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Reading Assignments. James B. Russell and J.L. Rychlik. 2001. Factors that alter rumen microbial ecology. Science 292:1119 J. Miron, D. Ben-Ghedalia and M. Morrison. 2001. Invited review: Adhesion mechanisms of rumen cellulolytic bacteria. J. Dairy Sci. 84:1294

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slide1

Reading Assignments

James B. Russell and J.L. Rychlik. 2001. Factors that alter rumen microbial ecology.

Science 292:1119

J. Miron, D. Ben-Ghedalia and M. Morrison. 2001. Invited review: Adhesion

mechanisms of rumen cellulolytic bacteria. J. Dairy Sci. 84:1294

Bryan A. White. 1991. Bichemistry and genetics of microbial degradation of the

plant cell wall. Rec. Adv. on the Nutr. Herbivores. pp 217-225

J.L. Rychlik and J.B. Russell. 2002. Bacteriocin-like activity of Butyrivibrio fibrisolvens

JL5 and its effect on other ruminal bacteria and ammonia production. Appl. And Environ.

Microbiol. 68:1040

H. Krajcaraski-Hunt, J.C. Plaizier, J.-P. Walton, R. Spratt and B.W. McBride. 2002.

Short communication: Effect of subacute ruminal acidosis on in situ fiber digestion

in lactating dairy cows. J. Dairy Sci. 85:570

A.L. Oliver, R.J. Grant, J.F. Pedersen and J.O. O’Rear. 2004. Comparison of brown

midrib-6 and -18 forage sorghum with conventional sorghum and corn silage in diets of

lactating dairy cows. J. Dairy Sci. 87:637

carbohydrates
Carbohydrates
  • Importance
    • Make up 60% to 70% of diet
    • Major source of energy
      • 1. Microbes
        • Energy for microbes
        • Metabolism, Growth, Protein synthesis
      • 2. Animal
        • End products of the fermentation
        • Digestible CHOC escaping the rumen
  • Classification
    • Nonstructural (NSC)
      • Cell contents - storage
    • Structural (SC)
      • Cell walls
chemistry of feed dry matter
Chemistry of Feed Dry Matter
  • Organic
    • Carbohydrates
      • Fiber
        • Cellulose, hemicellulose
      • Soluble fiber
        • Pectin, fructans, β-glucans
      • Starch
      • Free sugars
    • Lignin and other phenolics
    • Proteins
    • Lipids
  • Inorganic
plant carbohydrates
Plant Carbohydrates

Cell ContentCell Wall

Organic acids Pectins

Sugars β-glucans

Starches Hemicelluloses

Fructans Cellulose

Mammalian enzymes will digest starch and

sucrose (limited in ruminants)

Microbes digest the plant polysaccharides

slide5

Plant Cell Walls

Many plant cells have a primary cell wall, which accommodates the cell as it grows, and a secondary cell wall that develops inside the primary wall after the cell has stopped growing. The primary cell wall is thinner and more pliant than the secondary cell wall.

A specialized region of the cell walls of plants is the middle lamella. Rich in pectins, the middle lamella is shared by neighboring cells and cements them firmly together.

Secondary cell wall would develop

The main chemical components of the primary cell wall include cellulose and two groups of branched polysaccharides, the pectins and cross-linking glycans (hemicellulose). The secondary plant cell wall, which is often deposited inside the primary cell wall as a cell matures, contains lignin in addition to cellulose, but less hemicellulose and pectin.

carbohydrates6
Carbohydrates
  • Monosaccharides - one sugar molecule
    • Hexoses - 6 carbons
      • Glucose Fructose Galactose Mannose
    • Pentoses - 5 carbons
      • Arabinose Xylose Ribose
  • Disaccharides - two sugar molecules
    • Maltose = glucose + glucose
    • Cellobiose = glucose + glucose
    • Sucrose = glucose + fructose
    • Lactose = glucose + galactose
carbohydrates continued
Carbohydrates - Continued

3. Polysaccharides - polymers of sugar molecules

- Starch - polymer of glucose (plants)

  • Alpha 1- 4 linkages, branch at alpha 1-6
  • Amylose (unbranched) 20 to 30% of starch in grain
  • Amylopectin (branched) 70 to 80% of starch in grain

- Glycogen - polymer of glucose (animals)

  • Alpha 1- 4 linkages, branch at alpha 1- 6

- Cellulose - polymer of glucose (plants)

  • Beta 1- 4 linkages
slide8

Cellulose

Cellulose: A polymer of glucose units in β – 1,4 linkages. Cellulose is a linear molecule consisting of 1,000 to 10,000 β-D-glucose residues with no branching. Neighboring cellulose chains may form hydrogen bonds leading to the formation of microfibrils with partially crystalline parts. Hydrogen bonding among microfibrils can form microfibers and microfibers react to form cellulose fibers. Cellulose fibers usually consist of over 500,000 cellulose molecules.

β-1,4 linkage

slide9

Starch

Starch: A polymer of α-D-glucose in α-1, 4 linkages. Starch consists of two types of molecules, amylose and amylopectin. Amylose is a single chain of glucose units whereas in amylopectin at about every twenty glucose units there is a branch with an α-1, 6 linkage. The relative proportions of amylose to amylopectin depend on the source of the starch, e.g. normal corn contains over 50% amylose whereas 'waxy' corn has almost none (~3%). Amylose has lower molecular

weight with a relatively extended shape, whereas amylopectin has large but compact molecules.

Partial structure of amylose Partial structure of amylopectin

slide10

Starch

Amylose molecules consist of single mostly-unbranched chains with 500-20,000 α-(1, 4)-D-glucose units with a few α-1, 6 branches. Amylose can form an extended shape. Hydrogen bonding occurs between aligned chains. The aligned chains may form double stranded crystallites that are resistant to amylases.

Amylopectin is formed by non-random α-1, 6 branching of the amylose-type α-(1, 4)-D-glucose structure. This branching is determined by branching enzymes that leave each chain with up to 30 glucose residues. Each amylopectin molecule contains one to two million residues, about 5% of which form the branch points, in a compact structure forming granules. The molecules are oriented radially in the starch granule and as the radius increases so does the number of branches required to fill the space, resulting in concentric regions of alternating amorphous and crystalline structure.

slide11

Amylopectin

Corn starch

Potato starch

carbohydrates continued12
Carbohydrates - Continued
  • Polysaccharides

- Pentosans - polymers of 5-carbon sugars

- Fructans – Water soluble chains of fructose

β-2-6 with β-2-1 branching

Found in temperate grasses

β-2-1 Found in Jerusalem artichokes

- β-Glucans – Soluble chains of glucose

β-1-3 and β-1-4 chains not linear like cellulose

Found in oats & barley

carbohydrates continued13
Carbohydrates - Continued
  • Mixed polysaccharides
    • Hemicellulose
      • Branched polysaccharides that are structurally homologous to cellulose because they have a backbone composed of β-1, 4 linked sugar residues – Most often xylans, no exact structure
      • Hemicellulose is abundant in primary walls but is also found in secondary walls
      • Various side chains : arabinose, glucuronic acid, manose, glucose, 4-0-methylglucuronic acid – varies among species
      • In plant cell walls:
        • Close association with lignin – linkages to coumaric and ferulic acids
        • Xylan polymers may be crosslinked to other hemicellulose backbones
        • Bound to cellulose in plant cell wall
        • Ratio of cellulose to hemicellulose ranges from 0.8:1 to 1.6:1
slide14

Mixed Polysaccharides - Continued

  • Pectins
    • Pectins have a complex and not exact structure. Backbone is
    • most often α-1- 4 linked D-galacturonic acid
    • Rhamnose might be interspersed with galacturonic acid with
    • branch-points resulting in side chains (1 - 20 residues) of
    • mainly L-arabinose and D-galactose
    • Also contain ester linkages with methyl groups and sidechains
    • containing other residues such as D-xylose, L-frucose, D-
    • glucuronic acid, D-apiose, 3-deoxy-D-manno-2-octulosonic acid
    • and 3-deoxy-D-lyxo-2-heptulosonic acid attached to poly-α-(1,
    • 4)-D-galacturonic acid regions
    • Proteins called extensins are commonly found associated
    • with pectin in the cell wall
    • Commonly form crosslinkages and entrap other polymers
    • Composition varies among plants and parts of plants
      • Citrus pulp, beet pulp, soybean hulls have
      • high concentrations
      • Alfalfa intermediate concentrations of pectin
      • Grasses low concentrations of pectin
structural carbohydrates in plants
Structural Carbohydrates in Plants

Pectins less in grass than legumes.

Hemicellulose greater in grass than legumes.

Hemicellulose and cellulose increase with maturity.

slide16

Lignin

Lignin Monomers

  • Not a carbohydrate – does not contain sugars
  • Large phenolic three-dimensional polymers in secondary cell walls
  • The monomers are polymerized phenylpropane units, predominantly coumaryl alcohol [with an OH-group in position 4 of the phenyl ring], coniferyl alcohol (OH-group in position 4, -OCH3 in position 3) and sinapyl alcohol (OH-group in position 4, -OCH3 group in positions 3 and 5).
  • The side groups of the monomers are reactive forming poorly defined structures that are heavily cross linked.
  • Attach with hemicellulose and pectins
  • Not digested in the rumen
slide17

Relation of Lignin to Digestibility of Cell Walls

  • 1. A negative relationship usually observed
    • Encrustation of cell wall polysaccharides
    • Enzymes can not digest polysaccharides
      • However lignin content related to maturity
      • rather than digestibility of cell walls
  • 2. Ratio of monomers varies among plants
    • High concentrations of syringyl unit (sinapyl)
    • less digestible
      • However ratio of monomers not always
      • related to digestibility of cell walls
  • 3. Hydroxycinnamic acids (acid forms of monomers)
  • can form cross links among polysaccarides and
  • link polysaccarides with lignin
lignin and digestibility of cell walls
Lignin and Digestibility of Cell Walls
  • Cross links
  • Ferulic acid (acid form of coniferyl alcohol) is first
  • product synthesized
  • The ferulates (hydroxycinnamic acids)
    • 1. Can react with polysaccharides of cell wall
      • Reduces digestibility of cell wall polysaccharides
    • 2. Can link polysaccharides in cell wall with lignin
      • More dramatic reduction in digestibility of cell walls
      • Form early in the plant and become diluted with
      • maturity so negative relationship not always
      • apparent
slide19

Interaction of Lignin with

Polysaccharides

Core lignin

Non core lignin

slide20

Tannins

  • Not carbohydrate – do not contain sugars
  • Polyphenolic compounds of diverse nature
  • 1. Hydrolysable tannins
  • Residues of gallic acid that are
  • linked to glucose via glycosidic
  • bonds
  • 2. Condensed tannins (nonhydrolyzable)
  • Biphenyl condensates of phenols
  • Anti-nutrient effects
    • Combine with proteins, cellulose,
    • hemicellulose, pectin and minerals
    • Can inhibit microorganisms and enzymes
  • In plants
    • Most domesticated plants have been selectively bred for
    • low concentrations of tannins – bird resistant milo exception
    • Many warm season legumes and browses contain tannins
    • Colored seed coats indicative of tannins - Acorns
feed evaluation chemical
Feed Evaluation - Chemical
  • Sample feed
      • Need representative sample
  • Proximate analysis (Weende procedure)
      • Moisture - Residue is dry matter
        • Oven dry

Volatile components will be lost

Overheating causes reactions of carbohydrates with

proteins and changes solubility of carbohydrates

        • Freeze dry
        • Distill with toluene – Best for fermented feeds
        • Determine water with Karl Fischer reagent
      • Organic matter
        • Burn @ 6000C - Residue is ash
feed evaluation continued
Feed Evaluation - Continued
  • Crude protein
    • Kjeldahl N x 6.25
  • Ether extract
    • Lipids, waxes, pigments, fat soluble vitamins
    • Extract with ether or hexane
  • Crude fiber
    • Cellulose, hemicellulose, lignin
    • Boil in dilute acid and then dilute alkali, dry, weigh, ash (Wt loss is crude fiber)
  • Nitrogen-free extract

Starch & Sugars + Other

NFE = 100 - (moisture + ash + crude fiber + protein + ether extract)

Acid and sodium hydroxide used for crude fiber dissolve some cellulose, hemicellulose and lignin in cell walls which then are included in NFE.

slide23

Forage

(Neutral detergent solution)

Soluble Insoluble

Cell contents Cell walls (NDF)

Starch & Sugars Hemicellulose

(Pectin, β-glucans Cellulose

& fructans) Lignin

Soluble proteins Insoluble proteins

Lipids Insoluble minerals (dirt)

Organic acids

Fiber analysis - Detergent solutions (Van Soest)

slide24

Neutral Detergent Soluble CHOH

  • A calculated value:
  • NDSC = 100 - (%NDF+%CP+%Fat+%Ash)
    • NDF corrected for protein
      • 98% potentially digestible in the rumen
      • Rapidly fermented in the rumen
      • Diverse group and not easily measured
      • directly in feeds
      • Not all digested by mammalian enzymes
neutral detergent soluble choh
Neutral Detergent Soluble CHOH
  • Includes: Organic acids, sugars, disaccharides,
  • oligosaccharides, starches, fructans, pectins, β-glucans
  • Rate and extent of digestion of each will vary
    • Organic acids provide no energy to rumen microbes
    • Sugars rapidly fermented in rumen
    • Starch digestion varies with source, processing and
    • other dietary components
    • ND soluble fiber usually rapidly fermented, but not at
    • low rumen pH
  • Want to estimate:
    • 1. Digestibility of the feed (available energy)
    • 2. Microbial growth (microbial protein)
slide26

Neutral Detergent Soluble Fiber

  • Pectins
      • Galactans
      • β -glucans
    • Fructans – some lactic acid
  • Not digested by mammalian enzymes
  • Rapidly fermented in the rumen
    • 20 to 40% per hour
    • Produces mostly acetic acid – no lactate
    • Some byproduct feeds high in these soluble
    • fibers will be more rapidly fermented than
    • predicted from starch and free sugars
slide27

Fiber analysis - NDF

  • NDF (insoluble residue) of high starch
  • feeds may be contaminated with starch
  • if not predigested with -amylase
    • Treat sample with heat stable -amylase
  • Pectin is associated with cell walls
  • However soluble in NDF solution
  • Pectin insoluble in ADF solution
    • Extract samples high in pectin with
    • NDF solution before ADF extraction
fiber analysis van soest
Fiber analysis – (Van Soest)

NDF (Insoluble residue)

(Acid detergent solution)

SolubleInsoluble (ADF)

Hemicellulose Cellulose

Protein Lignin

Cutin

Insoluble minerals (soil)

Acid detergent insol N (ADIN)

ADIN is unavailable protein - not digested in rumen

or intestines

lignin assays
Lignin Assays

Klason Procedure (wood)

Feed (72% H2SO4) Lignin

Cellulose dissolved

Residue contains more than lignin

Protein, smaller molecular weight phenolics, cutin

Acid Detergent (proteins removed)

ADF (KMnO4) Lignin measured as

weight loss (Includes tannins

complexed with protein)

Cellulose, Cutin, minerals as residue

ADF (72% H2SO4) Cellulose measured as

weight loss

Lignin, cutin, minerals as residue

limitations of fiber analysis
Limitations of Fiber Analysis

NDF and ADF should be done sequentially on the same sample. Not done this way in most commercial labs. Pectin solubilized in ND soln, but not soluble in the AD soln.

Should report NDF and ADF on an organic basis. Minerals, especially soil, are not solubilized in the detergent solns.

Detergent system developed to measure fiber fractions in plant materials, not animal derived feeds.

Keratin proteins insoluble in ND soln. Add Na sulfite to dissolve

keratinized proteins but also attacks lignin.

Lipids interfere with NDF determination in feeds containing more than 10% lipids. ND is lipid soluble, so results in high NDF values.

starch analysis
Starch Analysis

Starch and cellulose both contain glucose.

1. Extract free sugars from the feed

2. Use enzymes specific for -linkage to digest

starch. (Amylase and Amyloglucosidase)

3. Measure glucose released

4. Starch = glucose x .9

Release of glucose following treatment of grain with amyloglucosidase provides an indication of availability starch in the rumen.

carbohydrate fractions in feeds computer models
Carbohydrate Fractions in FeedsComputer Models
  • Available fiber = NDF – NDF protein – (lignin*2.4)
  • Sugars = NFC (nonfiber) – (starch + pectin)
  • NFC = NDSC
    • CHOH fractions
      • CHO A = sugars
      • CHO B1 = starch & pectin
      • CHO B2 = available fiber
      • CHO C = unavailable fiber (lignin*2.4)
the rumen as a fermentation chamber contribution of the animal to the symbiotic relationship
The Rumen as a Fermentation ChamberContribution of the animal to the symbiotic relationship:
  • Open and continuous system

Open for inoculation from feed and water

Continuous passage

  • Constant supply of nutrients

Feed intake and feed retained in rumen and reticulum

  • Mixing of contents (Motility of rumen and reticulum)
  • Low oxygen concentration

Oxidation reduction potential –150 to –350 mv

  • Control of moisture content (85 - 90%)
  • Temperature control (38 - 40 Co)
  • pH control (5.5 – 7.0)

Saliva NaHCO3, VFA, less from HPO4= at rumen pH

  • Removal of end products (though acid concentrations are high)

Eructation of gases and absorption of end products

microbiology of the rumen
Microbiology of the Rumen
  • Relative stable population for a given feed (substrate)
  • Microorganisms adapted to rumen environment
  • Mostly obligate anaerobes
    • Bacteria - 1010 to 1011 cells/g
    • Protozoa - 105 to 106 cells/g
    • Fungi - 103 to 105 zoospores/ml
slide37

Groups of Bacteria in the Rumen

Habitats in the Rumen

  • Free-floating in the liquid phase
    • Maybe up to 50% of bacteria in rumen are free floating
    • Probably daughter cells of attached bacteria
        • Feed on solubles released by attached cells
  • 2. Associated with feed particles
    • Loosely associated with feed particles
    • Firmly adhered to feed particles
    • Up to 75% of bacteria associated with feed particles
        • Do most of the initial digestion of feed particles
  • 3. Associated with rumen epithelium
    • Similarities and differences from bacteria in the
      • rumen fluid
    • Suggested functions
        • Scavenging O2, tissue recycling, digest urea
  • 4. Other
    • Attached to surface of protozoa and fungi
    • Engulfed in protozoa
slide38

Bacteria Associated with Feed Particles

  • Groups 2 and 3
    • 75% of bacterial population in rumen
    • 90% of endoglucanase and xylanase activity
    • 70% of amylase activity
    • 75% or protease activity
slide39

Adherence of mixed rumen

bacteria to plant material.

Protuberances from cells

probably are binding factors.

slide40

Bacterial Adhesion to Plant Tissues

1. Transport of bacteria to fibrous substrate

Low numbers of free bacteria & poor mixing

2. Initial nonspecific adhesion

Electrostatic, hydrophobic, ionic

On cut or macerated surfaces

3. Specific adhesion to digestible tissue

Ligands or adhesins on bacterial cell surface

4.Proliferation of attached bacteria

Allows for colonization of available surfaces

slide41

Mechanisms of Bacterial Adhesion

  • Cellulosome paradigm 2 MDa
    • 1. Large multicomponent complexes
    • Multifunctional, multienzyme
  • Polycellulosomes up to 100 MDa
    • 1. Form protuberances on cell surface
    • 2. Cellulose binding proteins
    • 3. Enzyme binding domains
attachment of bacteria to fibers
Attachment of Bacteria to Fibers

Adherent cell Nonadherent cell

Glycocalyx (on outer membrane of cell)

Cellulose Cell Cell

Digested and fermented

Cellodextrins by adherent and

nonadherent cells

slide44

Carbohydrate epitopes of bacterial glycolcalyx

    • Slime layer surrounding bacteria composed of
    • glycoproteins
    • Proteins and carbohydrates involved in adhesion
  • Ruminococcus flavefaciens, Fibrobacter succinogenes
  • Cellulose-binding domains of cellulolytic
  • enzymes
    • Cellulase has two functional domains
      • Catalytic domain - hydrolysis of glycosidic bonds
      • Binding domain - binds enzyme to cellulose
  • Fibrobacter succinogenes
  • Ruminococcus flavefaciens (maybe)
slide47

Benefits of Bacterial Attachment

  • If attachment prevented or reduced digestion
  • of cellulose greatly reduced
  • Brings enzymes and substrate together in
  • a poorly mixed system
  • Protects enzymes from proteases in the rumen
  • Allows bacteria to colonize on the digestible
  • surface of feed particles
  • Retention in the rumen to prolong digestion
  • Reduces predatory activity of protozoa
slide48

Cellulose Digesting Bacteria

Predominant:

Ruminococcus flavefaciens

Gram+ cocci, usually in chains

Ferments cellulose, cellobiose & glucose

Produces acetic, formic, succinic, some lactic & H2

Fibrobacter succinogenes

Gram– rod

Ferments cellulose, cellobiose & glucose

Produces acetic, formic & succinic

Ruminococcus albus

Gram– cocci

Ferments cellulose, cellobiose, usually not sugars

Produces acetic, formic, lactic, ethanol & H2

Strict anaerobes

Tolerate narrow pH range (pH 6 to 7)

Attach to feed particles

slide49

Cellulose Digesting Bacteria

  • Secondary:
  • Eubacterium cellulosolvens Numbers usually low in rumen
  • Gram– rod
  • Ferments cellulose & soluble sugars
  • Produces mostly lactic acid
  • Butyrivibrio fibrisolvens Several strains in rumen
  • Gram– curved rod
  • Ferments cellulose (slow) & starch
  • Produces formic, butyric & lactic acids, ethanol & H2
    • Strict anaerobes
    • Tolerate narrow pH range (pH 6 to 7)
    • Attach to feed particles
slide50

Nutrient Requirements of Cellulose Digesters

  • Carbohydrates (source of energy)
  • Branched chain volatile fatty acids
    • Isobutyric, isovaleric, 2-methylbutyric
  • Needed for:
    • Synthesis of branched chain amino acids
    • Synthesis of branched chain fatty acids (phospholipids)
  • CO2
  • Minerals (PO4, Mg, Ca, K, Na, probably other trace minerals)
  • Nitrogen
    • Mostly NH3 rather than amino acids
  • Biotin is stimulatory in pure cultures
effects of sugar on cellulose digestion fibrobacter succinogenes hiltner and dehority 1983
Effects of Sugar on Cellulose DigestionFibrobacter succinogenesHiltner and Dehority, 1983

Added sugar was a source of readily available energy

from 0 to 24 h. Subsequent drop in pH after 24 h

limited the rate of cellulose digestion after 36 h.

effect of ph on cellulose digestion ruminococcus flavefaciens hiltner and dehority 1983
Effect of pH on Cellulose DigestionRuminococcus flavefaciensHiltner and Dehority, 1983

Low pH (6.0)decreased rate

of cellulose digestion, but

had little effect on subsequent

ability to digest cellulose.

Similar results observed

with Fibrobacter

succinogenes.

slide53

Regulation of Rumen pH

  • Dairy cow can produce up to 160 moles fermentation acids/d
  • Buffers secreted in saliva
    • Phosphate pK of 6.5
    • Bicarbonate pK of 6.4
  • Below 5.7 bicarbonate & phosphate not effective buffers
    • At low pH VFA become most effective buffer
  • Feeding effective fiber (forage) results in less acidic rumen
    • Increased saliva flow – but osmotic pressures in rumen
    • maintained close to that of blood and interstitial fluids so
    • bicarbonate concentrations in the rumen do not vary much
    • Only undissociated forms of VFA readily absorbed so rumen
    • has to be acidic for an increase in VFA absorption
    • More likely increased saliva flow increases fluid dilution rate
      • As high as 20% per h when forages fed
      • Compared with 5% per h when cattle fed grain
    • Increased amounts of VFA washed out of rumen
slide54

Effects of pH Gradient Across Microbial Cell Membrane

Out In

 pH

XCOO— XCOO—

H+ H+

XCOOH XCOOH

ATP

H+

ADP + Pi

Two methods to handle

Acidic pH:

Use energy to pump H+

out of the cell. Anion of

acid accumulates – toxic.

Let intracellular pH decline

to maintain a pH gradient.

Enzymes have to tolerate

low pH. S bovis produces

lactic acid.

slide55

Hemicellulose Digesting Bacteria

  • Butrivibrio fibrisolvens
  • Prevotella ruminicola
    • Gram– non motile rod
    • Digests starch, cellulose not digested
    • Produces succincic, formic, acetic and some strains propionic
  • Eubacterium ruminantium
    • Gram+ non motil rod
    • Ferments cellobiose, dextrins, maltose, glucose, fructose,
    • lactose, sucrose and 5-carbon sugars
    • Does not digest starch and cellulose
    • Produces lactic, formic, acetic & butyric acids
  • Ruminococcus flavefaciens
  • Ruminococcus albus
slide57

Pectin Digesting Bacteria

  • Lachnospira multiparus
    • Mostly gram– motile curved rod
    • Ferments pectin, glucose, fructose,
    • cellobiose & sucrose
    • Xylan, cellulose & starch not fermented
    • Produces acetic, formic, lactic,ethanol & H2
  • Treponemes
    • Anaerobic spiral organisms
    • Ferment pectin, arabinose, inulin and sucrose
    • Produces acetic and formic acids
  • B. fibrosolvens
  • P. ruminicola
  • R. flavefaciens and R. albus can degrade
    • pectins but not ferment the end products
slide59

Starch Digesting Bacteria

  • Streptococcus bovis
    • Gram+ spherical to ovoid in shape
    • Hydrolyzes starch and ferments glucose
    • Produces lactic acid, acetic, formic & ethanol
      • 80 to 85% of CHOH fermented converted to lactic acid
    • Tolerates low pH <5.0 and does not require low oxidation-
    • reduction potential
    • Rapid growth at low pH (25 to 30 min doubling time)
    • Low numbers in the rumen of hay-fed animals & numbers
    • remain low in grain adapted animals
    • If too much starch is available to animals not adapted:
      • pH drops, growth of S. bovis increases, production of lactic acid increased, further decrease in pH, loss of lactic acid
      • utilizers (Megasphaera elsdenii), lactic acid accumulates, further decrease in pH, all resulting in acute lactic acidosis
slide60

Starch Digesting Bacteria

  • Ruminobacter amylophilus
    • Gram– non motile rod, some are coccoid to oval in shape
    • Ferments starch & maltose Does not use glucose or cellobiose
    • Produces acetic, formic, succinic & ethanol
  • Nutritional interdependence
    • Medium containing starch, glucose and cellobiose
    • Inoculated with R. amylophilus, M. elsdenii & R. albus
      • Initially only R. amylophilus grows but when growth stops
      • cells undergo autolysis releasing amino acids
      • M. Elsdenii require branched chain amino acids can grow
      • M. Elsdenii produces branched chain fatty acids required
      • by R. albus that can now grow
slide61

Starch Digesting Bacteria

  • Succinomonas amylolytica
    • Gram– motile rod
    • Hydrolyzes starch and ferments dextrins, maltose & glucose
    • Produces succinic acid and small amounts of acetic and propionic
  • Selenomonas ruminantium
    • Gram– motile curved rod
    • Hydrolyzes starch and ferments soluble CHOH
    • Produces lactic, acetic & propionic, formic, butyric & H2
    • Also produces an intracellular polysaccharide (glycogen) that
    • is used when available energy is low
  • B. fibrisolvens
  • P. ruminicola
slide62

Sugar Utilizing Bacteria

  • Succinivibrio dextrinosolvens
    • Gram – helicoidal rod
    • Ferments sugars but does not hydrolyze starch,
    • cellulose or xylans
    • Produces succinic and acetic, formic & lactic
  • Eubacterium ruminantium
    • Gram+ non motile rod
    • Ferments glucose, cellobiose and fructose
    • Produces lactic, formic, acetic and butyric acids
slide63

Lactic Acid Utilizing Bacteria

  • Veillonella alcalescens
    • Gram– coccus
    • Does not ferment sugars but does ferment lactate
    • Produces propionic and acetic acids
  • Megasphaera elsdenii
    • Gram– coccus
    • Ferments lactate, sugars, glycerol and some amino acids
    • Produces propionic, acetic, butyric, valeric, caproic acids & H2
    • Increase in numbers during adaptation to grain
slide64

Methanogens

  • CO2 + 2 H2 CH4 + 2 H2O
  • Formic acid
  • Methanobrevibacter ruminantium
    • Gram+ non motile cocobacilli
    • Requires a low oxidation-reduction potential
  • Methanomicrobium mobile
    • Gram– rod
    • Uses formic, CO2 and H2
  • Methanosarcina barkeri
  • Methanobacterium formicicum
    • Have been isolated from the rumen but thought
    • To be of lesser importance
slide65

Acetogenic Bacteria

  • Reduce CO2 at expense of hydrogen
  • 2 CO2 CH3COOH + 2 H2O
  • Bacteria present in rumen and hind gut of several species
  • Do not compete with methanogens for hydrogen
    • H2 threshold 100 times greater
    • Only of significance if methanogens inhibited
    • If active would conserve energy loss from the fermentation
  • Fact they are present in the rumen indicates they might
  • use other substrates
rumen protozoa
Rumen Protozoa
  • Majority are ciliates
  • Low numbers of flagellates
  • Obligate anaerobes
  • 20 to 200 um length
  • Very motile
  • Not attached to feed particles
  • Calves isolated from birth do not become

faunated.

  • Counts up to 106 cells/g – can be up to

50% of microbial mass

rumen protozoa67
Rumen Protozoa
  • Isotricha
    • Starch, glucose, fructose, pectin
  • Dasytricha
    • Starch, glucose, maltose, cellobiose
  • Entodinium
    • Starch, maltose
    • Less use of cellobiose, sucrose & glucose
  • Diplodinium
    • Starch, pectin, maltose, glucose, sucrose
    • Cellulose not always hydrolyzed
  • Epidinium
    • Starch, hemicellulose, cellobiose, sucrose, maltose
    • Cellulose digested
  • Ophryoscolex
    • Pectin, starch
    • Moderate digestion of cellulose
role of protozoa in the rumen
Role of Protozoa in the Rumen
  • Digestion and fermentation
    • Carbohydrates and proteins
  • Ingest bacteria and feed particles
  • More of a digestive process.
  • Engulf feed particles and digest CHOH,

proteins and fats.

  • Produce volatile fatty acids, CO2, H2 & NH3
  • Make a type of starch (amylopectin) that is digested by the animal.
contribution protozoa to the animal
Contribution Protozoa to the animal
  • Observations
    • Numbers in increase when grain is added to
    • forage diets – up to 40 to 60% concentrate
    • Low rumen pH when high-grain diets are fed
    • results in loss of protozoa (Numbers decline below pH 5.6)
    • Only a slight decrease in digestion when defaunated
    • No change in growth of the host animal
  • Large mass
    • Mass of protozoa might equal mass of bacteria
      • Protein supply for animal
    • Number of bacteria declines in faunated animals
  • Some question how much of the protozoa mass leaves the rumen
    • Estimates range from 50 to 85% lyses in the rumen
      • Very sensitive to O2 and oxidation-reduction potential
    • Digestive enzymes probably remain active in the rumen
    • Provide nutrients for bacteria
rumen fungi
Rumen Fungi
  • Initially thought to be a flagellated protozoa. Later showed to contain chitin – representative of fungi
  • Five genera have been found in the rumen:
    • Neocallimastix
    • Piromyces
    • Caecomyces
    • Orpinomyces
    • Anaeromyces
  • Anaerobic flagellated organisms
  • Life cycle includes motile zoospores and non motile vegetative form
  • Zoospores attach to feed particles followed by encystment and germination
  • Counts range from 1.5X103 to 1.5x106 per g rumen contents
slide71

Role of Rumen Fungi

Fungi can degrade cellulose, starch, xylan, hemicellulose & pectin

Some evidence of esterases that free CHOH from lignin

Ferments cellobiose, maltose, sucrose, glucose, fructose & xylose

Role of the fungi not clearly established in mixed cultures with

bacteria. Bacteria seem to inhibit the fungi.

energy supply to ruminants contribution of the microbes to the symbiotic relationship
Energy Supply to Ruminants Contribution of the microbes to the symbiotic relationship:

VFA 70%

Microbial cells 10%

Digestible unfermented feed 20%

Concentration of VFA in the rumen =

50 to 125 uM/ml

amino acid supply to ruminants contribution of the microbes to the symbiotic relationship
Amino Acid Supply to RuminantsContribution of the microbes to the symbiotic relationship

Protein in microbial mass 65%

Undegraded feed proteins 30%

Recycled endogenous proteins 5%

Amino acid balance of microbial mass is

superior to that from undegraded feed

proteins when corn-based diets are fed.