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

J.L. Rychlik and J.B. Russell. 2002. Bacteriocin-like activity of Butyrivibrio fibrisolvensJL5 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 digestionin 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 brownmidrib-6 and -18 forage sorghum with conventional sorghum and corn silage in diets oflactating dairy cows. J. Dairy Sci. 87:637

CarbohydratesImportance

Make up 60% to 70% of dietMajor source of energy

1. Microbes Energy for microbes Metabolism, Growth, Protein synthesis

2. Animal End products of the fermentation Digestible CHOC escaping the rumen

ClassificationNonstructural (NSC)

Cell contents - storageStructural (SC)

Cell walls

Chemistry of Feed Dry Matter

1. Organic Carbohydrates

• Fiber Cellulose, hemicellulose

• Soluble fiber– Pectin, fructans, β-glucans

• Starch• Free sugars

Lignin and other phenolics Proteins Lipids

2. Inorganic

Plant Carbohydrates

Cell Content Cell Wall Organic acids Pectins Sugars β-glucans Starches Hemicelluloses Fructans Cellulose

Mammalian enzymes will digest starch andsucrose (limited in ruminants)Microbes digest the plant polysaccharides

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.

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.

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

Plant Cell Walls

Carbohydrates

1. Monosaccharides - one sugar molecule– Hexoses - 6 carbons

o Glucose Fructose Galactose Mannose– Pentoses - 5 carbons

o Arabinose Xylose Ribose

2. Disaccharides - two sugar molecules– Maltose = glucose + glucose– Cellobiose = glucose + glucose– Sucrose = glucose + fructose– Lactose = glucose + galactose

Carbohydrates - Continued

3. Polysaccharides - polymers of sugar molecules- Starch - polymer of glucose (plants)

o Alpha 1- 4 linkages, branch at alpha 1-6o Amylose (unbranched) 20 to 30% of starch

in graino Amylopectin (branched) 70 to 80% of starch

in grain- Glycogen - polymer of glucose (animals)

o Alpha 1- 4 linkages, branch at alpha 1- 6- Cellulose - polymer of glucose (plants)

o Beta 1- 4 linkages

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

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

Partial structure of amylose Partial structure of amylopectin

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.

Starch

Amylopectin

Corn starch

Potato starch

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 - 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:o Close association with lignin – linkages to coumaric

and ferulic acidso Xylan polymers may be crosslinked to other

hemicellulose backboneso Bound to cellulose in plant cell wallo Ratio of cellulose to hemicellulose ranges from 0.8:1 to

1.6:1

• 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

o Citrus pulp, beet pulp, soybean hulls have high concentrationso Alfalfa intermediate concentrations of pectino Grasses low concentrations of pectin

Structural Carbohydrates in Plants

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Young alfalfa Mature alfalfa

Pectin Hemicellulose Cellulose

Pectins less in grass than legumes.Hemicellulose greater in grass than legumes.Hemicellulose and cellulose increase with maturity.

Lignin MonomersLignin

• 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

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 maturityrather 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 alwaysrelated 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

Cross linksFerulic acid (acid form of coniferyl alcohol) is firstproduct synthesizedThe 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

Interaction of Lignin withPolysaccharides

Core lignin

Non core lignin

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

Tannins

Feed Evaluation - Chemical

Sample feed• Need representative sample

Proximate analysis (Weende procedure)• Moisture - Residue is dry matter

– Oven dryVolatile components will be lostOverheating 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

• 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 + OtherNFE = 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.

Fiber analysis - Detergent solutions (Van Soest)

Forage

(Neutral detergent solution)

Soluble InsolubleCell contents Cell walls (NDF) Starch & Sugars Hemicellulose (Pectin, β-glucans Cellulose & fructans) Lignin Soluble proteins Insoluble proteins Lipids Insoluble minerals (dirt) Organic acids

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

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)

PectinsGalactansβ -glucans

Fructans – some lactic acid

Not digested by mammalian enzymesRapidly 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

Neutral Detergent Soluble Fiber

Fiber analysis - NDF

NDF (insoluble residue) of high starchfeeds may be contaminated with starchif 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 solutionExtract samples high in pectin withNDF solution before ADF extraction

Fiber analysis – (Van Soest)

NDF (Insoluble residue)

(Acid detergent solution)

Soluble Insoluble (ADF)Hemicellulose CelluloseProtein Lignin

Cutin Insoluble minerals (soil)

Acid detergent insol N (ADIN)

ADIN is unavailable protein - not digested in rumenor intestines

Lignin Assays

Klason Procedure (wood)Feed (72% H2SO4) Lignin Cellulose dissolvedResidue contains more than ligninProtein, smaller molecular weight phenolics, cutin

Acid Detergent (proteins removed)ADF (KMnO4) Lignin measured as

weight loss (Includes tanninscomplexed with protein)

Cellulose, Cutin, minerals as residue

ADF (72% H2SO4) Cellulose measured asweight loss

Lignin, cutin, minerals as residue

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 dissolvekeratinized 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 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

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Cornsilage

Soybeanmeal

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NDFOrganic acidsSugarsStarchSol fiber

Carbohydrate Fractions in Feeds

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Alfalfasilage

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Soyhulls

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Organic acids

Sugars

Starch

Sol fiber

Carbohydrate Fractions in FeedsComputer Models

Available fiber = NDF – NDF protein – (lignin*2.4)Sugars = NFC (nonfiber) – (starch + pectin)

NFC = NDSC

CHOH fractionsCHO A = sugarsCHO B1 = starch & pectinCHO B2 = available fiberCHO C = unavailable fiber (lignin*2.4)

The Rumen as a Fermentation ChamberContribution of the animal to the symbiotic relationship:

• Open and continuous systemOpen for inoculation from feed and waterContinuous passage

• Constant supply of nutrientsFeed 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

• 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

1. 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 cells2. 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 particles3. Associated with rumen epithelium

• Similarities and differences from bacteria in therumen fluid

• Suggested functionsScavenging O2, tissue recycling, digest urea

4. Other• Attached to surface of protozoa and fungi• Engulfed in protozoa

Groups of Bacteria in the Rumen Habitats in the Rumen

Bacteria Associated with Feed Particles Groups 2 and 3

75% of bacterial population in rumen90% of endoglucanase and xylanase activity70% of amylase activity75% or protease activity

Adherence of mixed rumenbacteria to plant material.

Protuberances from cellsprobably are binding factors.

Bacterial Adhesion to Plant Tissues

1. Transport of bacteria to fibrous substrate Low numbers of free bacteria & poor mixing2. Initial nonspecific adhesion Electrostatic, hydrophobic, ionic On cut or macerated surfaces3. Specific adhesion to digestible tissue Ligands or adhesins on bacterial cell surface4.Proliferation of attached bacteria Allows for colonization of available surfaces

Mechanisms of Bacterial Adhesion

Cellulosome paradigm 2 MDa1. Large multicomponent complexes

Multifunctional, multienzymePolycellulosomes up to 100 MDa

1. Form protuberances on cell surface2. Cellulose binding proteins3. Enzyme binding domains

Attachment of Bacteria to Fibers

Adherent cell Nonadherent cell

Glycocalyx (on outer membrane of cell)

Cellulose Cell Cell

Digested and fermentedCellodextrins by adherent and

nonadherent cells

Cell Wall Structure of Bacteria

Gram +

Gram –

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 cellulolyticenzymes

Cellulase has two functional domains• Catalytic domain - hydrolysis of glycosidic bonds• Binding domain - binds enzyme to cellulose

Fibrobacter succinogenesRuminococcus flavefaciens (maybe)

Cellulosome – Multienzyme Complex

If attachment prevented or reduced digestionof 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

Benefits of Bacterial Attachment

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 & succinicRuminococcus 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

Cellulose Digesting Bacteria

Secondary:Eubacterium cellulosolvens Numbers usually low in rumen Gram– rod Ferments cellulose & soluble sugars Produces mostly lactic acidButyrivibrio fibrisolvens Several strains in rumen Gram– curved rod Ferments cellulose (slow) & starch Produces formic, butyric & lactic acids, ethanol & H2

Strict anaerobesTolerate narrow pH range (pH 6 to 7)Attach to feed particles

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 acidsSynthesis 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 DigestionFibrobacter succinogenes

Hiltner and Dehority, 1983

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Added sugar was a source of readily available energyfrom 0 to 24 h. Subsequent drop in pH after 24 hlimited the rate of cellulose digestion after 36 h.

Effect of pH on Cellulose DigestionRuminococcus flavefaciens

Hiltner and Dehority, 1983

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Low pH (6.0)decreased rateof cellulose digestion, buthad little effect on subsequentability to digest cellulose.

Similar results observedwith Fibrobactersuccinogenes.

Regulation of Rumen pH

Dairy cow can produce up to 160 moles fermentation acids/d

Buffers secreted in salivaPhosphate pK of 6.5Bicarbonate pK of 6.4

Below 5.7 bicarbonate & phosphate not effective buffersAt 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 rateAs high as 20% per h when forages fedCompared with 5% per h when cattle fed grain

Increased amounts of VFA washed out of rumen

Out In

pH XCOO— XCOO—

H+ H+

XCOOH XCOOH

ATPH+

ADP + Pi

Effects of pH Gradient Across Microbial Cell Membrane

Two methods to handleAcidic pH:

Use energy to pump H+

out of the cell. Anion ofacid accumulates – toxic.

Let intracellular pH declineto maintain a pH gradient.Enzymes have to toleratelow pH. S bovis produceslactic acid.

Hemicellulose Digesting Bacteria

Butrivibrio fibrisolvens

Prevotella ruminicolaGram– non motile rodDigests starch, cellulose not digestedProduces succincic, formic, acetic and some strains propionic

Eubacterium ruminantiumGram+ non motil rodFerments cellobiose, dextrins, maltose, glucose, fructose,lactose, sucrose and 5-carbon sugarsDoes not digest starch and celluloseProduces lactic, formic, acetic & butyric acids

Ruminococcus flavefaciens

Ruminococcus albus

Digestion of Forage HemicellulosePure cultures

Bromegrass Alfalfa

Boot Bloom Prebloom Late bloom

B. fibrisolvens 51.9/41.3 32.5/27.1 35.4/34.1 27.4/27.0

P. ruminicola 4.7/6.1 5.0/6.1 33.6/33.9 23.6/20.6

R. flavefaciens 56.6/23.0 34.7/17.1 44.6/10.1 23.6/0

F. succinogenes 77.3/3.0 62.0/2.4 62.1/0 28.7/0

R. albus 60.9/46.0 40.6/29.4 50.1/26.9 31.6/7.4

Degradation/Utilization

Pectin Digesting Bacteria

Lachnospira multiparusMostly gram– motile curved rodFerments pectin, glucose, fructose,cellobiose & sucroseXylan, cellulose & starch not fermentedProduces acetic, formic, lactic,ethanol & H2

TreponemesAnaerobic spiral organismsFerment pectin, arabinose, inulin and sucroseProduces acetic and formic acids

B. fibrosolvensP. ruminicolaR. flavefaciens and R. albus can degrade

pectins but not ferment the end products

Digestion of Forage PectinPure cultures

Bromegrass Alfalfa

Boot Bloom Prebloom Late bloom

B. fibrisolvens 55.3/49.7 46.7/45.3 67.5/57.3 54.4/53.1

P. ruminicola D31d 43.3/49.7 1.0/2.6 31.3/29.1 29.3/24.1

P. ruminicola 23 55.0/52.6 5.7/4.9 36.7/36.6 29.5/27.3

R. flavefaciens 71.3/29.8 35.5/8.1 70.5/30.4 54.3/26.6

L. multiparus 45.6/43.2 28.3/23.9 62.9/50.4 56.6/45.8

Degradation/Utilization

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 acidutilizers (Megasphaera elsdenii), lactic acid accumulates, further decrease in pH, all resulting in acute lactic acidosis

Ruminobacter amylophilusGram– non motile rod, some are coccoid to oval in shapeFerments starch & maltose Does not use glucose or cellobioseProduces 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 stopscells undergo autolysis releasing amino acidsM. Elsdenii require branched chain amino acids can growM. Elsdenii produces branched chain fatty acids requiredby R. albus that can now grow

Starch Digesting Bacteria

Starch Digesting Bacteria

Succinomonas amylolyticaGram– motile rodHydrolyzes starch and ferments dextrins, maltose & glucoseProduces succinic acid and small amounts of acetic and propionic

Selenomonas ruminantiumGram– motile curved rodHydrolyzes starch and ferments soluble CHOHProduces lactic, acetic & propionic, formic, butyric & H2

Also produces an intracellular polysaccharide (glycogen) thatis used when available energy is low

B. fibrisolvensP. ruminicola

Sugar Utilizing Bacteria

Succinivibrio dextrinosolvensGram – helicoidal rodFerments sugars but does not hydrolyze starch,cellulose or xylansProduces succinic and acetic, formic & lactic

Eubacterium ruminantiumGram+ non motile rodFerments glucose, cellobiose and fructoseProduces lactic, formic, acetic and butyric acids

Lactic Acid Utilizing Bacteria

Veillonella alcalescensGram– coccusDoes not ferment sugars but does ferment lactateProduces propionic and acetic acids

Megasphaera elsdeniiGram– coccusFerments lactate, sugars, glycerol and some amino acidsProduces propionic, acetic, butyric, valeric, caproic acids & H2

Increase in numbers during adaptation to grain

Methanogens

CO2 + 2 H2 CH4 + 2 H2O

Formic acid

Methanobrevibacter ruminantiumGram+ non motile cocobacilliRequires a low oxidation-reduction potential

Methanomicrobium mobileGram– rodUses formic, CO2 and H2

Methanosarcina barkeriMethanobacterium formicicum

Have been isolated from the rumen but thoughtTo be of lesser importance

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 hydrogenH2 threshold 100 times greaterOnly of significance if methanogens inhibitedIf active would conserve energy loss from the fermentation

Fact they are present in the rumen indicates they mightuse other substrates

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.faunated.• Counts up to 10Counts up to 1066 cells/g – can be up to cells/g – can be up to

50% of microbial mass50% of microbial mass

Rumen Protozoa

IsotrichaStarch, glucose, fructose, pectin

DasytrichaStarch, glucose, maltose, cellobiose

EntodiniumStarch, maltoseLess use of cellobiose, sucrose & glucose

DiplodiniumStarch, pectin, maltose, glucose, sucroseCellulose not always hydrolyzed

EpidiniumStarch, hemicellulose, cellobiose, sucrose, maltoseCellulose digested

OphryoscolexPectin, starchModerate digestion of cellulose

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

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

Initially thought to be a flagellated protozoa. Later showed to contain chitin – representative of fungiFive genera have been found in the rumen:

Neocallimastix PiromycesCaecomycesOrpinomycesAnaeromyces

Anaerobic flagellated organismsLife cycle includes motile zoospores and non motile vegetative formZoospores attach to feed particles followed by encystment and germinationCounts range from 1.5X103 to 1.5x106 per g rumen contents

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 Rumen Fungi

Digestion of wheat straw leaves in pure culture

Neocall. Pirom. Caeco.

DM, % 45.2 42.3 30.1

Cellulose, % 58.1 50.4 39.4

Hemicellulose, % 52.3 55.0 39.6

Pectin, % 20.5 47.3 16.3

Role of the fungi not clearly established in mixed cultures withbacteria. Bacteria seem to inhibit the fungi.

Composition of Rumen Microorganisms

Bacteria Protozoa

Nitrogen, % 7.8 6.4

CHOH, % 15.5 38.1

Lipids, % 10.1 9.1

Ash, % 16.8 6.4

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

Protein in microbial mass 65%

Undegraded feed proteins 30%

Recycled endogenous proteins 5%

Amino acid balance of microbial mass issuperior to that from undegraded feedproteins when corn-based diets are fed.