11/24/2019

Rumen Microbiology

M.A. Rashid
Deptt. Animal Nutrition

Ruminal Environment
• Self-contained, complex microbial ecosystem.
• Symbiotic relationship
• Microbial biomass is produced – provides
source of protein, CHO, and lipid for the
animal.

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• pH: 6.0 – 6.5 (well buffered between 5.5 and

7.0)
• Temperature: 38-42 °C
• Osmotic pressure: 250 mOsm/kg (normal)
• Anaerobic atmosphere: 65% CO2, 27% CH4,
7% N2, 0.6% O2, 0.2% H2, and 0.01% H2S.

• Rumen microorganisms are predominantly

strict anaerobes.
• Some O2 is tolerated.
– May diffuse across rumen wall and affect
organisms near the wall.
– Metabolized by facultative anaerobes.

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Ruminal Population
• Symbiotic relationship between:
– Many bacteria and protozoa
• Bacteria and host animal
– Digests feeds undigestible by animal in return for
providing anaerobic environment.

Ruminal Population
• Bacteria numbers: 1010 to 1011 / ml of rumen
contents.
• Probably > 200 species, but only a few seem
to have a quantitative significance
• 0.3 to 50 mm in size.
• Many unidentified.
Church, Ch. 7 – 22 genera, 63 species.

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Ruminal Population
• Protozoa numbers: 105 to 106/ ml of rumen
contents.
• Most are ciliates
• Flagellates
• 20 to 200 mm

Ruminal Population
• Bulk volume of protozoa @ bulk volume of
bacteria.
• Protozoal N ~ 40% of microbial N
• Most rumen microorganisms are specific to the
rumen.

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Bacterial Species of Rumen

• Classification
• Facultative anaerobes
– Usually, but not always, anaerobic
– Often adhere to rumen epithelium
– Scavenge for residual oxygen
– Small in number: 107 to 108 cells/g
– Example: Strep. bovis
• Starch digester

Bacterial Species of Rumen

• Classification
– Obligate anaerobes
• Most numerous

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Bacterial Species of Rumen

• By gram stain
– Most are gram negative (e.g. Bacteroides)
– Some important gram positive bacteria:
• Streptococcus bovis (starch digester)
• Ruminococcus sp. (fiber digester)

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• Classification by type of substrate fermented

or end product produced
• Cellulose Digesters
• Fibrobacter succinogenes (formerly Bacteroides s.)
• Ruminococcus flavefaciens
• Ruminococcus albus
• Butryrivibrio fibrisolvens
• Eubacterium cellosolvens, rods, motile, gram +, can be
quite abundant.

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Cellulose digesters
• Numbers usually increase on high roughage
diets.
• Adhere directly onto the surface of the fibers.
• Produces cellulase at or very near the cell
surface.
– Allows for hydrolysis of cellulose
– Ready access to soluble hydrolytic products
(cellodextrins).
– Surface bound nature of process reduces loss of
cellulases to proteolysis.
– Cellulase activity in ruminal fluid is minimal.

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Cellulolytic bacteria
Fibrobacter succinogenes
Ruminococcus flavefaciens
Ruminococcus albus
Strictly anaerobic
• Primarily produce succinate (first two) and
acetate (all) as end products and others – i.e.
CO2, H2, and formate (See Van Soest, Table 16.2).
• Require branched-chain VFA for growth, typically
supplied by AA fermenting bacteria.
• Require NH3 for nitrogen source.

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Hemicellulolytic bacteria
• Butyrivibrio fibrisolvens
• End products: butyrate, formate, acetate, and
lactate.
• Prevotella ruminicola (formerly Bacteroides r.)
– End products: succinate, acetate, and formate.
– Can ferment numerous substrates (can also
produce propionate).

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Pectinolytic digesters
• Butyrivibrio fibrisolvens
– Exopectate lyase: extracellular pectinolytic enzyme,
cleaves pectin chain at the terminal end.
• Prevotella ruminicola
– Endopectate lyase: cleaves randomly along pectin
chain.
• Lachnospira multiparus
• Succinivibrio dextrinosolvens
• Treponema bryantii
• Streptococcus bovis

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Amylolytic bacteria
• Ruminobacter amylophilus (formerly
Bacteroides a.)
– Ferments only starch and maltose
– Attach to starch granules, function via intracellular
a-amylase activity
• Streptococcus bovis
– Acid tolerant, major starch digester
– Do not seem to attach to starch, function via
extracellular a-amylase activity

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Sugar utilizing bacteria

• All bacteria capable of degrading complex
CHO can also degrade simple sugars.
• Exceptions:
– Ruminococcus flavefaciens cannot ferment
glucose, but can ferment cellobiose.
• Interspecies association:
– Treponema bryantii and Fibrobacter succinogenes
(cellulolytic).

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Intermediate acid-utilizing bacteria

• Secondary fermentation of end-products of
other bacteria.
– Lactate ………….. C2, C3 and larger FA.
• Megaspaera elsdenii, Selenomonas ruminantium
– Succinate ………… C3 + CO2
• Selenomonas ruminantium,
• Propionibacteria, etc.
– Formate is utilized as a precursor for methane
• Methanobrevibacter ruminantium

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Proteolytic bacteria
• Proteolytic activity is widely distributed, ~ 38%
of all species.
• None are solely dependent on protein as
energy source.
– Bacteroides amylophilus, Prevotella ruminicola,
Butyrivibrio fibrisolvens: trypsin like protease
associated with its cell wall.
– Many species also possess exopeptidases to
degrade oligopeptides to AA and shorter Peptides.

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Ammonia-producing bacteria
• Prevotella ruminicola, Megaspera elsdenii,
Selenomonas ruminantium
• Peptostreptococci anaerobius, Clostridium sp.
– Rapidly ferment AA to NH3
• Production of branched-chained VFA’s
– Oxidative deamination
• Valine ________ isobutyrate
• Leucine _______ isovalerate
• Isoleucine _____ 2-methylbutyrate

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Ammonia-producing bacteria
• Production of branched-chained VFA’s.

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Ammonia-producing bacteria
• Branched-chained VFA and NH3 are required
for many cellulolytic species.

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Ureolytic bacteria
• Urea 2NH3 + CO2
• Account for ~ 5% of all isolates from rumen
• Many are found near ruminal wall
• Many are facultative anaerobes

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Lipolytic bacteria
• Lipids – actively metabolized in the rumen
• Anaerovibrio lipolytica
– TG and phospholipids…. glycerol and FFA
– Extracellular and membrane bound lipases
• Butyrivibrio sp.
– Hydrolyzes galactolipids, phospolipids, sulfolipids
in forages.

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Lipolytic bacteria
• Hydrogenation of LCFA by rumen bacteria is the reason
for the relatively constant composition of:
– Body fat
– Milk fat
• Isomerization and partial hydrogenation of 18:3 and
18:2:
– Butyrivibrio fibrisolvens
– Treponema bryantii
– Eubacterium sp.
– Fusocillus sp.
– Micrococcus sp.
– Ruminococcus albus

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Isomerization & hydrogenation

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Methane Producing Bacteria

• Helps regulate overall fermentation by
removing H2
• Primary pathway in rumen is the production
of methane by the reduction of CO2 with H2
• 4 H2 + CO2 ______ CH4 + 2 H2O
• Strict anaerobes

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• Many major strains produce CH4 from CO2

and H2.
• The ability to use other substrates is restricted
to certain species of methanogens.
• Formate, methanol, methylamine, di- and
trimethylamine, and acetate.
– Methanosarcina barkerii uses methanol,
methylamine, acetate to produce CH4.
• Found in sheep on high molasses diets.

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Protozoa
• Comprise:
– ~ 2% of wt. of rumen contents
– Up to 40% of total microbial N
– Up to 60% of microbial fermentation products in the
rumen
• Protozoa cannot be maintained without bacteria
• Not essentially required for ruminal function
– Overall importance to ruminal fermentation is
uncertain.

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Protozoa
• Size: 20 to 200 mm
• All are strict anaerobes
• Mostly ciliates, some flagellates
• 30+ species identified in rumen fluid

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Chains of bacteria adhering to the surface of protozoan

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Collecting material and bacteria with its cilia

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• Classification: (2 major subclasses)

• 1) Holotrichs: family Isotrichidae
– Cilia over entire body surface
– Move more rapidly
• 2) Entodinomorphs: family Ophryoscolecidae
– Cilia is banded – usually around mouth

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Russell, 2003, p. 24.

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A Diplodinium with adherent bacteria on the surface, probably methanogens

which live symbiotically with their host (x1065). Van Soest, p. 263.

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Major Holotrichs
• Isotricha
– Utilizes starch and many sugars, but not maltose.
– Numbers high in high soluble sugar diets
– Largest in size
• Dasytricha
– Utilizes many sugars (maltose, cellobiose,
glucose), starch

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• Metabolism
– Ferments readily soluble CHO.
– Stores excess CHO as amylopectin (protozoal
starch).
• Unable to control sugar uptake – can become
so filled storage CHO that they burst

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Major holotrichs
• End-products
– Lactate, acetate, propionate (only in trace
amounts), butyrate, H2, and CO2.
– Symbiotic relationship with methanogens
– H2 needed for methane production.
– Some lipid

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Major Entodinomorphs
• Predominate on high forage diets
– Especially, Diplodinium, Epidinium, Ophryoscolex
– Most ferment cellulose, hemicellulose, starch
• Exception, Entodinium
– Predominates on high starch diets
• Ferments starch, hemicellulose, cellobiose, glucose,
maltose, and sucrose.
• Can tolerate acidity.

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• End-products
– Mostly acetate and butyrate
– Some lactate, H2, and CO2
– Entodinium, Diplodinium can produce
– Some Propionate.
– Some lipid

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Protozoa
• Proteolytic
– High amino-peptidase activity
– Very limited deaminase activity
– No urease, can’t use urea or NH3
– Excretes NH3 and AA
• Lipolytic
– Limited role in biohydrogenation, not conclusively
established.

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Predation by protozoa
• Actively ingest bacteria as source of protein
– Protozoa may contain 10 to 40% of total rumen N.
– Outflow of protozoal protein to SI is limited.
– Extensive recycling of N within the rumen.
• Compete with bacteria for substrates
– Bacteria usually with protozoa present.

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Mode of action
• Entodinium
– Engulfs, then digests internally
• Epidinium
– Lyse bacteria first, then ingests cell components
• Most clear any bacteria near them (not
selective)
• Some selectively cull certain bacteria
• Some eat other protozoa
– Polyplastron eats >1 Epidinium /d

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Fate of engulfed bacteria

• Rate of bacteria digestion varies with bacterial
species
– < 5 min for E. coli for most strains.
– Other strains viable > 150 minutes.
• Use of microbial components
– Protein and AA converted into protozoal protein N
– Purines and pyrimidines incorporated unchanged
into protozoal nucleic acids.

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• Are protozoa helpful?

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• Are protozoa helpful?

– Exact role is not certain
– Defaunation may be beneficial
• Esp. on high energy and high NPN diets
– starch digestibility
– Protozoa cause a lag in starch digestion.
– bacterial protein synthesis and flow to SI

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• Defaunation ?
• Kill off protozoa
• Methods
• Isolation at birth, raised in sterile environment
• Washing techniques and sterilization of rumen
contents
• Decrease pH (5.0-5.5), may not fully defaunate

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• Chemicals or dietary supplements

• Adding HCl to rumen
• Starvation for 6-7 days
• Copper sulfate with 3 d starvation
• Dioctyl sodium sulfosuccinate
• Triazine compounds
• Saponins from plant material
• Greater success in defaunating sheep compared
to cattle

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Effects of defaunation
• Bacteria numbers , population may differ
• Weight change
– Sheep: several studies
– ADG and feed efficiency
• Calves
– No change in ADG but change in physical
• appearance, pot-bellied and rougher hair coat

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Effects of Defaunation
• Ruminal fermentation
– in total VFA, especially butyrate
• Only minor changes in acetate, propionate
– in NH3 [ ]

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Effects of defaunation
• Ration digestibility
– Usually slight decrease in fiber digestibility
– Specific species have different effects:
• Polyplastron: DM and OM dig.
• Isotricha prostoma: DM and OM dig.
• In general, cellulose digestion with protozoa
• Starch digestion with protozoa, shifts site of
starch digestion to SI.

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Fat metabolism
• Lipids stimulate fermentation of some
protozoa
– Example, isotrichs: 9-11% lipid
• Protozoa present
– 18:0, 18:1, 18:2 in plasma and tissues of lambs

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Benefits of protozoa
• Stabilizing factor in rumen fermentation
– CHO storage
– Ingest bacteria
• Bacteria ferments small MW products than protozoa
– Continuous source of protein in rumen
– Stay in rumen longer (turnover is slow)
• Generation time of bacteria: ave. 3-7 hrs.
• Generation time of protozoa: ave. 16-24 hrs.

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Benefits of protozoa
• Possibly more important to wild ruminants
• Ruminants on low protein diet or short
periods of starvation
• Greater protein recycling
• Greater numbers in wild ruminants

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Establishing protozoa
• Need contact with other animals
– Isolation can prevent faunation
• Acidic conditions of lactic acid from milk often
delays establishment of protozoa
– pH > 6 or greater
– Sometimes detected as early as 1 wk, but
permanent establishment takes longer.
– Can reach adult protozoal populations by 5-9
weeks of age depending on the diet.

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Maintaining protozoa population

• Prevent low rumen pH.
– Low pH removes protozoa.
• Substrate availability
– Level of feed intake
• Feed intake protozoa numbers
– Rate of passage
• Rate of passage protozoa
• Protozoa washed out, slower generation time

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Maintaining protozoa population

• Particle size
– Protozoa attach to particles
– particle size protozoa numbers

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Factors affecting ruminal function

• Diet
– concentrate = protozoa (may persist)
– High energy levels = pH , protozoa
– Leveling effect on starch and protein fermentation
• Frequency of feeding
– Protozoan [ ] more stable as feedings increase

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Factors affecting ruminal function

• Level of intake
– Protozoa decrease at very high intake levels
• Age of animal
– Growing animal have different nutritional
requirements
– Microbial ecosystem may be different in young
and older ruminants

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Factors affecting ruminal function

• Animal host
– Variation in [ ] and occurrence of species
• Defaunation method
– Eliminate or change proportions of certain
bacteria or fungi
– In young animal, can cause partial shift in
digestion to hindgut

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