Transcript Microbiology of the Rumen - Iowa State University: Animal Science

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
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
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
Organic acids
Sugars
Starches
Fructans
Cell Wall
Pectins
β-glucans
Hemicelluloses
Cellulose
Mammalian enzymes will digest starch and
sucrose (limited in ruminants)
Microbes digest the plant polysaccharides
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.
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 1-6
o
Amylose (unbranched) 20 to 30% of starch
in grain
o
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 molecular
weight with a relatively extended shape, whereas amylopectin has
large but compact molecules.
Partial structure of amylose
Partial structure of amylopectin
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.
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 acids
o Xylan polymers may be crosslinked to other
hemicellulose backbones
o Bound to cellulose in plant cell wall
o 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, Dglucuronic 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 concentrations
o Alfalfa intermediate concentrations of pectin
o Grasses low concentrations of pectin
Structural Carbohydrates in Plants
Pectin
Hemicellulose
Cellulose
35
% Dry matter
30
25
20
15
10
5
0
Young
Orchardgrass
Young alfalfa
Mature alfalfa
Pectins less in grass than legumes.
Hemicellulose greater in grass than legumes.
Hemicellulose and cellulose increase with maturity.
Lignin
•
•
•
•
•
•
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
Lignin Monomers
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
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
Interaction of Lignin with
Polysaccharides
Core lignin
Non core lignin
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
 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
• 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.
Fiber analysis - Detergent solutions (Van Soest)
Forage
(Neutral detergent solution)
Soluble
Cell contents
Starch & Sugars
(Pectin, β-glucans
& fructans)
Soluble proteins
Lipids
Organic acids
Insoluble
Cell walls (NDF)
Hemicellulose
Cellulose
Lignin
Insoluble proteins
Insoluble minerals (dirt)
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)
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
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)
NDF (Insoluble residue)
(Acid detergent solution)
Soluble
Hemicellulose
Protein
Insoluble (ADF)
Cellulose
Lignin
Cutin
Insoluble minerals (soil)
Acid detergent insol N (ADIN)
ADIN is unavailable protein - not digested in rumen
or intestines
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
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 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
70
60
% of DM
50
NDF
Organic acids
Sugars
Starch
Sol fiber
40
30
20
10
0
Corn
grain
Corn
silage
Soybean
meal
Wheat
Carbohydrate Fractions in Feeds
70
60
% of DM
50
NDF
Organic acids
Sugars
Starch
Sol fiber
40
30
20
10
0
Alfalfa Alfalfa Citrus
hay silage pulp
Soy Wheat
hulls mids
Carbohydrate Fractions in Feeds
Computer 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:
• 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
• 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
Groups of Bacteria in the Rumen
Habitats in the Rumen
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 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
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
Adherence of mixed rumen
bacteria to plant material.
Protuberances from cells
probably are binding factors.
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
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
Adherent cell
Nonadherent cell
Glycocalyx (on outer membrane of cell)
Cellulose
Cell
Cellodextrins
Cell
Digested and fermented
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 cellulolytic
enzymes
Cellulase has two functional domains
• Catalytic domain - hydrolysis of glycosidic bonds
• Binding domain - binds enzyme to cellulose
Fibrobacter succinogenes
Ruminococcus flavefaciens (maybe)
Cellulosome – Multienzyme Complex
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
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
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
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
Cellulose remaining,
ug/tube
Control
+.15% Cellobiose
200
150
100
50
0
0
12
24
36
48
60
72
84
Hours
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
Control
Change pH
200
Low pH (6.0)decreased rate
of cellulose digestion, but
had little effect on subsequent
ability to digest cellulose.
100
0
0
24
48
72
96
0
24
48
72
96
7
pH
Cellulose remaining, ug/tube
Low pH
6
5
Hours
Similar results observed
with Fibrobacter
succinogenes.
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
Effects of pH Gradient Across Microbial Cell Membrane
Out
In
Two methods to handle
Acidic pH:
 pH
XCOO—
XCOO—
H+
XCOOH
H+
XCOOH
ATP
H+
ADP + Pi
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.
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
Digestion of Forage Hemicellulose
Pure 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 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
Digestion of Forage Pectin
Pure 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 oxidationreduction 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
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
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
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
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
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
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
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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 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
• 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 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
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
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 with
bacteria. 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 is
superior to that from undegraded feed
proteins when corn-based diets are fed.