d: Bacteria(1.0000), p: TM7(0.9963), g: TM7_genera_incertae_sedis(0.5795)


ascus_1801
0.87038
d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365),




o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047)


ascus_295
0.86724
d: Bacteria(1.0000), p: SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793)


ascus_1139
0.8598
d: Bacteria(1.0000), p: TM7(0.9951), g: TM7_genera_incertae_sedis(0.4747)


ascus_127
0.84082
d: Bacteria(1.0000), p: TM7(0.9992), g: TM7_genera_incertae_sedis(0.8035)


ascus_341
0.8348
d: Bacteria(1.0000), p: TM7(0.9992), g: TM7_genera_incertae_sedis(0.8035)


ascus_111
0.83358
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),




o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062), g: Papillibacter(0.0098)


ascus_288
0.82833
d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327),




o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042)









Integration of cell count data did not always affect the final MIC score assigned to each strain. This may be attributed to the fact that although the microbial population did shift within the rumen daily and over the course of the 38-day experiment, it was always within 107-108 cells per milliliter. Much larger shifts in population numbers would undoubtedly have a broader impact on final MIC scores.


Inactive Species vs. Active Species


In order to assess the impact of filtering strains based on activity data, target species were identified from a dataset that leveraged relative abundance with (Table 22) and without (Table 20) activity data as well as a dataset that leveraged absolute cell counts with (Table 23) and without (Table 21) activity data.


For the relative abundance case, ascus_126, ascus_1366, ascus_1780, ascus_299, ascus_1139, ascus_127, ascus_341, and ascus_252 were deemed target strains prior to applying activity data. These eight strains (53% of the initial top 15 targets) fell below rank 15 after integrating activity data. A similar trend was observed for the absolute cell count case. Ascus_126, ascus_1366, ascus_1780, ascus_299, ascus_1139, ascus_127, and ascus_341 (46% of the initial top 15 targets) fell below rank 15 after activity dataset integration.


The activity datasets had a much more severe effect on target rank and selection than the cell count datasets. When integrating these datasets together, if a sample is found to be inactive it is essentially changed to a “0” and not considered to be part of the analysis. Because of this, the distribution of points within a sample can become heavily altered or skewed after integration, which in turn greatly impacts the final MIC score and thus the rank order of target microorganisms.









TABLE 22







Top 15 Target Strains using Relative Abundance with Activity Filter









Target




Strain
MIC
Nearest Taxonomy












ascus_7
0.97384
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),




o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605)


ascus_82
0.93391
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),




o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073)


ascus_102
0.87095
d: Bacteria(1.0000), p: Firmicutes(0.9628), c: Clostridia(0.8317),




o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g: Saccharofermentans(0.0283)


ascus_209
0.84421
d: Bacteria(1.0000), p: TM7(0.9991), g: TM7_genera_incertae_sedis(0.8645)


ascus_1801
0.82398
d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365),




o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047)


ascus_372
0.81735
d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes(0.8623),




o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0190)


ascus_26
0.81081
d: Bacteria(1.0000), p: Firmicutes(0.9080), c: Clostridia(0.7704),




o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942), g: Clostridium_IV(0.0144)


ascus_180
0.80702
d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes(0.8623),




o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0237)


ascus_32
0.7846
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.4024),




o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883),




g: Hydrogenoanaerobacterium(0.0144)


ascus_288
0.78229
d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327),




o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042)


ascus_64
0.77514
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823),




o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605)


ascus_295
0.76639
d: Bacteria(1.0000), p: SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793)


ascus_546
0.76114
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851),




o: Clostridiales(0.1324), f: Clostridiaceae_1(0.0208), g: Clostridium_sensu_stricto(0.0066)


ascus_233
0.75779
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),




o: Clostridiales(0.5860), f: Ruminococcaceae(0.3642), g: Ruminococcus(0.0478)


ascus_651
0.74837
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),




o: Clostridiales(0.2335), f: Ruminococcaceae(0.0883), g: Clostridium_IV(0.0069)
















TABLE 23







Top 15 Target Strains using Absolute cell count with Activity Filter









Target




Strain
MIC
Nearest Taxonomy












ascus_7
0.97384
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),




o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605)


ascus_82
0.93391
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),




o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073)


ascus_209
0.84421
d: Bacteria(1.0000), p: TM7(0.9991), g: TM7_genera_incertae_sedis(0.8645)


ascus_1801
0.82398
d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365),




o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047)


ascus_372
0.81735
d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes(0.8623),




o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0190)


ascus_26
0.81081
d: Bacteria(1.0000), p: Firmicutes(0.9080), c: Clostridia(0.7704),




o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942), g: Clostridium_IV(0.0144)


ascus_102
0.81048
d: Bacteria(1.0000), p: Firmicutes(0.9628), c: Clostridia(0.8317),




o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g: Saccharofermentans(0.0283)


ascus_111
0.79035
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),




o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062), g: Papillibacter(0.0098)


ascus_288
0.78229
d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327),




o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042)


ascus_64
0.77514
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823),




o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605)


ascus_295
0.76639
d: Bacteria(1.0000), p: SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793)


ascus_546
0.76114
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851),




o: Clostridiales(0.1324), f: Clostridiaceae_1(0.0208),




g: Clostridium_sensu_stricto(0.0066)


ascus_32
0.75068
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.4024),




o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144)


ascus_651
0.74837
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),




o: Clostridiales(0.2335), f: Ruminococcaceae(0.0883), g: Clostridium_IV(0.0069)


ascus_233
0.74409
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),




o: Clostridiales(0.5860), f: Ruminococcaceae(0.3642), g: Ruminococcus(0.0478)










Relative Abundances and Inactive vs. Absolute Cell Counts and Active


Ultimately, the method defined here leverages both cell count data and activity data to identify microorganisms highly linked to relevant metadata characteristics. Within the top 15 targets selected using both methods (Table 23, Table 20), only 7 strains were found on both lists. Eight strains (53%) were unique to the absolute cell count and activity list. The top 3 targets on both lists matched in both strain as well as in rank. However, two of the three did not have the same MIC score on both lists, suggesting that they were influenced by activity dataset integration but not enough to upset their rank order.


Linear Correlations vs. Nonparametric Approaches


Pearson's coefficients and MIC scores were calculated between pounds of milk fat produced and the absolute cell count of active microorganisms within each sample (Table 24). Strains were ranked either by MIC (Table 24a) or Pearson coefficient (Table 24b) to select target strains most relevant to milk fat production. Both MIC score and Pearson coefficient are reported in each case. Six strains were found on both lists, meaning nine (60%) unique strains were identified using the MIC approach. The rank order of strains between lists did not match—the top 3 target strains identified by each method were also unique.


Like Pearson coefficients, the MIC score is reported over a range of 0 to 1, with 1 suggesting a very tight relationship between the two variables. Here, the top 15 targets exhibited MIC scores ranging from 0.97 to 0.74. The Pearson coefficients for the correlation test case, however, ranged from 0.53 to 0.45—substantially lower than the mutual information test case. This discrepancy may be due to the differences inherent to each analysis method. While correlations are a linear estimate that measures the dispersion of points around a line, mutual information leverages probability distributions and measures the similarity between two distributions. Over the course of the experiment, the pounds of milk fat produced changed nonlinearly (FIG. 18). This particular function may be better represented and approximated by mutual information than correlations. To investigate this, the top target strains identified using correlation and mutual information, Ascus_713 (FIG. 19) and Ascus_7 (FIG. 20) respectively, were plotted to determine how well each method predicted relationships between the strains and milk fat. If two variables exhibit strong correlation, they are represented by a line with little to no dispersion of points when plotted against each other. In FIG. 19, Ascus_713 correlates weakly with milk fat, as indicated by the broad spread of points. Mutual information, again, measures how similar two distributions of points are. When Ascus_7 is plotted with milk fat (FIG. 20), it is apparent that the two point distributions are very similar.


The Present Method in Entirety vs. Conventional Approaches


The conventional approach of analyzing microbial communities relies on the use of relative abundance data with no incorporation of activity information, and ultimately ends with a simple correlation of microbial species to metadata (see, e.g., U.S. Pat. No. 9,206,680, which is herein incorporated by reference in its entirety for all purposes). Here, we have shown how the incorporation of each dataset incrementally influences the final list of targets. When applied in its entirety, the method described herein selected a completely different set of targets when compared to the conventional method (Table 24a and Table 24c). Ascus_3038, the top target strain selected using the conventional approach, was plotted against milk fat to visualize the strength of the correlation (FIG. 21). Like the previous example, Ascus_3038 also exhibited a weak correlation to milk fat.


Table 24: Top 15 Target Strains using Mutual Information or Correlations









TABLE 24a







MIC using Absolute cell count with Activity Filter










Target

Pearson



Strain
MIC
Coefficient
Nearest Taxonomy













ascus_7
0.97384
0.25282502
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),





o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217),





g: Ruminococcus(0.0605)


ascus_82
0.93391
0.42776647
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),





o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062),





g: Saccharofermentans(0.0073)


ascus_209
0.84421
0.3036308
d: Bacteria(1.0000), p: TM7(0.9991), g: TM7_genera_incertae_sedis(0.8645)


ascus_1801
0.82398
0.5182261
d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365),





o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059),





g: Butyricimonas(0.0047)


ascus_372
0.81735
0.34172258
d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes(0.8623),





o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217),





g: Spirochaeta(0.0190)


ascus_26
0.81081
0.5300298
d: Bacteria(1.0000), p: Firmicutes(0.9080), c: Clostridia(0.7704),





o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942),





g: Clostridium_IV(0.0144)


ascus_102
0.81048
0.35456932
d: Bacteria(1.0000), p: Firmicutes(0.9628), c: Clostridia(0.8317),





o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367),





g: Saccharofermentans(0.0283)


ascus_111
0.79035
0.45881805
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),





o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062),





g: Papillibacter(0.0098)


ascus_288
0.78229
0.46522045
d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327),





o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050),





g: Butyricimonas(0.0042)


ascus_64
0.77514
0.45417055
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823),





o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792),





g: Ruminococcus(0.0605)


ascus_295
0.76639
0.24972263
d: Bacteria(1.0000), p: SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793)


ascus_546
0.76114
0.23819838
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851),





o: Clostridiales(0.1324), f: Clostridiaceae_1(0.0208),





g: Clostridium_sensu_stricto(0.0066)


ascus_32
0.75068
0.5179697
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.4024),





o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883),





g: Hydrogenoanaerobacterium(0.0144)


ascus_651
0.74837
0.27656645
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),





o: Clostridiales(0.2335), f: Ruminococcaceae(0.0883),





g: Clostridium_IV(0.0069)


ascus_233
0.74409
0.36095098
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),





o: Clostridiales(0.5860), f: Ruminococcaceae(0.3642),





g: Ruminococcus(0.0478)
















TABLE 24b







Correlation using Absolute cell count with Activity Filter










Target

Pearson



Strain
MIC
Coefficient
Nearest Taxonomy













ascus_713
0.71066
0.5305876
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),





o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062),





g: Saccharofermentans(0.0073)


ascus_26
0.81081
0.5300298
d: Bacteria(1.0000), p: Firmicutes(0.9080), c: Clostridia(0.7704),





o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942),





g: Clostridium_IV(0.0144)


ascus_1801
0.82398
0.5182261
d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365),





o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059),





g: Butyricimonas(0.0047)


ascus_32
0.75068
0.5179697
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.4024),





o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883),





g: Hydrogenoanaerobacterium(0.0144)


ascus_119
0.6974
0.4968678
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756),





o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217),





g: Ruminococcus(0.0478)


ascus_13899
0.64556
0.48739454
d: Bacteria(1.0000), p: Actinobacteria(0.1810), c: Actinobacteria(0.0365),





o: Actinomycetales(0.0179), f: Propionibacteriaceae(0.0075),





g: Microlunatus(0.0058)


ascus_906
0.49256
0.48418677
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),





o: Clostridiales(0.2714), f: Ruminococcaceae(0.1242),





g: Papillibacter(0.0098)


ascus_221
0.44006
0.47305903
d: Bacteria(1.0000), p: Bacteroidetes(0.9991), c: Bacteroidia(0.9088),





o: Bacteroidales(0.7898), f: Prevotellaceae(0.3217), g: Prevotella(0.0986)


ascus_1039
0.65629
0.46932846
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.2851),





o: Clostridiales(0.1324), f: Ruminococcaceae(0.0329),





g: Clostridium_IV(0.0069)


ascus_288
0.78229
0.46522045
d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327),





o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050),





g: Butyricimonas(0.0042)


ascus_589
0.40868
0.4651165
d: Bacteria(1.0000), p: Firmicutes(0.9981), c: Clostridia(0.9088),





o: Clostridiales(0.7898), f: Lachnospiraceae(0.5986),





g: Clostridium_XIVa(0.3698)


ascus_41
0.67227
0.46499047
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.3426),





o: Clostridiales(0.1618), f: Ruminococcaceae(0.0703),





g: Hydrogenoanaerobacterium(0.0098)


ascus_111
0.79035
0.45881805
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637),





o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062),





g: Papillibacter(0.0098)


ascus_205
0.72441
0.45684373
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.3426),





o: Clostridiales(0.1618), f: Peptococcaceae_2(0.0449),





g: Pelotomaculum(0.0069)


ascus_64
0.77514
0.45417055
d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823),





o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792),





g: Ruminococcus(0.0605)
















TABLE 24c







Correlation using Relative Abundance with no Activity Filter










Target

Pearson



Strain
MIC
Coefficient
Nearest Taxonomy













ascus_3038
0.56239
0.6007549
d: Bacteria(1.0000), p: Firmicutes(0.9945), c: Clostridia(0.8623),





o: Clostridiales(0.5044), f: Lachnospiraceae(0.2367), g: Clostridium_XIVa(0.0350)


ascus_1555
0.66965
0.59716415
d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.3426),





o: Clostridiales(0.1618), f: Ruminococcaceae(0.0449), g: Clostridium_IV(0.0073)


ascus_1039
0.68563
0.59292555
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.2851),





o: Clostridiales(0.1324), f: Ruminococcaceae(0.0329), g: Clostridium_IV(0.0069)


ascus_1424
0.55509
0.57589555
d: Bacteria(1.0000), p: Firmicutes(0.8897), c: Clostridia(0.7091),





o: Clostridiales(0.3851), f: Ruminococcaceae(0.1422), g: Papillibacter(0.0144)


ascus_378
0.77519
0.5671971
d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251),





o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073)


ascus_407
0.69783
0.56279755
d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.3426),





o: Clostridiales(0.1618), f: Clostridiaceae_1(0.0329),





g: Clostridium_sensu_stricto(0.0069)


ascus_1584
0.5193
0.5619939
d: Bacteria(1.0000), p: Firmicutes(0.9945), c: Clostridia(0.8756),





o: Clostridiales(0.5860), f: Lachnospiraceae(0.3217), g: Coprococcus(0.0605)


ascus_760
0.61363
0.55807924
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851),





o: Clostridiales(0.1324), f: Clostridiaceae_1(0.0208),





g: Clostridium_sensu_stricto(0.0066)


ascus_1184
0.70593
0.5578006
d: Bacteria(1.0000), p: “Bacteroidetes”(0.9992), c: “Bacteroidia”(0.8690),





o: “Bacteroidales”(0.5452), f: Bacteroidaceae(0.1062), g: Bacteroides(0.0237)


ascus_7394
0.6269
0.5557023
d: Bacteria(1.0000), p: Firmicutes(0.9939), c: Clostridia(0.7704),





o: Clostridiales(0.4230), f: Lachnospiraceae(0.1422), g: Clostridium_XIVa(0.0350)


ascus_1360
0.57343
0.5535785
d: Bacteria(1.0000), p: Firmicutes(0.9992), c: Clostridia(0.9351),





o: Clostridiales(0.8605), f: Lachnospiraceae(0.7052), g: Clostridium_XIVa(0.2649)


ascus_3175
0.53565
0.54864305
d: Bacteria(1.0000), p: “Bacteroidetes”(0.9991), c: “Bacteroidia”(0.8955),





o: “Bacteroidales”(0.7083), f: “Prevotellaceae”(0.1942), g: Prevotella(0.0605)


ascus_2581
0.68361
0.5454486
d: Bacteria(1.0000), p: “Spirochaetes”(0.9445), c: Spirochaetes(0.8623),





o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0237)


ascus_531
0.71315
0.5400517
d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851),





o: Clostridiales(0.1324), f: Clostridiaceae_1(0.0208),





g: Clostridium_sensu_stricto(0.0066)


ascus_1858
0.65165
0.5393882
d: Bacteria(1.0000), p: “Spirochaetes”(0.9263), c: Spirochaetes(0.8317),





o: Spirochaetales(0.4636), f: Spirochaetaceae(0.2792), g: Spirochaeta(0.0237)









Numbered Embodiments of the Disclosure

Subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

    • 1. A shelf-stable ruminant supplement capable of increasing milk production or improving milk compositional characteristics in a ruminant, comprising:
      • a) a purified population of Pichia fungi comprising a fungi with an ITS nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 32; and
      • b) a shelf-stable carrier suitable for ruminant administration,
      • wherein the purified population of Pichia fungi of a) is present in the supplement in an amount effective to increase milk production or improve milk compositional characteristics in a ruminant administered the supplement, as compared to a ruminant not administered the supplement.
    • 2. The shelf-stable ruminant supplement according to claim 1, wherein the purified population of Pichia fungi comprises a fungi with an ITS nucleic acid sequence that is at least about 99% identical to SEQ ID NO: 32.
    • 3. The shelf-stable ruminant supplement according to claim 1, wherein the purified population of Pichia fungi comprises a fungi with an ITS nucleic acid sequence comprising SEQ ID NO: 32.
    • 4. The shelf-stable ruminant supplement according to claim 1, wherein the purified population of Pichia fungi comprises a fungi as deposited at NRRL Y-67249.
    • 5. The shelf-stable ruminant supplement according to claim 1, further comprising:
      • i. a purified population of bacteria that comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103, and/or
      • ii. a purified population of fungi that comprises a fungi with an ITS nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31, 33-60 and 2104-2107.
    • 6. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of bacteria comprises a bacteria with a 16S nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
    • 7. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of fungi comprises a fungi with an ITS nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31, 33-60 and 2104-2107.
    • 8. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of bacteria comprises a bacteria with a 16S nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
    • 9. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of fungi comprises a fungi with an ITS nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31, 33-60 and 2104-2107.
    • 10. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of bacteria comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 28.
    • 11. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of bacteria comprises a bacteria with a 16S nucleic acid sequence that is at least about 99% identical to SEQ ID NO: 28.
    • 12. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of bacteria comprises a bacteria with a 16S nucleic acid sequence comprising SEQ ID NO: 28.
    • 13. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of bacteria comprises a bacteria as deposited at NRRL B-67248.
    • 14. The shelf-stable ruminant supplement according to claim 5, wherein both a purified population of bacteria i) and a purified population of fungi ii) are present in the supplement.
    • 15. The shelf-stable ruminant supplement according to claim 1, formulated for administration to a cow.
    • 16. The shelf-stable ruminant supplement according to claim 1, wherein the supplement is stable under ambient conditions for at least one week.
    • 17. The shelf-stable ruminant supplement according to claim 1, formulated as an: encapsulation, tablet, capsule, pill, feed additive, food ingredient, food additive, food preparation, food supplement, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, suppository, bolus, drench, or combinations thereof.
    • 18. The shelf-stable ruminant supplement according to claim 1, wherein the purified population of Pichia fungi is present in the ruminant supplement at a concentration of at least 102 cells.
    • 19. The shelf-stable ruminant supplement according to claim 1, wherein the ruminant administered the supplement exhibits an increase in milk production that leads to a measured increase in milk yield.
    • 20. The shelf-stable ruminant supplement according to claim 1, wherein the ruminant administered the supplement exhibits an increase in milk production and improved milk compositional characteristics that leads to a measured increase in energy-corrected milk.
    • 21. The shelf-stable ruminant supplement according to claim 1, wherein the ruminant administered the supplement exhibits an improved milk compositional characteristic selected from the group consisting of: an increase in milk fat(s), an increase in milk protein(s), an increase of carbohydrates in milk, an increase of vitamins in milk, an increase of minerals in milk, or combinations thereof.
    • 22. The shelf-stable ruminant supplement according to claim 1, wherein the ruminant administered the supplement exhibits at least a 1% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
    • 23. The shelf-stable ruminant supplement according to claim 1, wherein the ruminant administered the supplement exhibits at least a 10% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
    • 24. The shelf-stable ruminant supplement according to claim 1, wherein the ruminant administered the supplement exhibits at least a 20% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
    • 25. A composition suitable for administration to a ruminant and capable of increasing milk production or improving milk compositional characteristics in a ruminant, comprising:
      • a) a purified population of fungi as deposited at NRRL Y-67249; and
      • b) a carrier suitable for ruminant administration,
      • wherein the purified population of fungi of a) is present in the composition in an amount effective to increase milk production or improve milk compositional characteristics in a ruminant administered the composition, as compared to a ruminant not administered the composition.
    • 26. A composition suitable for administration to a ruminant and capable of increasing milk production or improving milk compositional characteristics in a ruminant, comprising:
      • a) a purified population of fungi as deposited at NRRL Y-67249;
      • b) a purified population of bacteria as deposited at NRRL B-67248; and
      • c) a carrier suitable for ruminant administration,
      • wherein the purified population of fungi of a) and purified population of bacteria of b) are present in the composition in an amount effective to increase milk production or improve milk compositional characteristics in a ruminant administered the composition, as compared to a ruminant not administered the composition.


The aforementioned compositions have markedly different characteristics and/or properties not possessed by any individual bacteria or fungi as they naturally exist in the rumen. The markedly different characteristics and/or properties possessed by the aforementioned compositions can be structural, functional, or both. For example, the compositions possess the markedly different functional property of being able to increase milk production or improve milk compositional characteristics, when administered to a ruminant, as taught herein. Furthermore, the compositions possess the markedly different functional property of being shelf-stable.


Numbered Embodiments of the Disclosure

Subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

    • 1. A composition capable of modulating the rumen microbiome of a ruminant, comprising:
      • a) a purified population of Pichia fungi comprising a fungi with an ITS nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 32; and
      • b) a carrier suitable for ruminant administration,
    • wherein the purified population of Pichia fungi of a) is present in the composition in an amount effective to cause a shift in the microbiome of the rumen of a ruminant administered the composition.
    • 2. The composition according to claim 1, wherein a population of microbes present in the ruminant's rumen before administration of the composition increase in abundance after administration of the composition.
    • 3. The composition according to claim 1, wherein a population of microbes present in the ruminant's rumen before administration of the composition decrease in abundance after administration of the composition.
    • 4. The composition according to claim 1, wherein a first population of microbes present in the ruminant's rumen before administration of the composition increase in abundance after administration of the composition and wherein a second population of microbes present in the ruminant's rumen before administration of the composition decrease in abundance after administration of the composition.
    • 5. The composition according to claim 1, wherein the rumen microbiome of the ruminant administered the composition is shifted to include an increased presence of fiber-degrading genera, volatile fatty acid-producing genera, structural carbohydrate-digesting genera, or combinations thereof.
    • 6. The composition according to claim 1, wherein the rumen microbiome of the ruminant administered the composition is shifted according to the disclosure and data presented in Example 6 and Table 13 or Table 14.
    • 7. A method for modulating the rumen microbiome of a ruminant, comprising administering to a ruminant an effective amount of a composition comprising:
      • a) a purified microbial population, said purified microbial population comprising:
        • i. a purified population of bacteria that comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103, and/or
        • ii. a purified population of fungi that comprises a fungi with an ITS nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107; and
      • b) a carrier suitable for ruminant administration,
      • wherein the ruminant administered the effective amount of the composition exhibits a shift in the microbiome of the rumen.
    • 8. The method according to claim 7, wherein a population of microbes present in the ruminant's rumen before administration of the composition increase in abundance after administration of the composition.
    • 9. The method according to claim 7, wherein a population of microbes present in the ruminant's rumen before administration of the composition decrease in abundance after administration of the composition.
    • 10. The method according to claim 7, wherein a first population of microbes present in the ruminant's rumen before administration of the composition increase in abundance after administration of the composition and wherein a second population of microbes present in the ruminant's rumen before administration of the composition decrease in abundance after administration of the composition.
    • 11. The method according to claim 7, wherein the rumen microbiome of the ruminant administered the composition is shifted to include an increased presence of fiber-degrading genera, volatile fatty acid-producing genera, structural carbohydrate-digesting genera, or combinations thereof.
    • 12. The method according to claim 7, wherein the rumen microbiome of the ruminant administered the composition is shifted according to the disclosure and data presented in Example 6 and Table 13 or Table 14.


The aforementioned compositions have markedly different characteristics and/or properties not possessed by any individual bacteria or fungi as they naturally exist in the rumen. The markedly different characteristics and/or properties possessed by the aforementioned compositions can be structural, functional, or both. For example, the compositions possess the markedly different functional property of being able to modulate the rumen microbiome, when administered to a ruminant, as taught herein.


Numbered Embodiments of the Disclosure

Subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

    • 1. A method for increasing milk production or improving milk compositional characteristics in a ruminant, comprising:
      • a) administering to a ruminant an effective amount of a shelf-stable ruminant supplement comprising:
        • i. a purified microbial population that comprises a bacteria with a 16S nucleic acid sequence, and/or a fungi with an ITS nucleic acid sequence, which is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-60 and 2045-2107, said bacteria having a MIC score of at least about 0.4 and said fungi having a MIC score of at least about 0.2; and
        • ii. a shelf-stable carrier suitable for ruminant administration,
      • wherein at least one of the bacteria or fungi are capable of converting a carbon source into a volatile fatty acid selected from the group consisting of: acetate, butyrate, propionate, or combinations thereof; and
      • wherein at least one of the bacteria or fungi are capable of degrading a soluble or insoluble carbon source; and
      • wherein the ruminant administered the effective amount of the shelf-stable ruminant supplement exhibits an increase in milk production or improved milk compositional characteristics, as compared to a ruminant not administered the ruminant supplement.
    • 2. The method according to claim 1, wherein the ruminant is a cow.
    • 3. The method according to claim 1, wherein the ruminant supplement is stable under ambient conditions for at least one week.
    • 4. The method according to claim 1, wherein the ruminant supplement is formulated as an: encapsulation, tablet, capsule, pill, feed additive, food ingredient, food additive, food preparation, food supplement, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, suppository, bolus, drench, or combinations thereof.
    • 5. The method according to claim 1, wherein the ruminant supplement is encapsulated in a polymer or carbohydrate.
    • 6. The method according to claim 1, wherein administering comprises: feeding the ruminant supplement to a ruminant.
    • 7. The method according to claim 1, wherein administering comprises: injecting the ruminant supplement into a ruminant.
    • 8. The method according to claim 1, wherein the purified microbial population is present in the ruminant supplement at a concentration of at least 102 cells.
    • 9. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
    • 10. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.
    • 11. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
    • 12. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.
    • 13. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
    • 14. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.
    • 15. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence and a fungi with an ITS nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-60 and 2045-2107.
    • 16. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 28.
    • 17. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 32.
    • 18. The method according to claim 1, wherein the purified microbial population comprises a Pichia fungi as deposited at NRRL Y-67249.
    • 19. The method according to claim 1, wherein the purified microbial population only contains organisms that are members of a group selected from:
      • Intestinimonas, Anaerolinea, Pseudobutyrivibrio, Olsenella, Eubacterium, Catenisphaera, Faecalibacterium, Solobacterium, Blautia, Ralsonia, Coprococcus, Casaltella, Anaeroplasma, Acholeplasma, Aminiphilus, Mitsuokella, Alistipes, Sharpea, Oscillibacter, Neocallimastix, Odoribacter, Pichia, Tannerella, Candida, Hydrogenoanaerobacterium, Orpinomyces, Succinivibrio, Sugiyamaella, Ruminobacter, Lachnospira, Caecomyces, Sinimarinibacterium, Tremella, Hydrogenoanaerobacterium, Turicibacter, Clostridium_XIVa, Anaerolinea, Saccharofermentans, Butyricicoccus, Olsenella, Papillibacter, Clostridium XIa, Pelotomaculum, Erysipelotrichaceae incertae sedis, Lachnospiracea_incertae_sedis, Solobacterium, Anaeroplasma, Ralstonia, Clostridium_sensu_stricto, Eubacterium, Rikenella, Lachnobacterium, Tannerella, Acholeplasma, Howardella, Selenomonas, Butyricimonas, Sharpea, Succinivibrio, Ruminobacter, Candida, Syntrophococcus, Pseudobtqrivibrio, Orpinomyces, Cyllamyces, Saccharomycetales, Phyllosticta, Ascomycota, and Piromyces.
    • 20. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits an increase in milk production that leads to a measured increase in milk yield.
    • 21. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits an increase in milk production and improved milk compositional characteristics that leads to a measured increase in energy-corrected milk.
    • 22. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits an improved milk compositional characteristic selected from the group consisting of: an increase in milk fat(s), an increase in milk protein(s), an increase of carbohydrates in milk, an increase of vitamins in milk, an increase of minerals in milk, or combinations thereof.
    • 23. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits at least a 1% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
    • 24. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits at least a 10% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
    • 25. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits at least a 20% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
    • 26. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits:
      • at least one improved phenotypic trait, selected from the group consisting of: an improved efficiency in feed utilization, improved digestibility, an increase in polysaccharide and lignin degradation, an increase in fatty acid concentration in the rumen, pH balance in the rumen, a reduction in methane emissions, a reduction in manure production, improved dry matter intake, an improved efficiency of nitrogen utilization, or combinations thereof.
    • 27. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen.
    • 28. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen,
      • wherein a population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement.
    • 29. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen,
      • wherein a population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.
    • 30. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen,
      • wherein a first population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement, and
      • wherein a second population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.


The aforementioned compositions, utilized in the described methods, have markedly different characteristics and/or properties not possessed by any individual bacteria or fungi as they naturally exist in the rumen. The markedly different characteristics and/or properties possessed by the aforementioned compositions, utilized in the described methods, can be structural, functional, or both. For example, the compositions, utilized in the described methods, possess the markedly different functional property of being able to increase milk production or improve milk compositional characteristics, when administered to a ruminant, as taught herein. Furthermore, the compositions, utilized in the described methods, possess the markedly different functional property of being shelf-stable.


In aspects, the aforementioned microbial species—that is, a purified microbial population that comprises a bacteria with a 16S nucleic acid sequence, and/or a fungi with an ITS nucleic acid sequence, which is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-60 and 2045-2107—are members of a Markush group, as the present disclosure illustrates that the members belong to a class of microbes characterized by various physical and functional attributes, which can include any of the following: a) the ability to convert a carbon source into a volatile fatty acid such as acetate, butyrate, propionate, or combinations thereof; b) the ability to degrade a soluble or insoluble carbon source; c) the ability to impart an increase in milk production or improved milk compositional characteristics to a ruminant administered the microbe; d) the ability to modulate the microbiome of the rumen of a ruminant administered the microbe; e) the ability to be formulated into a shelf-stable composition; and/or f) possessing a MIC score of at least about 0.4 if a bacteria and possessing a MIC score of at least about 0.2 if a fungi. Thus, the members of the Markush group possess at least one property in common, which can be responsible for their function in the claimed relationship.









TABLE 25







Budapest Treaty Deposits of the Disclosure











Depository
Accession Number
Date of Deposit







NRRL
NRRL Y-67249
Apr. 27, 2016



NRRL
NRRL B-67248
Apr. 27, 2016



NRRL
NRRL B-67347
Dec. 15, 2016



NRRL
NRRL B-67348
Dec. 15, 2016



NRRL
NRRL B-67349
Dec. 15, 2016



Bigelow
PATENT201612001
Dec. 12, 2016



Bigelow
PATENT201612002
Dec. 12, 2016



Bigelow
PATENT201612003
Dec. 12, 2016



Bigelow
PATENT201612004
Dec. 12, 2016



Bigelow
PATENT201612005
Dec. 12, 2016



Bigelow
PATENT201612006
Dec. 12, 2016



Bigelow
PATENT201612007
Dec. 15, 2016



Bigelow
PATENT201612008
Dec. 15, 2016



Bigelow
PATENT201612009
Dec. 15, 2016



Bigelow
PATENT201612010
Dec. 15, 2016



Bigelow
PATENT201612011
Dec. 15, 2016



Bigelow
PATENT201612012
Dec. 15, 2016



Bigelow
PATENT201612013
Dec. 19, 2016



Bigelow
PATENT201612014
Dec. 28, 2016










INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes.


However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims
  • 1. A method for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: a) administering to a ruminant an effective amount of a shelf-stable ruminant supplement comprising: i. a purified microbial population that comprises a bacteria with a 16S nucleic acid sequence, and/or a fungi with an ITS nucleic acid sequence, which is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-60 and 2045-2107, said bacteria having a MIC score of at least about 0.4 and said fungi having a MIC score of at least about 0.2; andii. a shelf-stable carrier suitable for ruminant administration,wherein at least one of the bacteria or fungi are capable of converting a carbon source into a volatile fatty acid selected from the group consisting of: acetate, butyrate, propionate, or combinations thereof; andwherein at least one of the bacteria or fungi are capable of degrading a soluble or insoluble carbon source; andwherein the ruminant administered the effective amount of the shelf-stable ruminant supplement exhibits an increase in milk production or improved milk compositional characteristics, as compared to a ruminant not administered the ruminant supplement.
  • 2. The method according to claim 1, wherein the ruminant is a cow.
  • 3. The method according to claim 1, wherein the ruminant supplement is stable under ambient conditions for at least one week.
  • 4. The method according to claim 1, wherein the ruminant supplement is formulated as an: encapsulation, tablet, capsule, pill, feed additive, food ingredient, food additive, food preparation, food supplement, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, suppository, bolus, drench, or combinations thereof.
  • 5. The method according to claim 1, wherein the ruminant supplement is encapsulated in a polymer or carbohydrate.
  • 6. The method according to claim 1, wherein administering comprises: feeding the ruminant supplement to a ruminant.
  • 7. The method according to claim 1, wherein administering comprises: injecting the ruminant supplement into a ruminant.
  • 8. The method according to claim 1, wherein the purified microbial population is present in the ruminant supplement at a concentration of at least 102 cells.
  • 9. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
  • 10. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.
  • 11. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
  • 12. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.
  • 13. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
  • 14. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.
  • 15. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence and a fungi with an ITS nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-60 and 2045-2107.
  • 16. The method according to claim 1, wherein the purified microbial population comprises a bacteria with a 16S nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 28.
  • 17. The method according to claim 1, wherein the purified microbial population comprises a fungi with an ITS nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 32.
  • 18. The method according to claim 1, wherein the purified microbial population comprises a Pichia fungi as deposited at NRRL Y-67249.
  • 19. The method according to claim 1, wherein the purified microbial population only contains organisms that are members of a group selected from: Intestinimonas, Anaerolinea, Pseudobutyrivibrio, Olsenella, Eubacterium, Catenisphaera, Faecalibacterium, Solobacterium, Blautia, Ralsonia, Coprococcus, Casaltella, Anaeroplasma, Acholeplasma, Aminiphilus, Mitsuokella, Alistipes, Sharpea, Oscillibacter, Neocallimastix, Odoribacter, Pichia, Tannerella, Candida, Hydrogenoanaerobacterium, Orpinomyces, Succinivibrio, Sugiyamaella, Ruminobacter, Lachnospira, Caecomyces, Sinimarinibacterium, Tremella, Hydrogenoanaerobacterium, Turicibacter, Clostridium XIVa, Anaerolinea, Saccharofermentans, Butyricicoccus, Olsenella, Papillibacter, Clostridium XIa, Pelotomaculum, Erysipelotrichaceae incertae sedis, Lachnospiracea incertae sedis, Solobacterium, Anaeroplasma, Ralstonia, Clostridium sensu stricto, Eubacterium, Rikenella, Lachnobacterium, Tannerella, Acholeplasma, Howardella, Selenomonas, Butyricimonas, Sharpea, Succinivibrio, Ruminobacter, Candida, Syntrophococcus, Pseudobtqrivibrio, Orpinomyces, Cyllamyces, Saccharomycetales, Phyllosticta, Ascomycota, and Piromyces.
  • 20. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits an increase in milk production that leads to a measured increase in milk yield.
  • 21. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits an increase in milk production and improved milk compositional characteristics that leads to a measured increase in energy-corrected milk.
  • 22. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits an improved milk compositional characteristic selected from the group consisting of: an increase in milk fat(s), an increase in milk protein(s), an increase of carbohydrates in milk, an increase of vitamins in milk, an increase of minerals in milk, or combinations thereof.
  • 23. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits at least a 1% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
  • 24. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits at least a 10% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
  • 25. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement exhibits at least a 20% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
  • 26. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: at least one improved phenotypic trait, selected from the group consisting of: an improved efficiency in feed utilization, improved digestibility, an increase in polysaccharide and lignin degradation, an increase in fatty acid concentration in the rumen, pH balance in the rumen, a reduction in methane emissions, a reduction in manure production, improved dry matter intake, an improved efficiency of nitrogen utilization, or combinations thereof.
  • 27. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen.
  • 28. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen, wherein a population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement.
  • 29. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen, wherein a population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.
  • 30. The method according to claim 1, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen, wherein a first population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement, andwherein a second population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 62/276,142, filed Jan. 7, 2016; U.S. Provisional Application No. 62/276,531, filed Jan. 8, 2016; U.S. Provisional Application No. 62/334,816, filed May 11, 2016; and U.S. Provisional Application No. 62/415,908, filed Nov. 1, 2016; each of which is herein incorporated by reference in its entirety.

Provisional Applications (4)
Number Date Country
62276142 Jan 2016 US
62276531 Jan 2016 US
62334816 May 2016 US
62415908 Nov 2016 US