Transcript Lecture Fermentation

Microbial Metabolism
Sugars
ADP
Catabolism
VFA
CO2
CH4
Heat
ATP
NADP+
NADPH
Growth
Maintenance
Transport
Fermentation in the Rumen
• Mostly fermentation of sugars from polysaccharides
• 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
Microbial Interactions
Secondary Fermentations
Cellulose
Fibrobacter
succinogenes
Cellulose fragments
Succinate + Acetate + Formate
Selenomonas
ruminantium
Lactic acid + Propionate + Acetate + Formate + H2
Megasphaera
elsdenii
Propionate + Acetate +H2
Fermentation of Six Carbon Sugars
(Glycolysis or Embden-Meyerhof)
Accounts for 90% of fermentation in the rumen
Glucose
Starch
Glu-1-P
Fructose
Glu-6-P
Fru-6-P
Fru-1,6-bisP
6 carbon
Fructose bisphosphate aldolase
Phospoenolpyruvate
Pyruvate
3 carbon
Dihydroxyacetone-P
Glyceraldehyde-3-P
Glycerol
Predominant pathway for six carbon sugars
(2 ATP + 2 NADH2)/Glucose
An Alternate Pathway of Glucose Metabolism
(Entner-Doudoroff & Pentose)
Gucose
Glu-6-P
6-P-Guconolactone
NADP NADPH
Ribulose-5-P
+ CO2
6-P-gluconate
Ribose-5-P
2-Keto-3-deoxy-6-P-gluconate
Pyruvate
Glyceraldehyde-3-P
Pyruvate
(1 ATP +1 NADPH)/Glucose
Source of five carbon sugars
Fermentation of Sugars
Hexose Monophosphate Pathway
75% of xylan fermented by these pathways
Gucose
Glu-6-P
6-P-Guconolactone
NADP+ NADPH
Glyceraldehyde-3-P
Ribulose-5-P
+ CO2
Xylulose-5-P
Ribose-5-P
Acetyl-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
Pyruvate production is a central intermediate
in ruminal bacteria and can be converted to
variety of fermentation end products.
The NADH produced during glycolysis must
be re-oxidized so fermentation can continue.
Acetic Acid
1. Pyruvate-formate lyase
Pyruvate
3 carbon
Acetyl COA
Acetate
2 carbon
Formate
6H
CH4 + 2H2O
2. Pyruvate oxidoreductase (Most common pathway)
FD
FDH2 (Flavin adenine dinucleotide)
Pyruvate
Acetyl COA
Acetate
CO2
Acetic Acid
One pathway for AcetylCoA
AcetylCoA
Acetyl-P
ADP
Phosphotransacetylase
Acetate kinase
ATP
Acetate
Butyric Acid
A second pathway for AcetylCoA
FD
Pyruvate
3 carbon CO2
FDH2 CO2
Acetyl COA
Acetaldyhyde
COA
Acetoacetyl CoA
Ethanol
Malonyl COA
NADH+H
Acetyl CoA
NAD
COA +B-hydroxybutyryl COA
ATP
Butyrate
Crotonyl COA
NADH+H
Butyryl COA NAD
ADP
Acetate
Butyrate-P
Acetyl COA
4 carbon
Propionic Acid
1. Succinate or dicarboxylic acid pathway
Accounts for about 60% of propionate production
ATP Pyruvate carboxylase
Uses H
Pyruvate
Oxaloacetate
Malate
CO2
ADP
Fumarate
NADH+H
Propionly COA
Succinate NAD
3 carbon
Propionate
Methylmalonly COA
Co
Vit B12
Succinyl COA
Propionic Acid
2. Acrylate pathway (mostly by Megasphaera elsdinii)
NADH
Pyruvate
NAD
Lactic acid
Propionate
Uses H
Acrylyl COA
NADH+H
NAD
Propionyl COA
This pathway becomes more important when
ruminants adjusted to high starch diets
Methane
CO2 + 4 H2
CH4 + 2H2O
The above is the overall reaction
There are a number of enzymes and cofactors involved
with combining CO2 and H2 to form CH4
Formate + 3 H2
CH4 + 2H2O
Lyase
Preferred pathway
CO2 + 2 H
3H2
Methane is the predominant hydrogen sink in the rumen
Methanogens use H2 as a source of energy
Fermentation of Glucose and Other Sugars
Glucose
Pyruvate
Formate
2H
Lactate
Acetyl-CoA
Acrylate
Acetoacetyl CoA
CO2
Oxaloacetate
Malate
Fumarate
Succinate
Methane Acetate Butyrate
Propionyl CoA
Co
Vit B12
Propionate Succinyl CoA
Methylmalonyl CoA
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
Fermentation
Low Acetate
Net: 3 Glucose
2 Acetate + Butyrate + 2 Propionate
+ 3 CO2 + CH4 + 2 H2O
(Acetate:Propionate = 1
High Acetate
Net: 5 Glucose
Methane:glucose = .33)
6 Acetate + Butyrate + 2 Propionate
+ 5 CO2 + 3 CH4 + 6 H2O
(Acetate:Propionate = 3
Methane:Glucose = .60)
Energetic Efficiency
VFA Production
Heat of combustion
kcal/mole
kcal/mole of
% of
of acid glucose fermented glucose
Acetate
209.4
Propionate 367.2
Butyrate
524.3
418.8
734.4
524.3
Glucose
673.0
62.2
109.1
77.9
Effect of Diet
VFA Ratios
Forage:Grain
100:0
75:25
50:50
40:60
20:80
-----Molar ratios----Acetate Propionate Butyrate
71.4
16.0
7.9
68.2
18.1
8.0
65.3
18.4
10.4
59.8
25.9
10.2
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 for
branched-chain fatty acids.
Rumen Acidosis
Animals gorge on grain
Decrease in rumen pH
Megasphaera elsdenii sensitive to acid pH
• Decreased utilization of lactic acid
Streptococcus bovis usually not present in
high numbers (107/ml)
• Grow very fast if sufficient glucose is present
• Double numbers within 20 min (up to 109/ml)
Produce lactic acid
• Lactobacillus ruminis & L. vitulinus also
produce some 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 of lactic acid produced
– D is poorly metabolized in the body
• Results in metabolic acidosis
Acidosis
Subacute acidosis
Decreased fiber digestion
Depressed appetite
Diarrhea
Liver abscess
Feedlot bloat
Decreased milk fat
Acute acidosis
Laminitis
Death
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 over 50%.
Feeding antibiotic Tylosin (10 g/ton of feed) reduces incidence
of liver abscess in feedlot cattle.
Acidosis
Laminitis (founder)
If rumen pH is chronically acidic
Epithelium releases metalloproteinases
Cause tissue degradation
If enter the blood stream causes inflammation
of laminae above the hoof
Feedlot bloat
Starch fermenting bacteria secrete polysaccharides
Produces 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
Acidosis
Diarrhea
Can be caused by some diseases
Often related to the diet in ruminats fed high-grain diets
Extensive fermentation in the hind gut
Acids produced
Absorbed but might cause damage to gut wall
Mucin secreted
Mucin casts can be observed in feces
Retention of water
Gas produced
Gas bubbles in feces
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
Adaptation to Grain Diets
Two 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, <5.5)
Ingest starch
Engulf bacteria producing lactic acid
Use glucose to make polysaccaride
Maintain methanogens
Use hydrogen
Growth of rumen papillae
Increased absorption of VFA
Action of Ionophores
Transmembrane Flux
Out
(High NA+, low K+)
IN
(High K+, low Na+)
ATP
H+
H+
ADP + Pi
H+
K+
Na+
H+
M
H+
K+
M
Na+
H+
Gram Negative
Ionophores Excluded
M
M
Gram - positive
Gram-negative
Effect of Ionophores
Carbohydrates
Sensitive to
ionophore
Resistant to
ionophore
Produce more
acetate & H
Produce more
propionate &
less acetate
CH4
Ionophores - Continued
Inhibit
Rumminococcus albus
Ruminococcus flavefaciens
Butrivibrio fibrisolvens
Increase
Bacteroides succinogenes
Bacteroides ruminicola
Selanomonas ruminantium
Also inhibit
Streptococci
Lactobacilli
No effect
Megasphaera
Selenomonas
Decreased acetate,
formate and CH4
Increase propionate
Decrease lactate
production
Utilize lactate
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
Effects of Rumensin on Rumen Propionate
Propionate production
moles/day
Roughage
5.96
Roughage + Rumensin
8.91
Concentrate
Concentrate + Rumensin
6.89
12.15