Groups of Bacteria in the Rumen 1. Free-living in the liquid phase 2. Loosely associated with feed particles 3. Firmly adhered to feed particles 4. Associated.

Groups of Bacteria in the Rumen

1. Free-living in the liquid phase2. Loosely associated with feed particles3. Firmly adhered to feed particles4. Associated with rumen epithelium5. Attached to surface of protozoa and fungi

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

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

Attachment ofBacteria to Fibers

Adherent cell Nonadherent cell

Glycocalyx

Cellulose Cell Cell

Digested and fermentedCellodextrins by adherent and

nonadherent cells

Mechanisms of Bacterial Adhesion

1. Large multicomponent complexesCellulosomes

2. Filamentous extracellular materialPili-protein complex

3. Carbohydrate epitopes of bacterial glycocalyx4. Enzyme binding domains

Benefits of Bacterial Attachment

If attachment prevented or reducedDigestion of cellulose greatly reduced

• Brings enzyme and substrate together in a poorly mixed system• Protects enzyme from proteases in the rumen• Allows bacteria to colonize the digestible surface of feed particles• Retention in the rumen to prolong digestion• Reduces predatory activity of protozoa

Microbiology of the Rumen Role of Protozoa

• 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.

ProtozoaContribution to the animal?

Disappear when high grain diets arefed if pH not controlled

Large massProtein

Produce some volatile fatty acids and NH3

Make a type of starch that is digested bythe animal.

Some question how much of the protozalmass leaves the rumen.

Rumen MicroorganismsNutritional Requirements

• CO2

• Energy– End products from digestion of CHOH

Fermentation of sugars• Nitrogen

– Ammonia (Majority of N needs)– Amino acids (nonstructural CHOH digesters)

• Minerals– Co, S, P, Na, K, Ca, Mg, Mn, Fe, Zn, Mo, Se

Rumen MicroorganismsNutritional Requirements - Continued

• Vitamins

– None required in mixed cultures

• Nutrient requirements of pure cultures more complex

Energy Supply to Ruminants

VFA 70%

Microbial cells 10%

Digestible unfermented feed 20%

Concentration of VFA in the rumen =50 to 125 uM/ml

Rumen Digestion

Cellulose Hemicellulose Pectin Starch

Uronic acids Galactose

Cellobiose Pentoses Pentose Dextrosepathway

Maltose

Glucose

Fermentation in the Rumen

• Mostly fermentation of sugars from polysaccarides• Rumen is an anaerobic habitat• Disposal of reducing equivalents is a critical feature of anaerobic fermentation

- Production of lactic acid and ethanol not extensively used in the rumen Production of VFA major pathway Hydrogenases produce hydrogen gas from reduced cofactors Methanogens use hydrogen to produce

methane

Rumen Digestion and Fermentation

CO2

VFADegradable Rumen Microbial cells Feed microbes NH3

CH4

Heat Long-chain fatty acids H2S

ADP

ATP

NADP+

NADPH

Sugars

Ca

tabo

lism

Biosyn

thesis

GrowthMaintenanceTransport

Microbial Metabolism

VFACO2

CH4

Heat

Microbial InteractionsSecondary Fermentations

Cellulose Fibrobacter Cellulose fragmentssuccinogenes

Succinate + Acetate + Formate

Selenomonas ruminantium

Lactic acid + Propionate + Acetate + Formate + H2

Megasphaeraelsdenii

Propionate + Acetate +H2

Fermentation of Six Carbon Sugars (Glycolysis or Embden-Meyerhof- Parnas)

Glucose Fructose

Starch Glu-1-P Glu-6-P Fru-6-P Fru-1,6-bisP

Dihydroxyacetone-PPhospoenolpyruvate Glyceraldehyde-3-P

Pyruvate GlycerolPredominant pathway for six carbon sugars(2 ATP + 2 NADH2)/Glucose

6 carbon

Fructose bisphosphate aldolase

3 carbon

An Alternate Pathway of Glucose Metabolism(Entner-Doudoroff & Pentose)

Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO2

6-P-gluconate Ribose-5-P

2-Keto-3-deoxy-6-P-gluconate

Pyruvate Glyceraldehyde-3-P

Pyruvate1 ATP, 1NADPH/GlucoseSource of five carbon sugars

NADP NADPH

Fermentation of SugarsHexose Monophosphate Pathway

Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO2

Xylulose-5-P Glyceraldehyde-3-P Ribose-5-P

Acetyl-PPyruvate Phosphoketolase Acetyl CoA

AcetateMajor pathway for five carbon sugarsSource of five carbon sugars for biosynthesis

2 ATP, 2 NADPH, 1 NADH/Glucose

NADP+ NADPH

Acetic Acid

1. Pyruvate-formate lyase

Pyruvate Acetyl COA AcetateFormate

6H CH4 + 2H2O

2. Pyruvate oxidoreductase (Most common pathway) FD FDH2 (Flavin adenine dinucleotide)

Pyruvate Acetyl COA AcetateCO2

3 carbon 2 carbon

Acetic Acid

AcetylCoA Acetyl-P ADP

PhosphotransacetylaseAcetate kinase

ATP

Acetate

Butyric Acid

Pyruvate Acetyl COA AcetaldyhydeCO2 COA

Acetoacetyl CoA EthanolMalonyl COA NADH+H

Acetyl CoA NADCOA B-hydroxybutyryl COA

Crotonyl COA NADH+H

Butyryl COA NADAcetate

Butyrate Butyrate-P Acetyl COA

FD FDH2 CO2

3 carbon

4 carbon

ATP ADP

Propionic Acid

1. Succinate or dicarboxylic acid pathwayAccounts for about 60% of propionate production

ATPPyruvate Oxaloacetate Malate

CO2 ADPFumarate

NADH+HPropionly COA Succinate NAD

PropionateMethylmalonly COA Succinyl COA

Co Vit B12

Pyruvate carboxylaseUses H

3 carbon

Propionic Acid

2. Acrylate pathway (mostly by Megasphaera elsdinii)

NADH NADPyruvate Lactic acid

Acrylyl COA NADH+H

Propionate NADPropionyl COA

This pathway becomes more important whenruminants adjusted to high starch diets

Uses H

Methane

CO2 + 4 H2 CH4 + 2H2O

The above is the overall reaction.There are a number of enzymes and cofactors involvedin combining CO2 and H2 to form CH4

Formate + 3 H2 CH4 + 2H2O

CO2 + 2 H 3H2

Methane is the predominant hydrogen sink in the rumenMethanogens use H2 as a source of energy

Methanogenic bacteriaMethanobacterium ruminantiumVibrio succinogenes

Lyase Preferred pathway

Fermentation of Glucose and Other SugarsGlucose

Pyruvate CO2

Formate Lactate Oxaloacetate2H

Acetyl-CoA Malate Acrylate Fumarate

Acetoacetyl CoASuccinate

Methane Acetate Butyrate Propionate Succinyl CoA

Propionyl CoA Methylmalonyl CoACo Vit B12

Fermentation Balance

Low Acetate (High grain)

Glucose 2 Acetate + 2 CO2 + 8 H

Glucose Butyrate + 2 CO2 + 4 H

Glucose 2 Propionate + 2 [O]

CO2 + 8 H CH4 + 2 H2O

Fermentation Balance

High Acetate (High forage)

3 Glucose 6 Acetate + 6 CO2 + 24 H

Glucose Butyrate + 2 CO2 + 4 H

Glucose 2 Propionate + 2 [O]

3 CO2 + 24 H 3 CH4 + 6 H2O

FermentationLow AcetateNet: 3 Glucose 2 Acetate + Butyrate + 2 Propionate

+ 3 CO2 + CH4 + 2 H2O

(Acetate:Propionate = 1 Methane:glucose = .33)

High AcetateNet: 5 Glucose 6 Acetate + Butyrate + 2 Propionate

+ 5 CO2 + 3 CH4 + 6 H2O

(Acetate:Propionate = 3 Methane:Glucose = .60)

Energetic EfficiencyVFA Production

Heat of combustion kcal/mole kcal/mole of % of of acid glucose fermented glucose

Acetate 209.4 418.8 62.2Propionate 367.2 734.4 109.1Butyrate 524.3 524.3 77.9

Glucose 673.0

Effect of DietVFA Ratios

Forage:Grain -----Molar ratios-----Acetate Propionate Butyrate

100:0 71.4 16.0 7.975:25 68.2 18.1 8.050:50 65.3 18.4 10.440:60 59.8 25.9 10.220:80 53.6 30.6 10.7

Branched-Chain Fatty Acids

Propionyl CoA + Acetyl CoA Valerate

Valine Isobutyrate + NH3 + CO2

Leucine Isovalerate + NH3 + CO2

Isoleucine 2-methylbutyrate + NH3 + CO2

Fiber digesting bacteria have a requirement forbranched-chain fatty acids.

Rumen AcidosisAnimals gorge on grain

Streptococcus bovis usually not present in high numbers (107/ml)

• Grow very fast if sufficient glucose is present• Double numbers within 12 min (up to 109/ml)

Produce lactic acid• Lactobacillus ruminis & L. vitulinus also Produce lactic acid

Methanobacter ruminantium in rumen (2 x 108/ml)• Sensitive to pH below 6.0• Have no capacity to utilize more H+

• Excess H+ accumulates

• Some formation of ethanol• Most is used to produce lactic acid

Rumen Acidosis

Increased production of lactic acid• Lactic acid poorly absorbed from rumen compared with other VFAs

Lactic acid is a relatively strong acid• pK: Lactic acid 3.08 A, P, & B 4.75 - 4.81

Very low rumen pH• Might be pH 5.5 or less

Both D and L isomers produced – D is poorlymetabolized in the body

• Results in metabolic acidosis

Acidosis

Subacute acidosisDecreased fiber digestionDepressed appetiteDiarrheaLiver abscessFeedlot bloatDecreased milk fat

Acute acidosisLaminitisDeath

Acidosis

Liver abscessRumen epithelium not protected by mucous

Acid causes inflammation and ulceration (rumenitis)Lactate promotes growth of Fusobacterium necrophorum

Fus. necrophorum infects ruminal ulcersIf Fus. necrophorum pass from rumen to blood, theycolonize in the liver causing abscesses

Incidence of liver abscess in feedlot cattle fed highconcentrate diets (60+ % grain) ranges from 10 to 50+%.

Feeding antibiotic Tylosin (10 g/ton of feed) reduces incidenceof liver abscess in feedlot cattle.

AcidosisLaminitis (founder)If rumen pH is chronically acidic

Epithelium releases metalloproteinasesCause tissue degradationIf enter the blood stream causes inflammationof laminae above the hoof

Feedlot bloatStarch fermenting bacteria secrete polysaccharidesProduce a foamGas trapped in foam

Sudden deathIf large amounts of starch escape the rumen

Overgrowth of Clostridium perfringens in the intestineProduce enterotoxin that might cause death

Acidosis

DiarrheaCan be caused by some diseasesOften related to the diet Extensive fermentation in the hind gut

Produces acidsAbsorbed but might cause damage to gut wall

Mucin secretedMucin casts can be observed in feces

Retention of water Produces gas

Gas bubbles in feces

Managing Acidosis

1. Allow time for adjustment to diets with grainGradually increase grain in the diet

Program “step up” rationsLimit intake until adjusted

2. Feed adequate roughageEffective fiber (eNDF)

3. Manage feed consumptionPrevent gorging of high starch feeds“Read bunks”

System for knowing when to changeamount of feed offered

4. Feed ionophores

Adaptation to Grain DietsTwo to Four Weeks

Allow lactic acid utilizers to increase in numbersMegasphaera elsdenii

Rarely present in rumen of hay fed animals

Selenomonas ruminantiumPropionibacter spp.

Not major populations in the rumenCommercial preparations available

Maintain protozoa (lost at low pH, <5.5)Ingest starchEngulf bacteria producing lactic acidUse glucose to make polysaccaride

Maintain methanogensUse hydrogen

Growth of rumen papillaeIncreased absorption of VFA

Action of IonophoresTransmembrane Flux

Out IN (High Na+, low K+) (High K+, low Na+)

ATP

H+ H+

ADP + Pi

H+ H+

K+ K+

Na+ Na+

H+ H+

M

M

Uses energy

Gram NegativeIonophores Excluded

M

M

Gram - positive Gram-negative

Effect of Ionophores

Carbohydrates

Sensitive to Resistant toionophore ionophore

Produce more Produce moreacetate & H propionate &

less acetate

CH4

Ionophores - ContinuedInhibit Result

Rumminococcus albus Decreased acetate,

Ruminococcus flavefaciens formate and CH4

Butrivibrio fibrisolvens

IncreaseBacteroides succinogenes Increased propionateBacteroides ruminicolaSelanomonas ruminantium

Also inhibitStreptococci Decreased lactateLactobacilli production

No effectMegasphaera Utilize lactateSelenomonas

Ionophores

Monensin sodium (Rumensin)

10 to 30 g per ton of 90% DM feedFeedlot: 27 to 28 g per ton

Lasalosid (Bovatec)

10 to 30 g per ton of 90% DM feedFeedlot: 30 g per ton

Laidlomycin propionate (Cattlyst)

5 to 10 g per ton of 90% DM feedFeedlot: 10 g per ton

Effects of Rumensin on Rumen Propionate

Propionate production moles/day

Roughage 5.96Roughage + Rumensin 8.91

Concentrate 6.89Concentrate + Rumensin 12.15

Predominant Microbial Populations1. pH Fiber digesters less competitive in acid environment - Active pH >6.22. Ionophores

Inhibits Gram + organisms3. Rate of passage

With increased rate of passage, organisms with longer generation time tend to be lost

ProtozoaFungi