Groups of Bacteria in the Rumen

1
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 with rumen epithelium 5.
Attached to surface of protozoa and fungi
2
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
3
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
4
Attachment ofBacteria to Fibers
Adherent cell Nonadherent
cell Glycocalyx Cellulose
Cell Cell Digested and
fermented Cellodextrins by adherent and
nonadherent cells
5
Mechanisms of Bacterial Adhesion 1. Large
multicomponent complexes Cellulosomes 2.
Filamentous extracellular material Pili-protein
complex 3. Carbohydrate epitopes of bacterial
glycocalyx 4. Enzyme binding domains
6

• Benefits of Bacterial Attachment
• If attachment prevented or reduced
• Digestion 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

7
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.

8
Protozoa
Contribution to the animal? Disappear when high
grain diets are fed if pH not controlled Large
mass Protein Produce some volatile fatty acids
and NH3 Make a type of starch that is digested
by the animal. Some question how much of the
protozal mass leaves the rumen.
9
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

10
Rumen MicroorganismsNutritional Requirements -
Continued

• Vitamins
• None required in mixed cultures
• Nutrient requirements of pure cultures more
  complex

11
Energy Supply to Ruminants
VFA 70 Microbial cells 10 Digestible
unfermented feed 20 Concentration of VFA in
the rumen 50 to 125 uM/ml
12
Rumen Digestion
Cellulose Hemicellulose Pectin Starch
Uronic acids Galactose Cellobiose Pentoses Pen
tose Dextrose pathway
Maltose Glucose
13
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

14
Rumen Digestion and Fermentation
CO2 VFA Degradable
Rumen Microbial cells Feed microbes
NH3 CH4 Heat
Long-chain fatty acids
H2S
15
Microbial Metabolism
Sugars
ADP ATP NADP NADPH
Biosynthesis
Catabolism
VFA CO2 CH4 Heat
Growth Maintenance Transport
16
Microbial Interactions Secondary Fermentations
Cellulose Fibrobacter Cellulose
fragments succinogenes Succinate
Acetate Formate Selenomonas
ruminantium Lactic acid
Propionate Acetate Formate
H2 Megasphaera elsdenii Prop
ionate Acetate H2
17
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-P Phospoenolpyruvate Glyceraldehy
de-3-P Pyruvate Glycerol Predominant
pathway for six carbon sugars (2 ATP 2
NADH2)/Glucose
6 carbon Fructose bisphosphate
aldolase 3 carbon
18
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-glucon
ate Pyruvate Glyceraldehyde-3-P Pyruvate
1 ATP, 1NADPH/Glucose Source of five carbon
sugars
NADP NADPH
19
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 Acety
l-P Pyruvate Phosphoketolase Acetyl
CoA Acetate Major pathway for five carbon
sugars Source of five carbon sugars for
biosynthesis 2 ATP, 2 NADPH, 1 NADH/Glucose
NADP NADPH
20
Acetic Acid
1. Pyruvate-formate lyase Pyruvate Acetyl
COA Acetate Formate 6H CH4 2H2O 2.
Pyruvate oxidoreductase (Most common pathway)
FD FDH2 (Flavin adenine
dinucleotide) Pyruvate Acetyl COA Acetate CO2
3 carbon 2 carbon
21
Acetic Acid
AcetylCoA Acetyl-P ADP
Phosphotransacetylase Acetate kinase ATP
Acetate
22
Butyric Acid
FD FDH2 CO2
Pyruvate Acetyl COA Acetaldyhyde CO2 COA
Acetoacetyl CoA Ethanol Malonyl COA
NADHH Acetyl CoA NAD COA B-hydroxybutyryl
COA Crotonyl COA NADHH Butyryl COA
NAD Acetate Butyrate
Butyrate-P Acetyl COA
3 carbon
ATP ADP
4 carbon
23
Propionic Acid
1. Succinate or dicarboxylic acid
pathway Accounts for about 60 of propionate
production ATP Pyruvate Oxaloacetate Malat
e CO2 ADP Fumarate
NADHH Propionly COA Succinate
NAD Propionate Methylmalonly COA Succinyl
COA Co Vit B12
Pyruvate carboxylase
Uses H
3 carbon
24
Propionic Acid
2. Acrylate pathway (mostly by Megasphaera
elsdinii) NADH NAD Pyruvate Lactic
acid Acrylyl COA
NADHH Propionate NAD Propionyl
COA This pathway becomes more important
when ruminants adjusted to high starch diets
Uses H
25
Methane
CO2 4 H2 CH4 2H2O The above is the overall
reaction. There are a number of enzymes and
cofactors involved in combining CO2 and H2 to
form CH4 Formate 3 H2 CH4 2H2O CO2 2 H
3H2 Methane is the predominant hydrogen
sink in the rumen Methanogens use H2 as a source
of energy Methanogenic bacteria Methanobacterium
ruminantium Vibrio succinogenes
Lyase Preferred pathway
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Fermentation of Glucose and Other Sugars
Glucose Pyruvate CO2 Formate Lactate O
xaloacetate 2H Acetyl-CoA Malate
Acrylate Fumarate Acetoacetyl
CoA Succinate Methane Acetate Butyrate
Propionate Succinyl CoA Propionyl CoA
Methylmalonyl CoA
Co Vit B12
27
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
28
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
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Fermentation
Low Acetate Net 3 Glucose 2 Acetate Butyrate
2 Propionate 3 CO2 CH4 2 H2O
(AcetatePropionate 1 Methaneglucose
.33) High Acetate Net 5 Glucose 6 Acetate
Butyrate 2 Propionate 5 CO2 3 CH4 6
H2O (AcetatePropionate 3
MethaneGlucose .60)
30
Energetic EfficiencyVFA Production
Heat of combustion kcal/mole
kcal/mole of of of acid glucose
fermented glucose Acetate 209.4
418.8 62.2 Propionate 367.2 734.4
109.1 Butyrate 524.3 524.3 77.9 Glucose
673.0
31
Effect of DietVFA Ratios
ForageGrain -----Molar ratios----- Acetate
Propionate Butyrate 1000 71.4 16.0
7.9 7525 68.2 18.1 8.0 5050 65.3
18.4 10.4 4060 59.8 25.9
10.2 2080 53.6 30.6 10.7
32
Branched-Chain Fatty Acids
Propionyl CoA Acetyl CoA Valerate Valine Is
obutyrate NH3 CO2 Leucine Isovalerate NH3
CO2 Isoleucine 2-methylbutyrate NH3
CO2 Fiber digesting bacteria have a requirement
for branched-chain fatty acids.
33
Rumen Acidosis

• Animals 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

34
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 poorly
• metabolized in the body
• Results in metabolic acidosis

35
Acidosis
Subacute acidosis Decreased fiber
digestion Depressed appetite Diarrhea Liver
abscess Feedlot bloat Decreased milk fat Acute
acidosis Laminitis Death
36
Acidosis
Liver abscess Rumen epithelium not protected by
mucous Acid causes inflammation and ulceration
(rumenitis) Lactate promotes growth of
Fusobacterium necrophorum Fus. necrophorum
infects ruminal ulcers If Fus. necrophorum pass
from rumen to blood, they colonize in the liver
causing abscesses Incidence of liver abscess in
feedlot cattle fed high concentrate diets (60
grain) ranges from 10 to 50. Feeding
antibiotic Tylosin (10 g/ton of feed) reduces
incidence of liver abscess in feedlot cattle.
37
Acidosis
Laminitis (founder) If rumen pH is chronically
acidic Epithelium releases metalloproteinases Caus
e tissue degradation If enter the blood stream
causes inflammation of laminae above the
hoof Feedlot bloat Starch fermenting bacteria
secrete polysaccharides Produce a foam Gas
trapped in foam Sudden death If large amounts of
starch escape the rumen Overgrowth of Clostridium
perfringens in the intestine Produce enterotoxin
that might cause death
38
Acidosis
Diarrhea Can be caused by some diseases Often
related to the diet Extensive fermentation in
the hind gut Produces acids Absorbed but might
cause damage to gut wall Mucin secreted Mucin
casts can be observed in feces Retention of
water Produces gas Gas bubbles in feces
39
Managing Acidosis
1. Allow time for adjustment to diets with
grain Gradually increase grain in the
diet Program step up rations Limit intake
until adjusted 2. Feed adequate
roughage Effective fiber (eNDF) 3. Manage feed
consumption Prevent gorging of high starch
feeds Read bunks System for knowing when to
change amount of feed offered 4. Feed ionophores
40
Adaptation to Grain DietsTwo to Four Weeks
Allow lactic acid utilizers to increase in
numbers Megasphaera elsdenii Rarely present in
rumen of hay fed animals Selenomonas
ruminantium Propionibacter spp. Not major
populations in the rumen Commercial preparations
available Maintain protozoa (lost at low pH,
lt5.5) Ingest starch Engulf bacteria producing
lactic acid Use glucose to make
polysaccaride Maintain methanogens Use
hydrogen Growth of rumen papillae Increased
absorption of VFA
41
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
Uses energy
M
M
42
Gram NegativeIonophores Excluded
M
M
Gram - positive Gram-negative
43
Effect of Ionophores
Carbohydrates Sensitive to Resistant
to ionophore ionophore Produce
more Produce more acetate H propionate
less acetate CH4
44
Ionophores - Continued
Inhibit Result Rumminococcus albus
Decreased acetate, Ruminococcus flavefaciens
formate and CH4 Butrivibrio fibrisolvens Increas
e Bacteroides succinogenes Increased
propionate Bacteroides ruminicola Selanomonas
ruminantium Also inhibit Streptococci
Decreased lactate Lactobacilli
production No effect Megasphaera Utilize
lactate Selenomonas
45
Ionophores
Monensin sodium (Rumensin) 10 to 30 g per ton of
90 DM feed Feedlot 27 to 28 g per
ton Lasalosid (Bovatec) 10 to 30 g per ton of
90 DM feed Feedlot 30 g per ton Laidlomycin
propionate (Cattlyst) 5 to 10 g per ton of 90 DM
feed Feedlot 10 g per ton
46
Effects of Rumensin on Rumen Propionate
Propionate production
moles/day Roughage 5.96 Roughage
Rumensin 8.91 Concentrate
6.89 Concentrate Rumensin 12.15
47
Predominant Microbial Populations
1. pH Fiber digesters less competitive in
acid environment - Active pH gt6.2 2.
Ionophores Inhibits Gram organisms 3. Rate of
passage With increased rate of passage, organisms
with longer generation time tend to be
lost Protozoa Fungi