THE ANAEROBIC MONOTRICHOUS
BUTYRIC ACID-PRODUCING
CURVED ROD-SHAPED BACTERIA
OF THE RUMEN
Marvin P. Bryant and Nola Small
J. Bacteriol. 1956, 72(1):16.
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THE ANAEROBIC MONOTRICHOUS BUTYRIC ACID-PRODUCING
CURVED ROD-SHAPED BACTERIA OF THE RUMEN
MARVIN P. BRYANT AND NOLA SMALL
Dairy Husbandry Research Branch, U. S. D. A., Beltsville, Maryland
Received for publication November 23, 1955
grain mixture alone. All strains were isolated
from 10 ml of rumen contents inoculated into
40 per cent rumen fluid-glucose-cellobiose agar
(RGCA) medium roll tubes (Bryant and Burkey,
1953a).
Stock cultures were maintained in slants of
the RGCA medium placed in a dry ice box at
-60 to -70 C. The cultures were transferred
every four to six months.
The purity of all strains was established by
serially diluting them in tubes of the RGCA
medium and picking isolated colonies from tubes
of the highest dilutions showing growth after 48
to 72 hr of incubation at 37 C.
Morphology was determined using both the
material from colonies that developed in RGCA
roll tubes after 48 to 72 hr of incubation and the
material from the water of syneresis of 18-hr
RGCA slant cultures. Wet mounts for motility
and morphological observations were observed
with a phase contrast microscope and morphology
was also observed in gram-stained smears. Approximations of cell size were made from the
producing organisms.
METHODS
gram-stained smears.
Fifteen strains failed to show motility in
preparations from the RGCA medium. However,
when cellobiose was omitted from the medium
and the glucose concentration reduced to 0.1
per cent, all strains showed active motility.
The type of flagellation was determined using
the 18-hr slant cultures and the staining procedure of Leifson (1951).
Descriptions of colony type were made from
observations on the original RGCA roll tubes
inoculated with rumen fluid and incubated 72
hr and from the 48 to 72-hr roll tube cultures
prepared to establish culture purity.
The basal medium to which various ingredients
were added to determine most cultural and
physiological characteristics contained 20 per
cent (v/v) of rumen fluid, 7.5 per cent (v/v)
each of mineral solutions 1 and 2 (Bryant and
Burkey, 1953a), 0.0001 per cent (w/v) of resa-
The anaerobic technique used throughout
the course of this study was that of Hungate
(1950). With this technique media were maintained in sterile rubber-stoppered culture tubes
with carbon dioxide or nitrogen gas displacing
air. Bicarbonate was added to all media held
under carbon dioxide to adjust the pH to about
6.7. Cystine-HCl was used as reducing agent
and resazurin was added as an oxidation-reduction indicator.
The cultures studied included 48 strains previously placed in the MR-GXC and MR-GXCS
groups (Bryant and Burkey, 1953a) and similar
cultures that varied from these groups in carbohydrates fermented, and in hydrogen sulfide
production. The strains were isolated from cows
fed various rations including alfalfa hay, alfalfa
hay plus grain, fresh green alfalfa, soy bean hay
plus grain, blue grass pasture plus grain, and
16
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During studies of bacteria isolated from rumen
contents, several workers have encountered
anaerobic, gram negative, butyric acid-producing
curved rods (Hungate, 1950; Huhtanen and
Gall, 1952; and Wilson, 1953).
Bryant and Burkey (1953a) published brief
descriptions of three groups of anaerobic, motile,
curved rods. Further studies have shown that
four fundamentally different kinds of bacteria
were included in these groups. They include
organisms producing large amounts of butyric
acid from glucose; organisms producing large
amounts of succinic acid from glucose; organisms
that belong in the species Selenomonas ruminantium because of the characteristic morphology,
arrangement of flagella, and habitat; and gram
positive organisms that ferment glucose with the
production of large amounts of ethanol and
lactic, formic and acetic acids.
The purpose of the present paper is to describe
the characteristics and to name the butyric acid-
19561
ANAEROBIC CURVED-ROD BACTERIA OF BOVINE RUMEN
added.
The medium used to determine if appreciable
amounts of bicarbonate were required for growth
of the organisms was the basal medium with
nitrogen in the gaseous phase in place of carbon
dioxide, with Na2CO3 omitted, and with 0.5
per cent of glucose added. This medium was adjusted to pH 6.7 before it was autoclaved.
Growth was determined by the increase in
visible turbidity and drop in pH after one week
of incubation. A control was the regular basal
medium with glucose added. One loop of inoculum was used to lessen the carryover of carbonate
from the inoculum medium.
The final pH of the above medium with nitrogen in the gaseous phase was recorded as the
minimum pH of the strain.
The basal medium with 0.5 per cent of glucose
added and 0.5 per cent of yeast extract and 1.5
per cent of trypticase added in place of the rumen
fluid was used to determine if strains would
grow in the absence of rumen fluid. Visible
turbidity and pH after one week of incubation
were compared with the regular basal medium
with glucose and rumen fluid. One loop of inoculum was used.
The basal medium with 0.5 per cent of glucose
added was used to determine the range of
temperatures at which growth occurred. Incubation was at 22, 30, 37, 45 and 50 C. Growth was
estimated by visible turbidity and lowering of
pH after one week of incubation.
The Voges-Proskauer test was determined in
week-old cultures in the basal medium plus
for one week. A black color indicated H2S production.
Tests for carbohydrates fermented, starch
hydrolysis, cellulose digestion, nitrate reduction,
indole production, gelatin liquefaction, and casein
digestion were performed as described by Bryant
and Doetsch (1954).
Analyses of fermentation products of strain
Dl were made as previously described (Bryant
and Doetsch, 1954) except that glucose was used
in the medium in place of cellulose and all acids
were determined chromatographically. In this
procedure succinic and lactic acids were not
separated. Therefore, lactic acid was determined
by the method of Friedemann and Graeser (1933)
and succinic acid, by difference. Analyses of
fermentation products of strain 28 were made in
the same manner except that the acids were
estimated using the chromatographic procedure
of Langston (1955). This procedure is a modification of the procedure of Neish (1950) and permits
the separation of lactic and succinic acids.
RESULTS AND DISCUSSION
There was considerable variation in characteristics between strains in the 48 studied. Most were
rather small slightly curved rods 0.4 to 0.6,i
by 2 to 5,u with somewhat tapered ends. Some
strains showed more pronounced curving and
some appeared to be cylindrical. A few strains
had bluntly pointed ends and were almost
spindle-shaped. A few strains were only 0.2 to
0.3,u in width and occasionally cultures contained
cells as much as 0.8,i in width. Cells usually
occurred singly, in pairs, and short chains but
it was not unusual to find long chains of cells. A
few strains contained some long slender rods
up to about 12,u in length. All were definitely
gram negative. Fifteen strains, representative
of the range of variability in morphological and
physiological characteristics found, showed monotrichous flagellation. The flagellum was usually
attached at the end of the cell but occasionally it
appeared to be attached laterallv. 'Motility was
observed in all strains.
Surface colonies observed after 48 to 72 hr
on
RGCA roll tubes were entire, slightly convex,
0.5 per cent of glucose.
and
translucent and were usually 2 to 4 mm in
The medium used to detect H2S production
diameter.
They were light tan in color. Colonies
was the basal medium plus 0.05 per cent (w/v)
strains
a
few
of
changed from smooth to rough
ferric ammonium citrate, 0.5 per cent agar, 0.1
These
colonies were more flat,
isolation.
after
1.5
cent
and
cent
per
glucose
per
trypticase.
This medium was stab-inoculated and incubated lighter in color and had filamentous margins.
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zurin, 0.05 per cent (w/v) of cysteine-HCl, and
0.4 per cent (w/v) of Na2CO3, and was maintained
under carbon dioxide gas. The media were prepared by the method described by Bryant and
Burkey (1953a).
Unless otherwise stated, media were inoculated
with one drop of a 24-hr culture in the basal
medium with 0.5 per cent of glucose added.
Except in temperature studies, incubation was
at 37 C.
Appearance of growth in liquid medium was
determined in 24-hr cultures of the basal medium
with 0.5 per cent of filter-sterilized glucose
17
18
BRYANT AND SMALL
showed zones in the second. None of the other
cultures showed any visible indication of cellulose
digestion after one year of incubation. Of particular interest was the fact that two of these cellulolytic cultures had not shown cellulose digestion
in any of the previous tests.
The results suggest that the ability of these
organisms to digest cellulose is a variable character and the maintenance of cultures in the
RGCA medium which contains glucose and
cellobiose as energy source may cause them to
lose their ability to visibly digest cellulose. It
appears that cellulolytic strains of these organisms should be maintained in cellulose medium
to be certain of retaining this character.
To determine whether fermentation products
from glucose were similar within the group,
rumen fluid-glucose medium cultures of seven
representative strains were analyzed for volatile
acids. Data in table 1 show that the production of
large amounts of butyric acid was characteristic
of all strains though they varied considerably in
other physiological characteristics. Some strains
showed an uptake of acetic acid while others
showed an overall production of this acid.
One of the most typical strains (D1) and one
of the most atypical strains (28) were analvzed
for other fermentation products (table 2). Both
strains produced carbon dioxide and butyric,
formic and lactic acids but differed in other
respects. Strain 28 did not produce hydrogen
and produced a much larger amount of formic
acid than strain D1. Strain 28 produced acetic
acid while strain D1 showed an uptake of this
acid.
Because of the variability in characteristics
found between strains of this group of organisms,
it will be difficult to determine what characteristics logically can be used to define natural species
specific patterns; this task is beyond the scope
of the present work. However, the characteristics
of the group indicated the necessity of creating
a new genus of anaerobic bacteria.
Butyrivibrio nov. gen. The name Butyrivibrio
is proposed for those anaerobic, nonsporeforming,
monotrichous, gram negative, curved rods that
ferment glucose with the production of large
amounts of butyric acid.
Because the genus includes nonsporeforming,
gram negative, curved rods, it appears to belong
in the tribe Spirilleae of the family Pseudomonadaceae as described by Breed et al. (1948). It
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Growth of 24-hr cultures in rumen fluidglucose liquid medium usually showed heavy
turbidity. However, some cultures had flocculent
sediment and others had granular sediment
which often adhered to the sides of the tube.
The organisms grew well in rumen fluid-glucose
medium with N2 added in place of CO2 and with
bicarbonate omitted, and grew well when rumen
fluid was replaced by yeast extract and trypticase
in the glucose medium.
The final pH in the lowly buffered rumen
fluid-glucose miedium was as follows: 38 strains,
pH 5.0 to 5.3; seven strains, pH 5.4 to 5.6; two
strains, pH 4.9; and one strain, pH 4.6.
None of the strains grew at 50 or 15 C and only
one strain grew at 22 C. Thirty-eight strains
grew at 45 C and ten did not. All strains grew
well at 30 and 37 C.
None of the strains produced catalase or indole
or reduced nitrate. Six strains produced hydrogen
sulfide and gelatin was liquefied by four strains.
Four strains gave positive Voges-Proskauer
reactions.
Fermentation of carbohydrates was quite
variable betw een strains, but most strains
fermented manv of them. The number of strains
that fermented certain carbohydrates was as
follows: glucose, 48; esculin, 46; xylose, maltose,
and cellobiose, 44; sucrose and salicin, 43;
fructose, 42; lactose and inulin, 40; dextrin, 37;
xylan, 34; trehalose, 16; mannitol, 1; and glycerol,
inositol, and gum arabic, 0. Starch was hvdrolyzed by 24 of the 48 strains.
When cultures wer e first isolate(l they were
tested for cellulose digestion in liquid medium.
Only 3 of the 48 strains showed a visible loss of
cellulose. When all strains were tested at a later
date none of them showed a visible loss of cellulose. There was no loss in dry weight of insoluble
residue after two weeks of incubation in 15 of
the cultures. These cultures included the three
strains which previously had shown a substantial
visible loss of cellulose from tubes of the medium.
All strains were then stab-inoculated into the
base of rumen fluid-cellulose agar slants and
incubated for a period of one year. Three strains
showed zones of cellulose digestion after three,
four, and five weeks of incubation. After five
weeks these strains and four others weie transferred to fresh cellulose agar slants. After one
week of incubation the three cultures which
showed cellulolytic zones in the first slant
[VOL. 72
1956]
ANAEROBIC CURVED-ROD BACTERIA OF BOVINE RUMEN
19
TABLE 1
Volatile acids produced in rumen-fluid glucose medium by strains of anaerobic monotrichous curved rods
showing differences in physiological characteristics
Strain
Volatile Acids*
Acetic ........................................
Butyric ...........................................
DI
C23
PC4
A38
49
1
28
-0.23
0.22
1.44
0.13
1.42
-0.18
1.30
-0.58
1.63
0.19
1.20
0.08
1.12
1.24
Some Variable Characteristics
.........
has manv similarities to other genera in this
tribe. It resembles the genus Vibrio and differs
from the genus Spirillum in being monotrichous.
It resembles the genus Desulfovibrio in that both
are strict anaerobes, and the genera Cellvibrio
and Cellfalcicula because some strains attack
cellulose. It differs from all other genera of the
tribe in that it produces substantial amounts
of butyric acid from glucose.
We propose the name Butyrivibrio fibrisolvens
nov. spec. for the type species to indicate the
organism's probable importance in the digestion
of fibrous constituents of the food in ruminant
rations. The following description of the species
is based on the characteristics of a type strain
(D1) which is among those with the most representative physiological characteristics. This
type strain has been deposited in the American
Type Culture Collection.
This organism is a gram negative, motile,
slightly curved rod, 0.4 to 0.6,u wide and 2 to 51u
long, with bluntly tapered ends. Arrangement
of cells includes singles and pairs to long chains.
Flagellation is monotrichous and polar.
Surface colonies in agar shortly after primary
isolation were entire, slightly convex, translucent,
and light tan in color and were 2 to 4 mm in
diameter. Deep colonies were thin and lensshaped. After several subcultures, colonies
became more flat, lighter in color and had filamentous margins.
TABLE 2
Some fermentation products produced in rumen
fluid-glucose medium by two strains of butyric
acid-producing monotrichous curved rods that
differed greatly in other physiological characteristics
Fermentation Products
mm Per 150 Ml of Medium
._
Strain DI
Strain 28
0 .00
Methane ...............
Hydrogen .............
0.76
Carbon dioxide ........
2.17
Butyric acid ...........
1.10
0.00
Propionic acid .........
Acetic acid ............
-0.14
Formic acid ...........
0.42
Succinic acid ........0.. .00
Lactic acid ............
Ethanol ...............
0.43
0.07
0.00
0.00
2.12
1.63
0.14
0.66
2.80
0.03
0.25
0.00
Growth in glucose liquid medium shows mainly
flocculent to granular sediment some of which
adheres to the sides of the tube.
Growth occurs at 30 and 45 C but not at 22 or
50 C.
Growth is good without CO2 or bicarbonate
added to the medium.
Final pH in glucose liquid medium is 5.1.
Glucose is fermented with the production of
H2, C02, and butyric, formic and lactic acids
and a small uptake of acetic acid.
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H2S production ...................... +
+
Gelatin liquefaction ..................
+
+ _
Casein digestion
.
+
+
+
+
+
+
V. P. reaction ........................
+
Acid from xylose, arabinose, maltose,
dextrin, inulin, xylan, and starch...
+
+
+
+
+
+
Acid from cellobiose and sucrose ........ +
+
+
+
+
+
Acid from fructose .....................
+
+
+
+
+
_
Digestion of cellulose..
.+
+
+
* Amounts expressed as mm per 100 ml of medium. There was no appreciable prodtiction of ipropiotiC or valeric and/or higher volatile fatty acids. Formic acid was not determined.
20
BRYANT AND SMALL[[VOI..
Motility and the type of flagellation were not
determined. The RO-H organisms were placed
in five types on the basis of the fermentation of
seven carbohydrates and reaction in litmus milk.
This grouping would not be satisfactory for the
present strains of Butyrivibrio. It seems probable
that the rich organic medium used for primary
isolation and study of cultures by Huhtanen
and Gall would account for many of the differences between RO-H organisms and the present
strains.
It is probable that members of the genus
Butyrivibrio contribute as much as any other
group of bacteria to the rumen fermentation.
This statement is based on the fact that they
have been found among the most numerous
bacteria cultured from cattle on a wide variety
of rations and because of the wide variety of
feed constituents that they attack.
They have been found among the most numerous organisms cultured from rumen contents of
animals on the following rations: alfalfa hay-grain
mixture, alfalfa hay, grain mixture, wheat straw,
alfalfa silage, fresh alfalfa, and bluegrass pasture
(Bryant and Burkey, 1953b,c).
The authors have isolated them from cattle
in Maryland. Hungate (1950, 1952) has isolated
them from cattle in Washington and from cattle
and sheep in New York. They were among the
more numerous saccharolytic bacteria in the
rumen of sheep studied by Wilson (1953) in
England.
They are of considerable importance in the
breakdown of fibrous constituents of cattle feed
as shown by the ability of some strains to digest
cellulose, and most strains digest xylan. The
culture of Hungate (1950) digested hemicellulose
isolated from various sources. Also, the genus
Butyrivibrio undoubtedly contributes to the
breakdown of starch and protein in the rumen.
Cultural studies suggest that this genus
produces a large portion of the butyric acid
found in the rumen. While other organisms
producing this acid have been isolated, they have
never been found in numbers approaching the
numbers of Butyrivibrio.
ACKNOWLEDGMENT
The authors wish to acknowledge the constructive criticism on the manuscript and helpful
suggestions on nomenclature so generously given
by Professor R. E. Hungate.
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D-Xylose, L-arabinose, galactose, fructose,
glucose, maltose, cellobiose, sucrose, lactose,
dextrin, inulin, salicin, esculin, pectin and xylan
are fermented. Trehalose, glycerol, mannitol,
inositol, gum arabic and cellulose are not fermented.
Starch is hydrolyzed.
Nitrate is not reduced.
Acetylmethylcarbinol, hydrogen sulfide, catalase and indole are not produced.
Gelatin is not liquefied.
Source: the reticulo-rumen of cattle.
The differences in characteristics between the
strain on which the species description is based
and most of the other strains are small and various
differences in character between strains failed
to correlate with each other. Therefore, it appears
impractical to establish more than one species
at the present time.
The authors regard strains with one or more
of the following differences in character from
the type strain as varieties of the species described. Fructose, cellobiose, sucrose, lactose or
inulin may not be fermented. Trehalose and
cellulose may be fermented. Starch may not
be hydrolyzed. Acetylmethylcarbinol and hydrogen sulfide may be produced and gelatin may be
liquefied. Many strains continued to produce
the smooth type of colony and even turbidity in
glucose liquid medium.
Disregarding the above variations in characteristics, 31 of the 48 strains studied would belong
to the species Butyrivibrio fibrisolvens and 9 of
these were identical in physiological characteristics with the type strain.
The "less actively cellulolytic" rumen rod
described by Hungate (1950) undoubtedly is a
member of this species. Wilson (1953) described
anaerobic, small slender, monotrichous rods that
produced acetic, butyric and lactic acid from
glucose. These organisms were believed to be a
stable part of the rumen flora of sheep and belong
in the genus Butyrivibrio.
The RO-H types of curved rods isolated from
the iumen of cattle and sheep by Huhtanen
and Gall (1952) are probably members of the
genus Butyrivibrio. These organisms differ from
those of the present study in being smaller than
most of the present strains and in frequently
showing deeply stained swellings. Also, they are
slower in growing, produce less turbidity and
acid in glucose medium and form smaller colonies.
72
1956]
ANAEROBIC CURVED-ROD BACTERIA OF BOVINE RUMEN
SUMMARY
REFERENCES
BREED, R. S., MURRAY, E. G. D. AND HITCHENS,
A. P. 1948 Bergey's manual of determinative
bacteriology, 6th ed. The Williams & Wilkins
Co., Baltimore.
BRYANT, M. P. AND BURKEY, L. A. 1953a Cultural methods and some characteristics of
some of the more numerous groups of bacteria
in the bovine rumen. J. Dairy Sci., 36, 205217.
BRYANT, M. P. AND BURKEY, L. A. 1953b Numbers and some predominant groups of bacteria
in the rumen of cows fed different rations.
J. Dairy Sci., 36, 218-224.
BRYANT, M. P. AND BURKEY, L. A. 1953c The
bacterial flora in the rumen of heifers fed a
ration of alfalfa silage. Bureau Dairy Industry Information, 151.
BRYANT, M. P. AND DOETSCH, R. N. 1954 A
study of actively cellulolytic rod-shaped
bacteria of the bovine rumen. J. Dairy Sci.,
37, 1176-1183.
FRIEDEMANN, T. E. AND GRAESER, J. B. 1933
Determination of lactic acid. J. Biol. Chem.,
100, 291-308.
HUHTANEN, C. N. AND GALL, L. S. 1952 Rumen
organisms I. Curved rods and a related rod
type. J. Bacteriol., 65, 548-553.
HUNGATE, R. E. 1950 The anaerobic mesophilic
cellulolytic bacteria. Bacteriol. Revs., 14,
1-49.
HUNGATE, R. E. 1952 Kinds of cellulolytic
cocci in the rumen of cattle and sheep. Bacteriol. Proc., 1952, p. 16.
LANGSTON, C. W. 1955 Microbiology and chemistry of grass silage. Thesis, University of
Wisconsin.
LEIFSON, E. 1951 Staining, shape, and arrangement of bacterial flagella. J. Bacteriol.,
62, 377-389.
NEISH, A. C. 1950 Analytical methods for bacterial fermentations, report no. 46-S-3 (revised). Nat. Research Council of Can.,
Saskatoon.
WILSON, S. N. 1953 Some carbohydrate fermenting organisms isolated from the rumen
of the sheep. J. Gen. Microbiol., 9, i-ii.
Downloaded from http://jb.asm.org/ on March 5, 2014 by PENN STATE UNIV
A study of many strains of anaerobic curved
rods isolated from the bovine rumen indicated
that four fundamentally different groups of
bacteria were included. The most numerous
group included monotrichous, nonsporeforming,
gram negative curved rods that ferment glucose
with the production of large amounts of butyric
acid. This group was placed in the new genus
Butyrivibrio, nov. glu.
There was considerable variation in morphological and physiological characteristics among
the 48 strains of the genus Butyrivibrio that were
studied. A type species, Butyrivibrio fibrisolvens
nov. spec., was described on the basis of characteristics of one of the most representative strains.
This species ferments glucose with the production
of carbon dioxide, hydrogen, and butyric, formic,
and lactic acids. It ferments a large number of
carbohydrates including hexoses, pentoses, disaccharides, glycerides, and polysaccharides.
Members of the genus not placed in the type
species differ mainly in the non-fermentation of
several of the carbohydrates fermented by the
type species. The fermentation products of one
of the more variant strains differed from the
type species in not producing hydrogen and in
producing a larger amount of formic acid.
The importance of the genus Butyrivibrio
in the rumen was indicated by the fact that
strains digest many of the major components of
ruminant rations including xylan, cellulose,
starch, and protein and appear to be among the
most numerous bacteria present in the rumen of
cattle and sheep fed a variety of rations.
21