Rumen

Secondary article
Article Contents

Roderick I Mackie, University of Illinios at Urbana-Champaign, Illinois, USA

Christopher S McSweeney, CSIRO Tropical Agriculture, Brisbane, Australia
Rustem I Aminov, University of Illinois at Urbana-Champaign, Illinois, USA

. Overview of Rumen Habitat

. Biodiversity of Rumen Microorganisms
. Fermentations in the Rumen

The rumen is a large pregastric fermentation compartment (foregut) which maintains a

diverse but concentrated population of anaerobic bacteria, protozoa and fungi that are
responsible for a variety of degradative and fermentative reactions. During this process
biodegradable organic matter, mainly plant cell wall polymers, are converted into volatile
fatty acids and microbial biomass which supply energy and protein to the host (ruminant)
animal.

Overview of Rumen Habitat

Many animals of a wide range of orders have a portion of
their digestive tract adapted to accommodate a microbial
population which aids in digestion and provides a variety
of nutritional and health benets. Microbial populations
have been described in the gut of herbivores, omnivores
and carnivores and in all zoological classes. This complex,
mixed, microbial culture (comprising bacteria, ciliate and
agellate protozoa, anaerobic phycomycete fungi and
bacteriophage) forms a closely integrated ecological unit
with each other and the host animal, as well as playing a
vital role in the normal nutritional, physiological, immunological and protective functions of the host. Development of microbial populations in the digestive tract of
higher animals commences soon after birth and involves a
complex process of microbial succession and many
microbial and host interactions, eventually resulting in
dense, stable populations inhabiting characteristic regions
of the gut. The rumen is the most extensively studied and
well-documented gut ecosystem because of the importance
of ruminants (cattle, sheep, goats and camels) to human
nutrition and the major role played by rumen microbes in
nutrition of the ruminant animal.
The ruminant foregut or stomach has evolved into three
pregastric fermentation chambers (rumen, reticulum and
omasum) of which the rumen is by far the largest. Ingested
plant material is hydrolysed and fermented in the rumen,
and microbial cells as well as undigested plant particles
pass into the abomasum where gastric digestion begins
(Figure 1). The most obvious feature of ruminants,
rumination, where foregut digesta is regurgitated, rechewed and reswallowed in a frequent regular pattern
repeated up to 500 times per day enables reduction in
particle size (comminution) and exposure of maximal
surface area to microbial attack. The mutualistic microbial
fermentation is based on digestion of the plant cell wall by
cellulases and hemicellulases, synthesis of microbial
proteins from poor quality dietary (forage) protein and
nonprotein nitrogen mainly via ammonia as precursor,

. Fermentation Pathways
. Methanogenesis

synthesis of vitamins B and K, as well as detoxication of

phytotoxins and mycotoxins. In turn, the host animal
provides a mechanism for the selection and harvesting of
feed, maintaining a high level of nutrient supply (1018%
dry matter), temperature regulation (38418C), pH control
(67) by buer in saliva, osmotic control (250350 mOsm)
and removal of soluble inhibitory end-products of digestion as well as undigested particulate matter (residence
time 12 days) and microbial cells, and provision of some
nutrients (urea, phosphate and bicarbonate through saliva
and the rumen wall) (Table 1).
The predominant microorganisms in the rumen are
obligate anaerobes. Fermentation of feedstus in the
rumen yields short-chain volatile fatty acids (VFAs),
primarily acetic, propionic and butyric acids, carbon
dioxide, methane, ammonia and occasionally lactic acid.
Some of the change in free energy is used to drive microbial
growth, but heat is also evolved. The overall fermentation
equation for an animal consuming a high roughage diet is
1 Glucose ! 1:13 Acetate 0:35 Propionate
0:26 Butyrate 1:04 CO2 0:61 CH4
0:44 H2 O
The molar proportions in which the principal VFAs are
formed in the rumen are 65 acetic, 20 propionic, 12 butyric
and 3 higher and branched-chain VFAs. Importantly,
VFAs provide 6080% of the daily metabolizable energy
intake in ruminant animals and provide the energetic
foundation for the mutually benecial association between
the rumen microbes and the ruminant animal. It follows
that ruminants are characterized by low blood glucose
levels and rely heavily on gluconeogenesis for provision of
glucogenic precursors. The quality and quantity of rumen
fermentation products is dependent on the types and
activities of the rumen microbes and this, in turn, has an
enormous impact on nutrient output and performance of
ruminant animals.

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Rumen

Oesophagus:
Regurgitation of
ingesta for rumination.
Escape of fermentation
gases by eructation
Ruminant:
Grazing, chewing, and
rumination. Secretion
of saliva containing
bicarbonate,
phosphate, and urea
Reticulum:
Rumination and
eructation
Omasum:
Absorption of water
and acetic, propionic,
and butyric acids
Abomasum:
Acid secretion kills
microbes. Peptic
digestion

Rumen tissues:
Oxidation of acetic, propionic,
and butyric acids. Work of
growth, milk production, etc.
Rumen microbial activities:
Digestion of cellulose,
hemicellulose, and starch.
Fermentation of sugars to
acetic, propionic, and butyric
acids, CO2 and methane.
Growth of microbial cell bodies
through the work of
fermentation
Large intestine:
Absorption of H2O
Small intestine: Tryptic
digestion of microbes.
Absorption of amino acids
Rumen host activities:
Mixing of rumen contents.
Absorption of acetic, propionic
and butyric acids, sodium and
other ions

Figure 1 Summary diagram describing interrelationships between the ruminant forestomach, its resident microbial population and the host animal. After
Hungate RE (1985) Anaerobic biotransformations of organic matter. In: Leadbetter ER and Poindexter JJ (eds) Bacteria in Nature, vol. 1, pp.3995. New
York: Plenum.

The microbial environment in the rumen has been

examined in great detail and allowing for variation in the
nature and amount of feed ingested, serves as a good model
for other gastrointestinal ecosystems in both herbivores
and nonherbivores. A summary of some of the approximate physical, chemical, and microbiological characteristics of grazing cattle and sheep is presented in Table 1.
Although the physical and chemical parameters of the
rumen environment illustrate the complexities that must be
considered in media selection and design, it is signicant
that some rumen bacteria still remain to be isolated and
characterized. Limitations of conventional culture-dependent techniques can be alleviated by modern molecular
techniques based on sequence comparisons of nucleic acids
and these are briey discussed in the following section.

Biodiversity of Rumen Microorganisms

The rumen is the most extensively studied gut community
and is characterized by its high population density, wide
diversity and complexity of interactions. Bacteria are
predominant (up to 1011 viable cells per g comprising 200
species) but a variety of ciliate protozoa occur widely (104
106 per g distributed over 25 genera). The anaerobic fungi
are also widely distributed (zoospore population densities
of 102 104 per g distributed over 5 genera). The occurrence
of bacteriophage is well documented (107 109 particles per
g). Importantly, the rumen contains representatives of the
three domains of life (Bacteria, Archaea and Eukarya)
articulated by Carl Woese and co-workers.
2

Taxonomic grouping of microbes is important for

identication and classication as well as for understanding metabolic activities and determining microbial
evolution. The classical bacterial taxonomic classication
of genera and species is based primarily on morphological
and physiological (phenotypic) features (see Bergeys
Manual of Systematic Bacteriology). In the classical
scheme, methanogens are grouped together with eubacteria as prokaryotes although they are physiologically and
phylogenetically dierent. Classication and taxonomy of
the ciliate protozoa and anaerobic fungi is still largely
based on morphological and ultrastructural features. Most
information, both phenotypic and genotypic, is available
for the ruminal bacteria based on greater numbers,
diversity, importance and relative ease of cultivation.

Anaerobic bacteria
Rumen bacteria generally range in size from 0.5 to 10 mm
and morphological shapes include cocci, cocco-bacilli,
spirals, crescents and rods. On roughage diets, the
predominant bacteria in the rumen are Gram-negative,
while Gram-positives are more prominent on concentrate
diets. A number of species are also Gram-variable
depending on growth conditions. The isolation of rumen
bacteria is usually carried out using habitat-simulating
media containing rumen uid and a range of fermentable
carbohydrates, or media tailored to the nutrient requirements of particular functional groups or species. Having
obtained a pure culture, identication usually begins with
an assessment of Gram reaction and cell morphology,

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Rumen

Table 1 Summary of physical, chemical, and microbiological characteristics of the rumen ecosystem
Characteristic

Property

Physical
pH
Redox potential
Temperature
Osmolality
Dry matter

5.56.9 (mean 6.4)

2 350 to 2 400 mV
38418C
250350 mOsmol/kg 2 1
1018%

Chemical
Gas phase (%)
Volatile fatty acids (mmol L 2 1)

Nonvolatile acids (mmol L 2 1)

Amino acids and oligopeptides
Ammonia
Soluble carbohydrates
Insoluble polysaccharides
Dietary (cellulose, hemicellulose, pectin)
Endogenous (mucopolysaccharides)
Lignin
Minerals
Trace elements/vitamins
Growth factors

Microbiological
Bacteria
Ciliate protozoa
Anaerobic fungi
Bacteriophage

CO2, 65; CH4, 27; N2, 7; O2, 0.6; H2, 0.2

Acetate 6090
Propionate 1530
Butyrate 1025
Branched-chain and higher 25
Lactate 5 10
5 1 mmol L 2 1 present 23 h postfeeding
212 mmol L 2 1
5 1 mmol L 2 1 present 23 h postfeeding
Always present
Always present
Always present
High Na; generally good supply
Always present; good supply of B vitamins
Good supply; branched-chain fatty acids, long-chain fatty
acids, purines, pyrimidines, others unknown
1010 1011 g 2 1 ( 4 200 species)
104 106 g 2 1 (25 genera)
103 105 g 2 1 (5 genera)
107 109 g particles mL 2 1

motility, range of substrates fermented, major and minor

end-products of fermentation as well as a diverse set of
biochemical reactions including nitrate reduction, production of H2S or indole, and urease activity. Pioneering work
in the laboratories of Robert E. Hungate and Marvin P.
Bryant and others since the early 1950s have provided the
basis for our current understanding of the major culturable
bacteria in the rumen, their metabolic features and
contributions to rumen fermentation.
However, despite this vast amount of knowledge
generated for the ruminal and other gut ecosystems using
traditional techniques, the basic prerequisites for ecological studies, namely enumeration and identication of all
community members have limitations. The two major
limitations faced by gut microbial ecologists include the
inevitable bias introduced by culture-based enumeration
and characterization techniques and the lack of phylogenetically based classication schemes. Modern molecular
ecology techniques based on sequence comparisons of
nucleic acids (DNA or RNA) can be used to provide

molecular characterization while at the same time providing a classication scheme that predicts natural evolutionary relationships. These molecular methods provide
results that are independent of growth conditions and
media used. Also using these techniques, microbes can be
classied and identied before they can be grown in pure
culture. An example is Quinella ovalis, a morphologically
distinctive but uncultivable rumen bacteria, which based
on 16S rRNA phylogeny was most closely related to the
SelenomonasMegasphaeraSporomusa group in the
Gram-positive phylum. Application of both DNA- and
RNA-based molecular methodology to investigate species
diversity has resulted in revision and reclassication in the
predominant ruminal genera Bacteroides, Prevotella,
Fibrobacter, Butyrivibrio and Ruminococcus. For example,
Fibrobacter (formerly Bacteroides) succinogenes from
culture collections actually represents two distinct species
within a genetically diverse yet phylogenetically coherent
genus based on comparative 16S rRNA sequence analysis.
Also, application of 16S rRNA-targeted oligonucleotide

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Rumen

probes revealed a large proportion of F. succinogenes-like

organisms that were not accounted for and remain to be
characterized. Finally, quantitative estimates of population levels using probe technology demonstrated levels 10fold higher than studies using habitat simulating culture
techniques. Examination and alignment of 16S rRNA
sequences deposited in databases allows the construction
of large phylogenetic trees (or phylograms) showing all the
major branches of this tree for Gram-negative and Grampositive bacterial taxonomic groups represented in the
rumen (Figure 2).
Although these molecular methods provide a phylogenetic description of bacteria, this has yet to be clearly
integrated into microbial systematics and ecology and the
denition of species, genus or higher taxonomic units
based on molecular characterization remain to be dened.
The successful development and application of these
methods will provide the link between distribution and
identity of bacteria in their natural environment with their
genetic potential and in situ activities the ultimate goal of
microbial ecologists.

tricha are currently in the Isotrichidae family (order

Vestibuliferida). The remaining protozoa are placed in
the order Entodiniomorphida (family Ophryoscolecidae)
and include the Entodiniinae, Diplodiniinae and Ophryoscolecinae. Phylogenetic placement of 18S rDNA sequences
from the rumen ciliate protozoa are presented in Figure 4.
Ciliate protozoa range in size from 20 to 200 mm. The
Isotrichidae have cilia covering the entire surface and
utilize soluble carbohydrates. The ophryoscolecid protozoa have localized bands of cilia especially surrounding the
oropharynx and utilize both particulate as well as soluble
substrates. Rumen protozoa are proteolytic and actively
ingest bacteria as a protein source. This predatory
relationship results in inverse relative numbers of ciliate
protozoa and anaerobic bacteria. In defaunated animals,
bacterial numbers are higher, as are NH3 and total VFA
concentrations as well as numbers of fungal zoospores.
Rumen protozoa sequester in particulate matter through
chemotaxis and exit the rumen at a slower rate than the
bacteria.

Archaea

Anaerobic fungi

The domain Archaea is represented in the rumen by the

methanogens, which are unied and dened as a group by
methanogenesis but are otherwise extremely diverse
(Figure 3). The methanogens also have many other unique
biological features, including cell wall and membrane
lipids, presence of unique coenzymes and electron carriers,
as well as certain molecular features related to RNA and
DNA. The methanogens are perhaps the strictest anaerobes known, requiring special techniques for their
cultivation and study. Despite much research, few ruminal
methanogens have been described. Methanobrevibacter
ruminantium is predominant in the cattle rumen. Methanobacterium formicicum and Methanomicrobium mobile
have also been isolated. An aceticlastic methanogen,
Methanosarcina barkeri (also Ms. mazei) has also been
isolated from the rumen. Anaerobic ciliate protozoa
produce hydrogen and have methanogens attached to the
cell surface in an episymbiotic mutualistic association. The
importance of gut methanogens in greenhouse gas emissions is likely to stimulate the study of biodiversity in
ruminal methanogens and result in isolation of fresh
strains.

Eukarya
Ciliate protozoa
Early classication of the ciliate protozoa split them into
Holotrichs (Isotricha and Dasytricha) and Entodiniomorphs (Oligotrich subclass). In more current classications the rumen protozoa have been placed in the subclass
Trichostomatia (phylum Ciliophora). Isotricha and Dasy4

Initially, agellated protozoa were described in the rumen

but these were subsequently recognized as fungal zoospores as recently as 1973.
In 1975, Orpin rst succeeded in culturing one of these
polyagellated organisms under anaerobic conditions. The
demonstration that the cell walls of these zoosporic species
contained chitin and chitin synthetase proved that they
were true members of the kingdom Fungi despite being
strict anaerobes and limited in distribution to the guts of
herbivores. The taxonomic position of these organisms is
the focus of considerable debate but presently ve genera
(Neocallimastix, Caecomyces, Piromyces, Orpinomyces
and Ruminomyces; class Chytridiomycota (Chytridiomycetes); order Neocallimasticales) are recognized based on
morphological, metabolic and life cycle characteristics.
The rumen fungi have a specialized life cycle that includes a
free-swimming agellated zoospore stage that attaches to
plant particles, germinates, followed by rhizoid (mycelial)
growth. A sporangium forms allowing zoospores to
develop. At maturity the sporangium bursts, releasing
motile zoospores to begin the life cycle again. The
anaerobic fungi are involved in carbohydrate degradation
and produce a range of VFA end-products. Molecular
phylogenetic studies based on 18S ribosomal RNA show
this class to be monophyletic (Figure 5).

Bacteriophage
More than 125 dierent morphological forms have been
identied. More recent molecular quantitation shows the
presence of 107 109 phage particles per mL rumen uid.
The majority of phage are temperate. The signicance of

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Rumen
0.1
Selenomonas ruminantium
Selenomonas ruminantium ssp lactilytica
Schwartzia succinivorans
Quinella ovalis
Megasphaera elsdenii ATCC 17752
999
Veillonella parvula
Succiniclasticum ruminis
Desulfotomaculum ruminis
Ruminococcus albus ATCC 27211
Ruminococcus flavefaciens ATCC 49949
RF26
RF6
RC5
Anaeroplasma abactoclasticum
1000
Anaeroplasma bactoclasticum
Lactobacillus vitulinus
Lactobacillus ruminis
Streptococcus bovis ATCC 27960
Mycoplasma mycoides ssp mycoides
Clostridium aerotolerans
1000
Clostridium celerecrescens
Clostridium clostridiiforme
Pseudobutyrivibrio ruminis
Clostridium proteoclasticum
1000
Butyrivibrio fibrisolvens Bu43
1000
Butyrivibrio fibrisolvens ATCC 19171
Clostridium aminovalericum
Eubacterium ruminantium GA195
Acetitomaculum ruminis
Clostridium polysaccharolyticum DSM 1801
Cowdria ruminantium
RF32
Oxalobacter formigenes
Ruminobacter amylophilus
Desulfovibrio desulfuricans
Wolinella succinogenes
989

461
1000
599

967

1000
861
598

173

795

512
976
950
627

951
717
381
371

464
132

380
1000
502

997

700
669

Proteobacteria

976
615

Low-G+C Gram-positive bacteria

382

Prevotella ruminicola
Prevotella bryantii
Bacteroides fragilis
366
RF17
411
Bacteroides splanchnicus
740
Porphyromonas asaccharolytica
932
Porphyromonas levii
282
866
Porphyromonas ruminis
Rikenella microfusus
RF2
1000
633
RC9
382
RF15
609
RC2
1000
RC16
1000
1000
RF36
RF14
Cytophaga heparina
420
Cytophaga hutchinsonii
Saprospira grandis
Flavobacterium gleum
Chlorobium vibrioforme
Treponema bryantii
Spirochaetales
Treponema saccharophilum
Fibrobacter succinogenes
Fibrobacteria
Actinomyces bovis
1000
Actinomyces sp.
Actinomycetales
RFP12
Aquifex pyrophilus
1000

858

CFB phylum

395
167

1000
600

587
308

1000

600
526

Figure 2 Phylogenetic placement of 16S rDNA sequences of rumen bacteria. An Aquifex pyrophilus sequence is used as the outgroup for rooting the tree.
Numbers by each node are confidence levels generated from 1000 bootstrap trees. The scale bar is in fixed nucleotide substitutions per sequence position.
Sequences retrieved from 16S rDNA clone libraries from uncultivated bacteria have the prefix RF or RC. Sequences of nonruminal bacteria (nonitalicized)
are inserted into the tree to improve resolution.

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Rumen

0.1

878

878

Methanobacteriaceae

Methanomicrobiaceae

Ruminal archaea

734

Methanobacterium sp. BRM12

Methanobacterium sp. BRM9
1000
Methanobacterium formicicum DSM 1312
1000
Methanobacterium formicicum DSM 3636
1000
Methanobrevibacter ruminantium
563
Methanobacterium mobile DSM 1539
1000
Methanomicrobium sp. BRM16
668
995
Halobacterium salinarum
Thermoplasma acidophilum
Archaeoglobus fulgidus
Methanococcus jannaschii
Pyrococcus furiosus
Methanopyrus kandleri
Pyrolobus fumarius

Halobacteriales
Thermoplasmales
Archaeoglobales
Methanococcales
Thermococcales
Methanopyrales
Crenarchaeota

Figure 3 Phylogenetic placement of 16S rDNA sequences from the rumen archaea. The sequence of the Crenarchaeota representative, Pyrolobus
fumarius, is used as the outgroup for rooting the tree. Numbers by each node are confidence levels generated from 1000 bootstrap trees. The scale bar is in
fixed nucleotide substitutions per sequence position. All rumen archaeal sequences available in databases are included in the tree to infer their phylogenetic
positions. Other taxonomic groups of Euryarchaeota are represented by a single sequence from each order.

879

Rumen ciliates

Litostomatea

Haptorida

Vestibuliferida

1000

Entodiniomorphida

Subclass Class
Eudiplodinium maggii
Polyplastron multivesiculatum
805
Diplodinium dentatum
650
Epidinium caudatum
972
Ophryoscolex purkynjei
1000
Entodinium caudatum
998
Dasytricha ruminantium
845
Isotricha intestinalis
1000
Homalozoon vermiculare
732
Spathidium sp.
478
713
Loxophyllum utriculariae
Trithigmostoma steini Phyllopharyngea
643
Colpoda inflata
Colpodea
767
Prorodon teres
Prostomatea
953
Paramecium tetraurelia
Nassophorea
1000
867
Tetrahymena corlissi
Oligohymenophorea
Oxytricha granulifera
Hypotrichs
673
Protocruzia sp.
Protocruzia
Tracheloraphis sp.
Karyorelictea
Spirotrichea
Blepharisma americanum
0.1

Figure 4 Phylogenetic placement of 18S rDNA sequences from the rumen ciliate protozoa. The Blepharisma americanum sequence is used as the
outgroup for rooting the tree. Numbers above each node are confidence levels generated from 1000 bootstrap trees. The scale bar is in fixed nucleotide
substitutions per sequence position. All rumen protozoan sequences available in databases are included in the tree to infer their positions in the ciliate
phylogenetic tree. Also, the tree includes the three sequences of the free-living haptorian ciliates, close relatives of the rumen ciliates. All other classes are
represented by a single sequence.

phage in controlling specic bacterial populations and

uctuation is largely unexplored.

Fermentations in the Rumen

Fermentative microbes, mainly bacteria, hydrolyse plant
polymers (starch, cellulose, hemicellulose, pectins and
protein) to short oligomers and monomers. These soluble
substrates are transported into the cell by specic transport
6

mechanisms and fermented, resulting in synthesis of

microbial cells and production of fermentation endproducts (acetate, propionate, butyrate, carbon dioxide
and hydrogen). Hydrogen and formate are produced by
many microbes in the rumen where the hydrogen is
quantitatively converted to methane by methanogenic
bacteria, resulting in undetectable levels of free hydrogen
in the gas phase (see Methanogenesis). Although acetogenic and syntrophic bacteria have been isolated, they are
of minor quantitative importance in the rumen.

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Rumen

Neocallimastix joyonii NJ1

Piromonas communis FL
999
Neocallimastix frontalis MCH3
1000
Neocallimastix frontalis L2
Neocallimastix sp. LM-2
Basidiobolus ranarum IFO9117
Endogone pisiformis
Spizellomyces acuminatus 62A
Chytridium confervae 81-1
Dermocystidium sp.
842

272
430
347
430

Ruminal fungi

0.01

Figure 5 Phylogenetic placement of 18S rDNA sequences from the

rumen fungi. The Dermocystidium sp. sequence which represents a
protistan clade near the animalfungal divergence is used as the outgroup
for rooting the tree. Numbers by each node are confidence levels
generated from 1000 bootstrap trees. The scale bar is in fixed nucleotide
substitutions per sequence position. All rumen fungal sequences available
in databases are included into the tree to infer their positions in the fungal
phylogenetic tree. Other fungal sequences include the closest relatives,
namely the Zygomycetes (Basidiobolus ranarum and Endogone pisiformis)
and Chytridiomycetes (Spizellomyces acuminatus and Chytridium confervae).

The major components of organic matter are plant cell

wall components (cellulose, hemicellulose, pectin and
lignin), starch, proteins, lipids and lower amounts of
soluble compounds (oligomers and organic acids).

Fibre degradation
Plant cell wall hydrolysis is carried out by specialist
bacteria (mainly the genera Ruminococcus and Fibrobacter), ciliate protozoa and anaerobic fungi. Bacteria are the
most important group involved in bre degradation
although indirect estimates suggest that protozoa may be
responsible for 3040% of overall bre digestion under
certain conditions, while the role of fungi is unclear.
Cellulase enzyme systems are complex and comprise a
number of endo- and exocellulases, cellodextrinases and bglucosidase activities. The rst step in the degradation of
an insoluble substrate, such as the plant cell wall or
cellulose, is attachment, and factors that regulate this are
under investigation. Also, molecular mechanisms involved
in adherence of bre-degrading bacteria and their enzymes
to insoluble substrates are being determined. Xylan is a
more heterogeneous polymer than cellulose and is broken
down by a variety of enzymes having endo- and
exoxylanase, b-glucosidase and a range of debranching
activities. This hemicellulose-degrading ability is more
widely distributed among rumen microbes than cellulolytic
activity.

Starch and pectin degradation

Starch is rapidly and extensively degraded in the rumen.
Starch granules are rapidly engulfed by the Entodiniomorphid protozoa and converted to an iodophilic storage
polymer, as are soluble sugars by the Holotrich protozoa.

Degradation of dietary starch by bacteria, protozoa and

fungi occurs by combined activity of debranching, a-linked
endo- and exo-amylase and glucosidase enzymes. Limit
dextrins, maltose and glucose are the products of
enzymatic starch hydrolysis. Pectin (polygalacturonic
acid) is hydrolysed by pectin esterase and polygalacturonase enzymes of bacteria and protozoa. Anaerobic fungi
are weakly pectinolytic.

Lipid degradation
Dietary lipids (triglycerides, galactolipids and phospholipids) are rapidly hydrolysed in the rumen to glycerol, free
fatty acids (FFAs) and galactose. Butyrivibrio brisolvens
and Anaerovibrio lipolytica are actively lipolytic while the
long-chain FAs are isomerized and hydrogenated by a
range of bacteria (B. brisolvens, Treponema bryantii,
Eubacterium sp. and Ruminococcus albus). Protozoa are
also active in lipid hydrolysis and may be responsible for
3040% of the total ruminal activity although this may be
confounded by activities of attached and engulfed bacteria.
Hydrogenation (saturation) of unsaturated FAs serves as
an electron sink but importantly results in detoxication of
inhibitory (uncoupling) eects of unsaturated FAs. On
roughage diets hydrogenation capacity results in a high
proportion of saturated FAs in body (depot) fat but this
capacity may be exceeded with high intakes of unsaturated
FAs resulting in an increase in quantity of unsaturated FAs
deposited. Further anaerobic degradation of long-chain
FAs to acetate requires longer residence times (slower
turnover) than occurs in the rumen, resulting in their
outow and absorption in the small intestine.

Protein degradation
Proteolytic activity is widely distributed among the
predominant ruminal bacteria including Prevotella ruminicola, B. brisolvens, S. bovis and Ruminobacter amylophilus. Rumen bacterial proteases are primarily of the
cysteine (6580%) and serine (3040%) types based on
inhibitor studies. Protein breakdown results in production
of peptides and amino acids. Amino acids produced in
excess of the amount incorporated into microbial protein
are rapidly deaminated to produce ammonia, carbon
dioxide and corresponding FA. Ammonia and branchedchain VFAs are essential nutrients, especially for cellulolytic bacteria. Protozoa engulf bacteria, fungi and other
smaller protozoa. This activity plays a signicant role in
intraruminal nitrogen recycling and the eciency of
protein synthesis in the rumen. Protozoa play a major role
in the ingestion of particulate protein, including plant
(supplementary) protein and a lesser role in uptake of
soluble protein, peptides and amino acids. Protozoa have
mixed protease activity similar to the bacteria and rapidly
deaminate amino acids. Isolation and characterization of

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Rumen

ammonia hyperproducing bacteria and investigation of

their role in rumen fermentation of peptides and amino
acids are current research topics. Fungi also have
proteolytic activity, mostly trypsin-like metalloprotease.
Recent evidence suggests a role for plant proteases in initial
proteolysis of plant proteins.
Many rumen bacteria utilize ammonia, urea or other
nonprotein nitrogenous compounds as sole nitrogen
source and 6080% of bacterial protein is synthesized
from ammonia as precursor. Oligopeptides (di- and
tripeptides), rather than amino acids, account for the
remaining 2030% of bacterial protein synthesized.
Characterization of the enzymes of ammonia assimilation
(glutamate dehydrogenase and glutamine synthetase) and
their regulatory mechanisms are currently being studied.

The types of fermentation products produced from

pyruvate depend on the metabolic pathways of individual
organisms, substrate level and type, as well as growth
conditions, including whether the organism is grown in
pure culture or in the presence of hydrogen-utilizing
bacteria (see Methanogenesis).

Acetate
Most anaerobes oxidize pyruvate to acetate via acetyl
CoA as an intermediate. AcetylCoA is converted to
acetylphosphate and subsequently to acetate, with the
production of 1ATP per acetate produced from pyruvate.
Because 2 acetate molecules are produced per molecule of
glucose, there is net production of 4ATP and 4 reducing
equivalents per glucose molecule (Table 2).

Detoxification of phytotoxins and mycotoxins

An important reason for the evolution of foregut
fermentation is detoxication of phytotoxins (of plant
origin) and mycotoxins (of fungal origin). Phytotoxins
occur in many common feeds, including grain, protein
supplements and forages. They range from tannins,
alkaloids, goitrogens, gossypol, saponins, glucosinolates,
mimosine and cyanogens to nitrate and oxalate. In many
instances the rumen microbiota provide a protective
function and eectively modify or degrade a wide variety
of toxic compounds. In some cases the opposite can occur,
with production of toxic metabolites from innocuous
compounds. However, prior exposure of rumen bacteria to
many of the plant toxicants increases the rate of subsequent
detoxication and adaptation is an important factor to
consider. This ability can be modied and deliberately
managed as a system to detoxify feedstus both naturally
by adaptation or inoculation, and through modern genetic
engineering technology.

Succinate and propionate

Succinate production involves reversal of the tricarboxylic
acid (TCA) cycle with carbon entering either (1) by
formation of oxaloacetate (via carboxylation of pyruvate
or phosphoenolpyruvate) followed by reduction to malate
or (2) by reductive carboxylation of pyruvate to malate.
Two reducing equivalents are utilized in succinate production. However, succinate does not accumulate in the rumen
and bacteria such as Selenomonas ruminantium, and others,
rapidly decarboxylate succinate to propionate. The succinylCoA (randomizing) pathway is quantitatively the
most important pathway of propionate production.
Propionate may also be produced by direct reduction of
lactate via the acrylate pathway. This fermentation is
characteristic of Megasphaera elsdenii and results in
utilization of 4 reducing equivalents with a yield of only
2ATP (Table 2).

Butyrate

Fermentation Pathways
Glycolysis
Glucose is quantitatively the most important monomeric
substrate and is fermented in the glycolytic (Embden
MeyerhoParnas) pathway. The use of this pathway is
advantageous to anaerobic bacteria because it maximizes
the yield of ATP. Pentose metabolism proceeds mainly via
the pentose phosphate cycle. Pyruvate is the central
intermediary metabolite in rumen fermentation and is the
branch point where pathways diverge to form fermentation end-products (acetate, propionate, butyrate and
lactate). During glycolysis, nicotinamideadenine dinucleotide (NAD) is converted to the reduced form (NADH)
and it is essential that pyruvate metabolism results in the
reoxidation of NADH so that fermentation can continue.
8

Butyrate is produced by condensation of 2 molecules of

acetylCoA to acetoacetylCoA and then reduced in a
series of reactions, similar to reverse b-oxidation, to
butyrylCoA. Conversion of glucose to butyrate and
2CO2 results in net production of 3ATP and 2 reducing
equivalents (Table 2).

Lactate and ethanol

Lactate is produced by direct reduction of pyruvate by
lactate dehydrogenase. In this reaction no additional ATP
is produced, however all reducing equivalents produced in
glycolysis are utilized. Thus from glucose there is a net
production of 2ATP with the production of 2 molecules of
lactate (Table 2). Ethanol is produced by oxidation of
pyruvate to acetylCoA followed by reduction to acetaldehyde and then ethanol. Many species of rumen

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Rumen

Table 2 Equations of major fermentation reactions a that occur in the rumen

ATP
Substrates

Products

Rxn

Glu

Glycolysis
Glucose + 2NAD+
Pyruvate oxidation

2 Pyruvate + 2NADH + 4H+

Pyruvate + 2H2O

Acetate + HCO3 + H+ + H2

1
1
0

3
4
4

2
2
0

2 Pyruvate + 2H2O
Pyruvate + HCO3 + 2NADH + 2H+

Butyrate + 2HCO3 +
Succinate + 2NAD + 2H2O

Succinate

Propionate + HCO3
Propionate
Ethanol + HCO3 + NAD+

Lactate
Pyruvate + H2O + NADH + H+
H+

Pyruvate + NADH +
Methanogenesis
4H2 + HCO3 + H+

Lactate +

NAD+

CH4 + 3H2O
CH4 + 2CO2 + 3H2O

4Formate
a

H+

Net [2H]

1
1

4
4

Rxn equals net ATP per reaction from pyruvate and Glu equals net ATP from glucose including ATP produced during glycolysis; Net [2H] equals
net reducing equivalents produced or consumed during the fermentation of glucose.

bacteria produce lactate in pure culture but accumulations

in the rumen are transient unless pH drops rapidly and
lactate utilization is reduced resulting in accumulation and
subsequent lactic acidosis. Similarly, ethanol is formed in
pure culture as an alternative electron sink product but is
rarely detected in the rumen.

Methanogenesis
Biological methanogenesis is an important component of
the carbon cycle in a variety of habitats, including the
rumen and large intestines of various animals. Currently,
there is great interest in global warming forced by
greenhouse gases and it is estimated that ruminants and
other gastrointestinal fermentations account for over 50%
of the biogenic methane emissions amounting to 70
100 Tg per year. It is thought that a 50% reduction in
methane emission from ruminants will contribute about
5075% of the emissions reductions needed to stabilize
atmospheric methane concentrations.

Substrates for methanogenesis

The methanogenic bacteria are perhaps the strictest
anaerobes known, requiring not only oxygen-free conditions but also a redox potential lower than 2 330 mV.
Because of these strict growth conditions, special techniques have been developed to study the methanogens. A

salient feature of methanogenic bacteria is that they are

capable of using only a limited number of simple substrates
in methane generation. An important consequence of this
limited metabolic capacity is that in most habitats,
methanogens depend on consortia of other interacting
anaerobes to convert more complex organic matter into
substrates for methanogenesis. Furthermore, the catabolic
reactions that they carry out yield very little energy,
resulting in slow growth rates and low growth yields of
methanogenic bacteria.
The hydrogen that methanogens utilize in nature is
normally the product of metabolism of organic matter by
many anaerobes including bacteria, fungi and protozoa.
Nearly all known species of methanogens can use hydrogen
to reduce carbon dioxide to methane. For some species
hydrogencarbon dioxide is the only substrate they can
use, whereas many can use formate and a few species can
also use acetate, methanol or methylamines.

Biochemistry of methanogenesis
The biochemistry and enzymology of the seven-step
pathway for methane biosynthesis from carbon dioxide
plus hydrogen has been described in several recent reviews.
This unique metabolic pathway has in common the
terminal, major energy-yielding step associated with the
reduction of the methyl group to methane, although
electrons for this reductive step may be obtained from the
oxidation of hydrogen, formate, methanol, methylamines

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Rumen

CO2
COOH

COOH

COOH

CH2 NH2

HOOCCH2 CH2 CHCHCH2 CH2 CNHCHCH2 CH2 CNHCHCH2 CH2 CNHCH2 CH2

OCH2

XH 2 + MF
1 Formylmethanofuran
dehydrogenase

COOH

X + H2 O

methanofuran (MF)

formyl-MF
H
H

H H H H
C C C C CH2
H OHOHOH O

CH
H
HCH3
CH3
H

H2 N

N10

HN

N
H

H 4 MPT

CO2 H
O
OH HO

CH2
HH O
CH2
C O P O CH

H
H

OH

2
MF

Formylmethanofuran:
tetrahydromethanopterin
formyltransferase

CO2 H

formyl-H4MPT

tetrahydromethanopterin (H 4 MPT)

3 N 5N 10 -Methylenetetrahydromethanopterin cyclohydrolase
H2O

HO

COO

9
6

5a

1 2
3

4a

O
Oxidized

CH2 CH CH CH CH2 O P O CH C NH CH CH2 CH2

OH OH OH
R
H
O
N 10a N
O
HO
N
N
O
10

9a
8
7

CH3 O

COO
C NH CH
CH2
CH2

methenyl-H4MPT
F420.H2 or H2
4 N 5N 10 -Methylenetetrahydromethanopterin dehydrogenase

COO
H

F420

Reduced (F420 H2 )

methylene-H4MPT

coenzyme F420

F420 . H2
5 10
5 N N -Methylenetetrahydromethanopterin reductase

HS CH2 CH2 SO3

coenzyme M (HS-CoM)

F420
methyl-H4MPT

CH3 O

HS CH2 CH2 CH2 CH2 CH2 CH2 CNHCHCHOPOH

COOH OH
7-mercaptoheptanoylthreonine phosphate
(HS-HTP)

CoM-SH
6 N 5-Methylenetetrahydromethanopterin:methyl
transferase

H 4 MPT

H2 NOC H2 C
H3 C

H
H
OOC CH2

COO
CH2 O
H CH2
HHN

methyl-S-CoM
CH3

N
Ni
N

CH2 CH2 COO

H

HTP-SH
7

N
12
13

H
O

H
CH2
CH2

CH2 COO

Methyl coenzyme M
reductase

CoM-S-S-HTP
CH4

COO
Factor F430

Figure 6 Biochemical pathway of hydrogen-dependent reduction of carbon dioxide to methane. The C1 unit is sequentially modified, reduced and
transferred bound to novel coenzymes which participate in the reaction cycle. The chemical structures of the unusual methanogenic coenzymes are
located adjacent to the reaction steps in which they participate.

10

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

Rumen

or acetate depending on substrate and species. The central

pathway is illustrated in Figure 6. Structures of six new
unique coenzymes discovered so far during the elucidation
of this central pathway are also presented. The reduction of
carbon dioxide occurs by a novel cycle in which the C1
group is passed, bound to a coenzyme, as it is sequentially
reduced through the formyl, methenyl, methylene, and
methyl stages to methane. The terminal reaction in
methanogenesis is catalysed by the methylreductase system
and involves activation of the formyl methanofuran with
methylCoM (CH3-S-CoM). The presence of a methylreductase denes a cell as a methanogen and historically
these enzymes have been the most well studied enzymes of
methanogenesis.
Methanogenic bacteria conserve energy through electron transport phosphorylation involving a membraneassociated, protein-dependent ATPase coupled to a Na 1 /
H 1 antiporter, although there are some dierences for the
various genera. It is likely that the terminal step in
methanogenesis, the methylreductase reaction, is the site
where ATP synthesis is coupled to methanogenesis, since
this step is universal for all methanogens regardless of
substrate, environment or phylogenetic position.

Microbial interactions and interspecies

hydrogen transfer
The two primary groups that compete with methanogens
are hydrogen-consuming acetogens and sulfate reducers.
Sulfate reducers are capable of using hydrogen at lower
partial pressures than methanogens. However in habitats
receiving large amounts of organic matter, such as
anaerobic digestors, low sulfate concentrations usually
limit sulfate reducers and competition for hydrogen is not
as keen. The acetogens are at a thermodynamic disadvantage to hydrogen-using methanogens and are often outcompeted by methanogens in anaerobic habitats. In 1966
M.P. Bryant and co-workers described the concept of
interspecies hydrogen transfer dened as the mutually
benecial unidirectional transfer of hydrogen from a
hydrogen-producing to a hydrogen-utilizing bacteria. This
may be obligate or facultative depending on whether or not
the hydrogen producer can grow in the absence of
hydrogen consumer. Central to this concept is the

maintenance of low partial pressures of hydrogen by the

hydrogen consumer, which allows the coupled reaction to
be thermodynamically feasible.

Carbon flow in methanogenic habitats

During anaerobic degradation in a bioreactor or waste
digestion there is almost complete conversion of biodegradable organic matter to methane and carbon dioxide
(biogas). Conversion is a multistep process involving many
dierent kinds of interacting microbial species, mainly
bacteria. In the last step, methanogens reduce carbon
dioxide to methane using hydrogen produced by other
bacteria and also cleave acetate to methane and carbon
dioxide. Quantitatively, some 70% of methane is derived
from the methyl group of acetate. However, only a partial
methane fermentation occurs in the rumen and gastrointestinal tract due to the short retention time and thus
fatty acids are not usually degraded but are instead
absorbed and used by the host as a source of oxidizable
energy as well as biosynthetic precursor.
In the rumen, methanogenesis from carbon dioxide
reduction dominates terminal electron ow where the
predominant genus is usually Methanobrevibacter. The
methane produced is eructated (belched) and a typical cow
produces 400 L of methane per day, which is released into
the atmosphere. Because this represents a loss of energy (5
15% gross energy in the feed), strategies to manipulate this
process are under investigation either by feeding concentrate diets, by inclusion of ionophores in the diet or
alternative strategies.

Further Reading
Ferry JM (1993) Methanogenesis. Ecology, Physiology, Biochemistry and
Genetics. New York: Chapman and Hall.
Hobson PN and Stewart CS (1997) The Rumen Microbial Ecosystem, 2nd
edn. New York: Chapman and Hall.
Mackie RI and White BA (1997) Gastrointestinal Microbiology, vol. 1,
Gastrointestinal Ecosystems and Fermentations. New York: Chapman
and Hall.
Mackie RI, White BA and Isaacson RE (1997) Gastrointestinal
Microbiology, vol. 2, Gastrointestinal Microbes and Host Interactions.
New York: Chapman and Hall.

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