normal flora rumen bacteria

1440

THE AMERICAN JOURNAL OF CLINICAL NUTRITIoN

Vol. 23, No. 11, November, 1970, pp. 1440-1450

Printed in U.S.A.

Normal Flora Rumen Bacteria1

M. P. BRYANT,2 PH.D.

LL HERBIVOROUS ANIMALS have an ex-

panded part of the alimentary tract

where bulky fibrous foods, which are rich

in cellulose and similar polysaccharides,

can be delayed in passage in order to

undergo the extensive microbial fermenta-

tion that is necessary for utilization. In

ruminants the expanded part of the tract

is represented by the rumen. Ingested food

passes directly from the esophagus to the

reticulorumen, which represents about 85%

of the total stomach capacity and contains

digesta equal to about 10-20% of the

animal’s weight. The food stays in the

rumen until the microbial fermentation

has brought about the digestion of about

70-85% of the total digestible dry matter.

The undigested food residues and microbial

cells produced in the fermentation con-

tinuously pass out of the reticulum and

into the omasum and, hence, to the

abomasum, or true stomach and intestine,

where digestion of microbes and feed

residues proceeds much as it does in

nonruminant animals.

The fermentation represents a very com-

plex symbiotic association involving the

animal and specific groups of microorga-

nisms that have evolved with the animal.

The chief groups of microorganisms in-

clude certain ciliate protozoa, which are

not found elsewhere in nature, some

flagellates, and a very complex mixture of

bacteria consisting mainly of nonspore-

forming anaerobes. Although the protozoa

contribute significantly to the fermentation,

they are not essential. My discussion will

1 From the Departments of Dairy Science and

Microbiology, University of Illinois, Urbana, Illinois

61801.2 Professor of Microbiology.

mainly concern the small bacteria that

always account for much of the activity.

Most of the information has been obtained

from cattle and sheep. Other ruminants

appear to support a similar flora but

relatively little information on these has

been obtained. A number of comprehen-

sive reviews on various aspects of rumen

microbiology are available. (See (1, 2, and

3) for further references.) Thus, no attempt

will be made to present complete references

in this paper.

The Rumen Environment

The rumen can be likened to a highly

efficient continuous culture system for

growth of anaerobic microorganisms. Food

and water supply, temperature and osmotic

pressure, mixing, and outflow of undigested

residues and microorganisms are relatively

constant. The pH is held relatively con-

stant, usually 6-7, by the buffering action

of a large amount of secreted saliva, which

is high in sodium and potassium bicarbo-

nate and urea, by absorption through the

rumen wall into the blood stream of acids,

and by ammonia produced in the fermen-

tation. The oxidation-reduction potential

is maintained at about -400 my because

of the intensive microbial activity and the

entrance of only a small amount of

oxygen. Gas production is rapid and con-

sists of about 50-70% of CO2 and the rest

is mainly methane. Because of the very

rapid fermentation of soluble proteins and

carbohydrates of the feed, carbohydrate

energy sources for microbes and protein

are mainly present in the particulate frac-

tion of the digesta during the greater part

of time between feedings. Soluble materials

in rumen fluid include a large amount of

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

Normal Flora-Rumen Bacteria 1441

ammonia available as a nitrogen source for

bacteria and the volatile acids and some

other organic acids available as carbon

sources, but not usually available as energy

sources, for the bacteria. Minerals and

vitamins are also present in the fluids.

One of the major ways in which the

rumen differs from a continuous culture

apparatus is that the ingesta is quite

stratified (4). Thus, much more of the

fibrous materials and digestible dry matter

are present in dorsal contents than in

ventral contents. Also, the turnover time

of particles such as hay is longer, i.e.,

about 1-2 days, as compared with 0.3-0.8

day for materials in the fluids (1).

Based on turnover times one can see that

the bacteria in the rumen, even if associ-

ated solely with the more rapidly passing

liquid phase, would only have to grow

with a very slow mean generation time,

about 7 hr, to be maintained. However,

about twice as many bacteria per unit

weight are present in the dorsal contents,

which contains most of the undigested

fibrous particulate materials, rather than

the more liquid ventral material (4). Thus,

the mean generation time needed to main-

tain bacteria in the rumen is probably

much longer than 7 hr even under condi-

tions when rates of passage are very rapid.

Although maximum growth rates of few

rumen bacteria have been determined, it

seems certain that they almost always grow

at a much slower rate than is their poten-

tial rate.

General Functions of the Bacteria

A typical balance for the fermentation

of carbohydrate in the rumen as calculated

by Wolin (5) is given in the following

equation:

57.5(C,H120,) -* 65 acetate + 20 propionate

+ 15 butyratc + 60 CO2 + 35 CH4 + 25 H20

Small amounts of n-valerate and, sometimes,

n-caproate are also produced. Energy con-

version allows microbial growth (dry

weight) equal to about 10-20% of the

carbohydrate fermented (1, 6). The volatile

acids produced are the main source of

energy for the ruminant whereas the

methane, containing about 8% of the gross

energy of the animal’s diet, is lost.

Protein synthesis by rumen bacteria is

very important to the animal (7). The more

soluble proteins of the diet are very rapidly

hydrolyzed to peptides and amino acids

and either converted to microbial protein

or further catabolized to ammonia, volatile

and other organic acids (i.e., the aromatic

acids produced from aromatic amino acids),

carbon dioxide, and sulfide. Practically all

of the bacteria can utilize ammonia as the

main source of nitrogen, and those bacteria

requiring amino acids require only one or

a few. If too little energy is available for

efficient microbial growth, much of the

ammonia is absorbed into the blood

stream, converted to urea, and excreted.

Thus, a proper balance of carbohydrate

energy source and nitrogen is necessary in

the diet in order for the animal to utilize

nitrogen efficiently and to obtain a proper

supply of essential amino acids.

A significant amount of urea from the

blood and saliva is returned to the rumen

and a very active microbial urease converts

it to ammonia and carbon dioxide.

Because under usual conditions most of

the digestible dietary protein is converted

to microbial protein in the rumen, the

distribution of essential amino acids in the

diet of ruminants is of relatively little con-

sequence as compared to nonruminant

animals. In fact, due to the ability of the

bacteria to synthesize amino acids using

ammonia nitrogen, the ruminant has no

requirement for amino acids, and most of

the nitrogen in the diet can be supplied as

urea or other nonprotein nitrogen com-

pounds.

It is of interest that a significant number

of the rumen bacterial species cannot

efficiently utilize amino acids or peptides

but require ammonia as the nitrogen

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

1442 Bryant

source (8, 9), whereas some others utilize

peptides or ammonia but do not effectively

utilize free amino acids (10).

Rumen bacteria also are important in

ruminant fat metabolism. Glycerides and

other lipids such as the galactosyl glyc-

eiides of clover lipids are rapidly hydro-

lyzed and the glycerol or galactose is

fermented, the former mainly to propionic

acid (11). The unsaturated fatty acids are

largely hydrogenated but fatty acids are

not degraded by rumen bacteria. Rumen

bacteria contain relatively large amounts of

odd-numbered carbon straight-chain and

branched-chain saturated fatty acids (11)

and these are biosynthesized by some

species from carbohydrate or amino acid

carbon, but a number of species require

straight-chain volatile acids such as n-

vales-ate or branched-chain acids such as

isovalera te, isobutyrate, or 2-methylbuty-

rate, or both, in order to grow and bio-

synthesize the longer chained acids (12).

Relatively large amounts of these acids are

presellt in ruminant fat (11).The ruminant animal is not dependent

on an exogenous supply of B-vitamins or

vitamin K as they are all biosynthesized by

runien bacteria (13).

Culture and Nutrition of the Bacteria

Dus-ing the first half of the century many

workers attempted the pure culture of

bacteria, functional in the rumen, with

maj 01� emphasis on cellulolytic bacteria;

however, it was not until 1946 that Hun-

gate (14) had unqualified success in ob-

taining pure cultures of the important

cellulolytic bacteria, Bacteroides succino-

genes and members of the genus Rum mo-

coccus. The success of the methods was due

to the development of the roll-tube

technique, which allows very good anaero-

bic conditions to be continuously main-

tamed, and to the use of a prereduced

medium with a composition similar to that

of the natural environment of the orga-

nisms. Although Hungate was, at first,

mainly interested in cellulolytic bacteria

and added finely dispersed cellulose as

the main energy source in the media, he

found that very large numbers of noncellu-

lolytic bacteria grew. This was especially

true when a sugar such as glucose was

added.

It is surprising that little definitive work

has been published on the agar media best

suited for the relatively nonselective erni-

meration and isolation of bacteria in the

various anaerobic habitats in nature and!,

also, that anaerobic techniques often have

been inadequate. However, after working

in Hungate’s laboratory during the time

cellulolytic anaerobes and treponemes

(Borrelia sp.) were routinely isolated!

(14, 15), we modified slightly the anaerobic

techniques and developed the RGCA

medium (16) and, latem-, medium 98-5 that

appears to be as good as any medium for

isolation and enumeration of rumen bac-

teria (17). The latter medium is only

slightly modified from that of Hungate

(14), and similar media have been indi-

cated to be the best available for intestinal

and fecal anaerobes of man and various

animals (18), total anaerobic counts of

anaerobic sewage digestors (19), and the

most sensitive anaerobes known, i.e., the

methanogenic bacteria (20).

Medium 98-5 was developed on the bases

of a large number of experiments in which

various additions, dieletions, and levels of

ingredients were te,ted via colony counts

of rumen contents and isolation and pre-

sumptive identification of large numbers of

strains of rumen bacteria (17). The medium

contains small amounts of glucose, cello-

biose, and starch as addled! energy sources.

The three are necessary because some

carbohydrate-fermenting an aerobes, as i ndi-

cated later, utilize only one of these.

Addition of other energy souices such as

lactate or glycerol would be necessary for

growth of a few species. Energy sous-ces

should be added at a low level to keep gas

production low and! colonies and spreading

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org


F,, = observed difference in electromotive force

between electrode and normal hydrogen electrode.

small so that accurate counts and effective

isolation can be made from tubes contain-

ing many colonies or incubated for long

times. Clarified rumen fluid is addled as a

source of many growth factors including

some that are not found in the usual

bacteriological media. The main buffer in

the medium (pH about 6.7) is bicarbonate-

carbonic acid. This is provided by the

addition of NaHCO3 and maintenance of

a CO2 or 1: 1 C02-H2 gas phase. A carbon

dioxidle gas phase of 50% or more is

1-ecommended because many of the rumen

anaerobes, and especially those that pro-

duce succinate as a major product, require

substrate levels for growth (16, 21). A low

oxidation-redluction potential is obtained

by adding a combination of cysteine and

sulfide as reducing agents. In our hands,

inclusion of 0.025% of Na2S�9H2O in addi-

tion to cysteinc approximately doubles

colony counts (17). Resazurin, 0.0001%, is

usually added as a crude indicator of

anaerobiosis. If reduced to the colorless

state, it indicates an Eh (see footnote 3)

more negative than about -75 my. Actis-

ally, the medium rapidly attains an E,, of

about -250 my or lower, i.e., the Eh at

which phenosafranine is partially ieduced

and indligo disulfonate is completely re-

ducedl (2).

Many of the rumen anaerobes were

shown to require factors present in rumen

fluid i)ut not found in sufficient amounts

in ingredients commonly added to bacteri-

ological media or in feeds consumed by

ruminants. Work done mainly at Beltsville

in the 1950’s and early 1960’s established

the nature and functions of most of these

growth factors and showed that the

greater number of strains of most species

of rumen anaerohes could be grown in

defined media (2, 3, 9, 12, 21). An exception

is the methanogenic bacterium that requires

an as yet unidentified growth factor (22).

The work suggested that rumen fluid in the

medium for enumeration and isolation of

most bacteria could be replaced by more

readily available and better standardized

ingredients; and this was subsequently

shown to be possible (23).

Medium 10 was essentially identical with

the rumen fluid medium (17) except that

the rumen fluid was replaced with a number

of ingredients (23). Small amounts of

trypticase and yeast extract were adided as

sources of vitamins, peptides, and! amino

acids. For most species, these could prob-

ably be replaced by B-vitamins alone, or

B-vitamins plus one or a few amino acids

(9). Very few rumen bacteria i-equire

nucleic acid degradation products. Hemin

was added! because it is essential foi growth

of most strains of Bacteroides ruminicola,

which is usually very numerous (24). The

volatile fatty acids, acetate, n-valerate,

isovalerate, 2-methylbutyrate, and isobuty-

i-ate were added as one or more of these

are either essential or highly stimulator) to

growth of many species of rumen anaei-obes

(2, 9, 22). Vitamin K, or menad!ione, is

essential to growth of some strains of

Bacteroides melaninogenicus (25) but was

not addled to the medium as this species

is a minor component of the rumen floia.

The establishment of the requirement of

volatile fatty acids and! heme for growth

of many rumen bacteria allowed recogni-

tion of important mici-obial interactions in

that these factors are produced by other

species of rumen bacteria. For example, the

branched!-chain volatile acids required by

many species are prod!uced by oxidative

decarboxylation of the corresponding

branched-chain amino acids by other spe-

cies (3).

Some Species of Rumen Bacteria

The rumen contains a very large number

of species (1). Many aeiobic and faculta-

tively anaerobic species are present in small

numbers and! are not metabolically active

un(lel riimen conditions, or are present in

too small numbers to be functionally

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

1444 Bryant

significant. For example, coliforms are

usually present in numbers of about 1,000-

10,000/g and Streptococcus bovis, often the

most numerous facultative anaerobe, is

usually present in numbers of l05-lOT/g.

Many of the more important species, the

nonsporeforming anaerobes, are usually

present in numbers of 108-1010/g in very

young animals (26) as well as in adults (2).

Under usual conditions, no single species

dominates the flora. For example, among

about 50 randomly, pure cultured strains

from well isolated colonies from high

dilutions of rumen contents, at least 10-

15 quite different species representing

about 6-10 genera are commonly ob-

tained. Under a few conditions such as

certain regimes with very high grain feed-

ing a few species may dominate (see (23)).

Many of the dominant species utilize

TABLE I

Some features of more numerous rumen

anaerobic bacteria-gram-negative, non-

motile rods of the genus Bacteroides

F eature B.ruminicola

B.succinogenes

B.amylop/sUus

Energy sources

Glucose + +

Maltose-starch + ZF +Cellulose - +Xylan

Others

Fermentation

products”

Cytochrome b

NH3 from amino

+ManyS,A,F,P(-C02)

+

+

-

FewS,A,F(-C02)

+-

-

NoneS,A,F

(-C02)

-

-

acids

Some nutrient re-

quirements

Heme required

Acids requiredb

±

Stimula-

lation

-

+-

-

NH3 required

Animal diet where

numerous

-

Many

+Many

+High

grain

= succinate, A = acetate, F = formate,

P = propionate, (-CO2) = CO2 uptake. b

Valerate or longer straight-chain acid and/or

isobutyrate or 2-methylbutyrate.

one or more of the polysaccharides such as

cellulose, pentosan, pectin, or starch as the

energy source, and most of the others

utilize hydrolytic products of these. How-

ever, a few species utilize only energy

sources such as glycerol (27) or, in the case

of methanogenic bacteria, C02-H2 or for-

mate (28). Relatively few ferment amino

acids and none utilize fatty acids such as

acetate or palmitate as an energy source.

A few selected features of 15 species repre-

senting 12 genera of representative rumen

bacteria are shown in Tables I-V. For

detailed references on these species, various

reviews are available (1, 2).

Nonmotile members of the genus

Bacteriodes are very numerous. The three

species shown (Table i) all produce suc-

cinate, acetate, and formate from carbo-

hydrate in a CO2-dependent fermentation

(21). Of interest is the fact that Bacteroides

ruminicola produces a small amount of

propionate via the acrylate pathway even

though succinate is the major fermentation

product. The B. ruminicola is the most

versatile member of this group in that it

ferments a large number of carbohydrates

and grows either with ammonia or pep-

tides, but not with amino acids, as the

main source of nitrogen (10). The other

two species require ammonia as the source

of nitrogen and utilize few energy

sources. Bacteroides succinogenes is prob-

ably the most actively cellulolytic bacterium

in the rumen (14) and is of further

interest in that it requires a straight-chain

acid such as n-valerate and a branched-

chain acid, isobutyrate or 2-methylbuty-

rate, for growth. Bacteroides amylophilus

ferments only starch and its hydrolytic

products such as maltose but not glucose

or other carbohydrates and has the greatest

biosynthetic capabilities of any known

rumen species. It has no requirement for

any carbon compounds or organic growth

factors other than the energy source and

CO2.Although the mineral requirements of

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

Feature Succini-monas

Oval

+

+

None

S,A

(-CO2)

Forage-

grain

Helical

+

Few

S,A,F

(-CO2)

High

grain

Bulyrivibrio”

Curved

+

±

±

Many

B,A,F,L,EH2,CO2

9:

Many


few anaerobes have been studied, it is of

interest that both B. succinogenes (29) and

B. amylophilus (30) require Na+ for

growth. Although few nonmarine bacteria

are known to require Na+ and no other

anaerobes have been studied for this

feature, it is possible that many rumen

anaerobes require Na+ and that this

feature might serve as a convenient tool

for separation of these bacteria from non-

rumen anaerobes.

Some features of three of the most

important species of gram-negative rods

with monotrichous polar flagella are

shown in Table II. Two of these, Succi-

n ivibrio dextinosolvens and Succinimonas

amylolytica, are found among the domi-

nant species only when grain is included

in the diet and both produce succinate and

acetate as major products. Succinimonas

amylolytica ferments only starch and its

hydrolytic products. Succinivibrio dextrino-

solvens is often the most numerous bac-

terium when diets high in starch are fed.

Although it does not hydrolyze all com-

ponents of starch, all strains actively fer-

ment dextrin. The species Butyrivibrio

fibrisolvens is one of the most numerous

and ves-satile bacteria present in the rumen

and wide variations occur in features within

the group suggesting that more than one

species may be represented. This is the

major butyric acid-producing species in

the rumen and various strains ferment a

wide variety of energy sources including

compounds such as cellulose, starch, vari-

ous pentosans, pectin, and saponins. Re-

cent work with this species presented the

first demonstration of fermentation and

degradation of the heterocyclic ring struc-

ture of rutin and other bioflavonoids by

pure cultures of anaerobic bacteria (31).

It also hydrogenates trienoic and dienoic

18 carbon fatty acids to monoenoic acid.

In addition to butyrate, this organism may

either produce or fix acetate and produces

formate, lactate, ethanol, CO2 and H2 in

carbohydrate fermentation.

TABLE II

Some features of the more numerous rumen

anaerobic bacteria-gram-negative, motile

rods with monotrichous polar flagella

Shape

Energy sources

Glucose

Cellulose

Starch

Xylan

Others

Fermentation

products

NH3 from

amino acids

Animal diet

where num-

erous

Succini-vibrio

“Another unnamed organism (B-385-like) with

tufts of polar flagella and similar physiologically to

Butyrivibrio is often numerous, especially when the

rumen is somewhat acid. b Starch is not com-

pletely hydrolyzed but dextrin is fermented.S = succinate, A = acetate, F = forniate, B =

butyrate, L = lactate, E = ethanol, (-CO2) =

CO2 uptake.

Some features of other motile bacteria of

the rumen are shown in Table us. Seleno-

inonas ruminantium is a relatively large

crescentic bacterium with tufts of flagella

emanating from the concave side. Many

early workers thought it to be a flagellate

protozoan. It is very versatile in that it

ferments many carbohydrates and is one

of the major lactate- and amino acid-fer-

menting bacteria of the rumen. Its fermen-

tation of carbohydrate is unusual among

rumen bacteria in that propionic acid is

a major product, whereas most of the

rumen propionate is produced via succi-

nate production by many species and

succinate decarboxylation to propionate by

others (32). Lachnospira multiparus is

unusual in that it is a gram-positive curved

rod with lateral monotrichous flagella. It

is a very dominant member of the rumen

flora when legume pasture, high in pectin,

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

Shape

Grain stain

Flagella

Spiro-

chete

Curved,

cres-

cent

Tuft

Concave

side

+

±

Many

P,A,L

CO2+

Many

Curved

+Mono-

trichous

Lateral

++

Few

A,F,L,E

CO2,H2

Legume

pasture

1446 Bryant

TABLE III

Some features of more numerous rumen

anaerobic bacteria-other motile bacteria

Feature Selenomonas Lachnospira Treponema

Energy sources

GlucosePectin

Lactate

OthersFerinen tation

products”

NH3 from amino

acids

Animal diet where

n imerous

= propionate, A = acetate, L =

F = formate, E = ethanol.

is fed and it appears to function primarily

in pectin fermentation. The rumen spiro-

chete, Treponema sp. (Borrelia sp.) is

almost always present but usually repre-

sents only about 4% or less of the total

cultured bacteria. It produces succinate as

a major product and appears to function

mainly in the fermentation of sugars pro-

duced during the hydrolysis of cellulose and

other polysaccharides by other species. This

was the first treponeme to be cultured in

a completely defined medium and was

shown to require large amounts of CO2

(16) and acids, such as valerate and iso-

butyrate, for growth (33).

Some of the major gram-positive non-

motile bacteria include Eubacterium rumi-

nan/in in, a bu tyrate-produci ng organism,

and an as yet unnamed strictly anaerobic

Lactobacillus sp. (Table iv). The latter

organism is homofermentative and pro-

duces n (-) lactic acid (34). It ferments

many sugars and is only numerous when

high grain rations or lush pasture is fed.

Under many conditions lactobacilli are

insignificant in the rumen.

The methanogenic bacteria are very

important members of the rumen fermen-

tation in that they utilize most of the

hydrogen and, possibly, formate, produced

by carbohydrate-fermenting bacteria, to re-

duce carbon dioxide to methane and to

- obtain energy for growth. They do not

None ferment other materials. Met hanoba cterium

ruminantium is the most numerous spe-

cies, although a new species, Met hano-

bacterium mobilis (28), also quite num-

+ erous under some condlitions, was recently? described. The M. ruminantium requires

ammonia as the main source of nitrogenMany

S,A,F,L and acetate and 2-methylbutyrate are essen-E tial as carbon sources (22).

- Table v shows some features of three of

the more important species of cocci. TheMany . -

ruminococci are usually present in large

numbers and are primarily concerned withlactate, fermentation of the polysaccharides, cellu-

lose, and xylan. It is of interest that most

strains do not ferment the monosaccharid!e

TABLE IV


anaerobic bacteria-gram-positive

nonmotile rods

FeatureEubacierium

ruminan-hum

Laclobacillussp.

Meihano-baclerium

ru�ni-nanhium

Energy sources

Glucose + +Starch, cellulose, - - -

lactate

Xylan ± - -

H2-C02, formate - - +Fermentation B,F,L,A L CH4

products” CO2, H2

NH3 from amino - - -

acids

Animal diet where Forage Lush pas- Many

numerous ture, high

grain

“B = butyrate, F = formate, A = acetate,

L = lactate. The Laclobacillus sp. produces D (-)lactic acid.

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

FeatureRumi-

nococcusalbus

0.7-1 .2

:1:

±

Few

A,F,E

CO2,H2

+

Many

Rumi-nococcus

flavefaciens

0.7-1.2

+

9:

±

Few

S,A,F

H2,

(-CO2)

+

Many

Pepto-siref’tococcus

elsdenii

1.2-2.4

+

+

+Few

A,P,B,V

C,C02,H2

+

High grain


hydrolytic products of these polysaccharides,

glucose, or xylose, although the disaccha-

rides, cellobiose, or xylobiose, are fer-

mented. This is presumably due to lack of

permeases for the monosaccharide (35).

The ruminococci require ammonia as the

main nitrogen source and most require one

or moie of the acids, isovalerate, isobuty-

rate, and 2-methylbutyrate for growth. The

two species are separated mainly on the

basis of fermentation products and chain

formation.

The major lactate fermenting species in

the rumen, other than Selenomonas rum i-

nantiu m, is Peptostreptococcus elsdenii

(Table v). This species is a major organism

in the rumen only when high grain diets,

or other diets that result in relatively low

pH, are fed. It is primarily functional in

lactate fermentation and is of particular

interest in that it is the major species

responsible for the production in the rumen

of larges- amounts of valeric and! caproic

acids from carbohydrate when high grain

diets are fed.

The question arises as to whether many of

the dominant species of rumen bacteria

are present in other habitats. Although

earlier workers often believed them to be,

for the most part, restricted to the rumen,

it is now evident that many identical or

closely related species are present in other

habitats. For example, ruminococci have

been isolated from the rabbit and guinea

pig cecum. Met han obacterium rum man-

tium is one of the dominant methane

bacteria involved in the anaerobic diges-

tion of sewage (20). It is the only methane

bacterium so far isolated from human

feces (36). Butyrivibrio sp. were isolated

from feces of humans, rabbits, and horses

(37). Both S. ruminantiuin and S. sputi-

genum from the human oral cavity have

recently been shown to be distinct species

(38), but it is possible that the guinea pig

cecal organism, Selenomonas palpitans,

which has not yet been isolated, and

selenonomads from the ceca of other

TABLE V


anaerobic bacteria-gram-variable cocci

Size (�sm)

Chains

Energy sources

Glucose

Cellobiose, cellu-

lose, xylan

Lactate

OthersFermentation

products”

NH3 from amino

acids

NH3 and VFA re-

quired�

Animal diet where

numerous

“A = acetate, F = formate, E = ethanol, S =

succinate, P = propionate, B = butyrate, V =

valerate, C = caproate. b One or more of the

acids, isobutyrate, isovalerate, and 2-niethylbu-

tyrate ar� essential for growth.

rodlents are identical with S. ruminantium.

Studies on DNA homology may be neces-

sary in order to be certain that B. rumini-

cola is definitely different from some mem-

bers of the Bacteroides fragilis group

found in the intestinal tract of animals and

man. Further studies of habitats other than

the rumen and involving adequate isolation

techniques and definitive methods of iden-

tification such as those in use in the

Anaerobe Laboratory at Virginia Poly-

technic Institute (18) will materially aid in

solving this question.

Pathway of Carbohydrate Fermentation

Dr. Baldwin discusses the pathways of

fermentation in relation to energy genera-

tion (6); however, to close my discussion,

brief mention should be made of the path-

ways of carbohydrate fermentation in the

rumen in relationship to kinds of bactei-ia

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

1448 Bryant

Polysaccharide - Fermenting Species

5: Sugar - Fermenting Species

C Lactate - Fermenting Species

D: Methanogenic Species

FIG. 1. Schematic presentation of kinds of bacte-ria, major end products (underlined), and majorextracellular intermediates (enclosed) formed in

fermentation of polysaccharides by rumen bacteria.

and extracellular intermediates. Figure 1

shows a simplified scheme of the fermenta-

tion of polysaccharides. Studies of fermen-

tations carried out by pure cultures and by

whole rumen contents indicate that poly-

saccharides are hydrolyzed to soluble oligo-

saccharides and sugars by polysaccharide-

hydrolyzing organisms. Then, these extra-

cellular intermediates are assimilated by

cells of these organisms and other sugar-

fermenting species and fermented with

production of some rumen end products

such as acetate, propionate, butyrate, and

CO2. However, these organisms usually also

produce end products that are further

metabolized by other species and, therefore,

are not rumen end products. These prod-

ucts, or extracellular intermediates, include

H2, formate, succinate, and lactate. The

formate is rapidly degraded to CO2 and

H2 by species of carbohydrate-fermenting

bacteria and methanogenic bacteria (39),

and the latter species very rapidly utilize

H2 to reduce CO2 to methane. Thus, one

almost never finds a significant concentra-

tion of formate or H2 in the rumen. The

succinate produced by many species is

rapidly decarboxylated with production of

propionate and CO2 by other species (32),

and lactate produced by some is fermented

by species such as P. elsdenii and S. rumi-

nantium with production of the acid end

products plus H2.

Pure culture studies suggested that lac-

tate and ethanol were important extra-

cellular intermediates in the rumen, but

studies of pooi sizes and turnover rates of

these compounds in whole rumen contents

indicate that very little of the carbohydrate

is fermented via lactate and essentially

none via ethanol under usual conditions

(1). Conversion via lactate increases if high

grain diets are fed, and under very unusual

conditions ethanol may be produced.

The discrepancy between the production

by pure cultures of much ethanol and

lactate and the fact that these compounds

are not significant intermediates in the

rumen under many conditions is probably

best explained by the fact that the methane

bacteria nearly always maintain a very low

partial pressure of H2. Most of the carbo-

hydrate-fermenting bacteria that produce

lactate or ethanol also produce acetate and

hydrogen. In pure culture fermentations,

hydrogen accumulates and the energetics

of electron flow from low-potential reduced

carriers, such as pyridine nucleotides and

ferredoxin generated in glycolysis and

pyruvate metabolism, to H2 become much

less favorable (40). Thus, in the pure

cultures more lactate and ethanol and less

acetate is probably produced in response to

an increased need for disposal of these

electrons. In the rumen and some other

anaerobic environments, the methane bac-

teria maintain a low partial pressure of

H2 that obviates the tendency for the

carbohydrate-fermenting bacteria to pro-

duce ethanol or lactate, and they probably

produce a larger amount of acetate. Acetate

formation from pyruvate generates adeno-

sine triphosphate, which provides energy

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org


1. HUNGATE, R. E. The Rumen and Its Microbes.

New York: Academic, 1966.

for growth of the organism, but lactate or

ethanol formation does not. Thus, the

probable importance of the methane bac-

teria in the fermentation becomes more

clear. They not only obtain energy for

growth by utilization of H2 but allow

hydrogen-producing bacteria to ferment

organic matter via pathways involving

more efficient energy generation (1, 40).

SUMMARY

The digestion of feeds, which usually

contain large amounts of cellulose and

similar polysaccharides, by the ruminant

animal is largely dependent on an intense

anaerobic microbial fermentation in the

rumen. Nonsporeforming anaerobic bac-

teria are mainly responsible for the fermen-

tation. The carbohydrates of the diet are

fermented mainly to acetate, propionate,

butyrate, CO2, and CH4, and these volatile

acids are a major source of energy for the

ruminant animal. Major extracellular in-

termediates, i.e., products of some micro-

bial species that are utilized by others in

the fermentation of polysaccharides include

oligosaccharides and sugars, H2, formate,

succinate and, under some conditions,

lactate. Microbial protein from the rumen

is usually the chief source of essential

amino acids for the ruminant regardless

of the type of protein in the diet and most

species of rumen bacteria can utilize

ammonia as the main nitrogen source. The

microorganisms also metabolize dietary

lipids and synthesize B-vitamins. The

methods of culture and some taxonomic,

nutritional, and metabolic characteristics

of some representative rumen anaerobic

bacteria are briefly discussed. These bac-

teria, in general, are better characterized

and their functions better understood than

those of any other anaerobic habitat.

REFERENCES

2. HUNGATE, R. E., M. P. BRYANT AND R. A. MAH.The rumen bacteria and protozoa. Ann. Rev.

Microbiol. 18: 131, 1964.

3. ALLISON, M. J. Biosynthesis of amino acids by

ruminal microorganisms. J. Animal Sci. 29: 797,

1969.

4. BRYANT, M. P., AND I. M. ROBINSON. Effects ofdiet, time after feeding, and position sampled on

numbers of viable bacteria in the bovine rumen.

J. Dairy Sci. 51: 1950, 1968.

5. W0LIN, M. J. A theoretical rumen fermentation

balance. I. Dairy Sci. 43: 1452, 1960.6. BALDWIN, R. L. Energy metabolism in anaerobes.

Am. J. Clin. Nutr. 23: 1508, 1970.

7. ALLISON, M. J. Nitrogen metabolism of ruminalmicroorganisms. In: Third International Sympo.

sium on the Physiology of Digestion in the

Ruminant, edited by E. F. Annison. Newcastleupon Tyne, England: Oriel, 1970.

8. BRYANT, M. P., AND I. M. ROBINSON. Apparent

incorporation of ammonia and amino acid car-

bon during growth of selected species of ruminal

bacteria. 1. Dairy Sci. 46: 150, 1963.

9. BRYANT, M. P., AND I. M. ROBSINSON. Some

nutritional characteristics of predominantculturable ruminal bacteria. I. Bacteriol. 84:

605, 1962.

10. PrrrMAN, K. A., S. LAKSMANAN AND M. P.BRYANT. Oligopeptide uptake by Bacteroides

ruminicola. J. Bacteriol. 93: 1499, 1967.

11. GARTON, G. A. The digestion and assimilation of

lipids. In: Physiology of Digestion in the Rumi-

nant, edited by R. W. Dougherty. Washington,

D. C.: Butterworths, 1965, p. 390.12. ALLIsoN, M. J. Nutrition of rumen bacteria. In:

Physiology of Digestion in the Ruminant, edited

by R. W. Dougherty. Washington, D. C.: But-

terworths, 1965, p. 369.

13. PORTER, J. W. G. Vitamin synthesis in the rumen.

In: Digestive Physiology and Nutrition of the

Ruminant, edited by D. Lewis. London: Butter-

worths, 1961, p. 226.

14. HUNGATE, R. E. The anaerobic mesophilic

cellulolytic bacteria. Bacteriol. Rev. 14: 1, 1950.

15. BRYANT, M. P. The isolation and characteristics

of a spirochete from the bovine rumen. J.

Bacteriol. 64: 325, 1952.

16. BRYANT, M. P., AND L. A. BURKEY. Culturalmethods and some characteristics of some of the

more numerous groups of bacteria in the bovinerumen. 1. Dairy Sci. 36: 205, 1953.

17. BRYANT, M. P., AND I. M. ROBINSON. An improved

non-selective culture medium for ruminal bacte-

ria and its use in determining diurnal variation

in numbers of bacteria in the rumen. I. Dairy

Sci.44: 1446, 1961.

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

1450 Bryant

18. Virginia Polytechnic Institute, Anaerobe Lab-oratory. Outline of Clinical Methods in

Anaerobic Bacteriology. Blacksburg, Virginia:

Virginia Polytech. Inst. 1969, p. 35.

19. MAH, R. A., AND C. SUSSMAN. Microbiology of

anaerobic sludge fermentation. I. Enumeration

of the nonmethanogenic anaerobic bacteria.AppI. Microbiol. 16: 358, 1968.

20. SMITH, P. H. The microbial ecology of sludge

methanogenesis. In: Developments in Indwstrial

Microbiology. Washington, D. C.: Am. Inst.

Biol. Sci. 1966, vol. 7, p. 156.

21. WHITE, D. C., M. P. BRYANT AND D. R. CALDWELL.

Cytochrome-linked fermentation in Bacteroides

ruminicola. J. Bacteriol. 84: 822, 1962.

22. BRYANT, M. P. Rumen methanogenic bacteria.

In: Physiology of Digestion in the Ruminant,

edited by R. W. Dougherty. Washington, D. C.:

Butterworths, 1965, p. 411.

23. CALDWELL, D. R., AND M. P. BRYANT. Mediumwithout rumen fluid for nonselective enumer-

ation and isolation of rumen bacteria. Appl.

Microbiol. 14: 794, 1966.

24. CALDWELL, D. R., D. C. WHITE, M. P. BRYANT

AND R. N. DOETScH. Specificity of the heme re-

quirement for growth of Bacteroides ruminicola.

J. Bacteriol. 90: 1645, 1965.

25. LEV, M. The growth-promoting activity of com-

pounds of the vitamin K group and analogues

for a rumen strain of Fusiformis nigrescens. J.

Geni. Microbiol. 20: 697, 1959.

26. BRYANT, M. P., N. SMALL, C. BOUMA AND I. M.ROBINSON. Studies on the composition of the

ruminal flora and fauna of young calves. I.Dairy Sci. 41: 1747, 1958.

27. HoBsoN, P. N., AND S. 0. MANN. The isolation of

glycerol-fermenting and lipolytic bacteria from

the rumen of the sheep. J. Gen. Microbiol. 25:

227, 1961.

28. PAYNTER, M. J. B., AND R. E. HUNGATE.

Characterization of Methanobacterium mobilis,

sp.n., isolated from the bovine rumen. J. Bacte-

riol. 95: 1943, 1968.

29. BRYANT, M. P., I. M. RoBINsON AND H. CHU. Ob-servations on the nutrition of Bacteroides suc-

cinogenes. J. Dairy Sci. 42: 1831, 1959.

30. CALDWELL, D. R., J. S. BARTON AND M. KEENEY.

Mineral Requirements of Bacteroides amylo-

philus. Bacteriol. Proc. 1969, p. 31.

31. CHENG, K. J., G. A. JONES, F. J. SIMPSON AND

M. P. BRYANT. Isolation and identification of

ruinen bacteria capable of anaerobic rutin deg-radation. Can. J. Microbiol. 15: 1365, 1969.

32. BLACKBURN, T. H., AND R. E. HUNGATE. Succinic

acid turnover and propionate production in the

bovine rumen. Appl. Micro biol. 11: 132, 1963.33. WEGNER, G. H. Metabolic Fate of Fatty Acids Re-

quired by Certain Rumen Bacteria. (Ph.D.Thesis). Madison, Wis.: Univ. of Wis., 1962.

34. BRYANT, M. P. Bacterial species of the rumen.

Bacteriol. Rev. 23: 125, 1959.

35. AYRES, W. A. Phosphorylation of cellobiose and

glucose by Ruminococcus flavefaciens. I. Bacte-

riol. 76: 515, 1958.

36. NOTTINGHAM, P. M., AND R. E. HUNGATE. Iso-

lation of methanogenic bacteria from feces ofman. J. Bacteriol. 96: 2178, 1968.

37. BROWN, D. W., AND W. E. C. Mooit�. Distribution

of Butyrivibrio fibrisolvens in nature. I. Dairy

Sd. 43: 1570, 1960.

38. KINGsLEY, V. V. Investigation into the Structure

and Classification of Selenomonads. (Ph.D.

Thesis). Toronto, Canada: Univ. of Toronto,

1968.

39. HUNGATE, R. E., W. SMITH, T. BAUcHOP, I.Vu AND J. C. RABINOWITZ. Formate as an inter-

mediate in the bovine rumen fermentation. 1.Bacteriol. 102: 389, 1970.

40. W0LIN, M. J. Fermentation interactions in micro-

bial ecosystems. Abstracts of Papers, 158th Nat.Meeting. Am. Chem. Soc., Washington, D. C.,MICR19, 1969.

by on Decem

ber 22, 2006 w

ww

.ajcn.orgD

ownloaded from

http://www.ajcn.org

normal flora rumen bacteria

Documents