INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1986, p. 275-277
0020-7713/86/040275-03$02.00/0
Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 2
Description and Designation of a Neotype Strain of Eubacterium
cellulosolvens (Cillobacterium cellulosolvens Bryant, Small, Bouma
and Robinson) Holdeman and Moore
N. 0. VAN GYLSWYK AND J. J. T. K. VAN DER TOORN
Anaerobic Microbiology Division, Council for Scientijic and Industrial Research Laboratory for Molecular and Cell
Biology, Onderstepoort 0110, South Africa
Since type strain B348 of Eubacterium cellulosolvens (Cillobacterium cellulosolvens Bryant, Small, Bouma,
and Robinson) was lost, we propose that van Gylswyk and Hoffman strain 6 (= ATCC 00000) be designated
the neotype strain of this species. The characteristics of the neotype strain are compared with the characteristics
of strains described elsewhere and with some of the most important characteristics of strains recently isolated
by us from sheep rumina.
positions of deoxyribonucleic acids from the ratios of absorbance at 245 nm to absorbance at 270 nm of purified
deoxyribonucleic acids from selected strains were done by
methods described previously (6).
Cillobacterium ceilulosolvens was first described by Bryant et al. (1) in 1958 and was subsequently named
Eubacterium cellulosolvens by Holdeman and Moore (2) in
1972. There have been two reports (3,5) of the occurrence of
E. cellulosolvens in rumina since it was first described, and
based on the work of Moore and Holdeman (W. E. C. Moore
and L. V. Holdeman, in Bergey’s Manual of Systematic
Bacteriology), it appears that at least one strain has been
isolated from the intestinal tract of a hog. The original
description of the species was based on the characteristics of
a single strain, B348, which was also the type strain. Since
this strain was lost soon after isolation (1)and since some of
the characteristics of most strains studied subsequently
differ from characteristics of the type strain, it was deemed
appropriate to designate a neotype strain. In this paper we
also describe some of the most important characteristics of
E. cellulosolvens strains isolated during a recent study (4) in
which the predominant fiber-digesting bacteria in rumina of
sheep fed corn stover-based rations were examined.
RESULTS
The 11 strains studied conformed in morphology, dimensions, and Gram reaction to previous descriptions of E.
cellulosolvens (5). Strains C3E34 and X4B54 were both
motile. The latter strain was examined by transmission
electron microscopy, and up to five flagella per cell were
observed. These flagella were arranged peritrichously.
The ability to produce acid from different carbon and
energy sources is shown in Table 2. Strain X4B54 did not
ferment cellulose. The end products of cellulose or xylan
fermentation are shown in Table 3. Because strain X4B54
grew poorly in xylan medium, producing low concentrations
of end products (Table 3), the production of lactic acid was
assessed in medium containing cellobiose. This strain did not
produce L-lactic acid but produced 2.4 mmol of D-lactic acid
per 100 ml of medium. In a previous study (5) we determined
the total lactic acid production by nine very similar strains.
Hence, strains 2 and 6T were examined for the production of
the two isomers after growth in medium containing
cellobiose. No L-lactic acid was produced, but 1.2 and 3.3
mmol of D-lactic acid per 100 ml of medium were produced
by strains 2 and 6=, respectively.
The guanine-plus-cytosine content of the deoxyribonucleic acid was found to be between 49 and 51 mol% for
strains ST and C3E34 tested in three separate experiments.
MATERIALS AND METHODS
Bacterial strains. The origins of 10 strains identified as E.
cellulosolvens and one strain (strain X4B54) which was
placed in a group called “coccoid rods” by van der Linden
et al. (4) are shown in Table 1.
Methods. The anaerobic techniques and basal medium
used have been described previously (6). The strains were
maintained on rumen fluid (40%) agar (15 g/liter) slopes
containing 15 g of ball-milled Whatman no. 1filter paper per
liter (or 15 g of xylan [Fluka] per liter in the case of strain
X4B54). The motility of strains C3E34 and X4B54 was
determined microscopically after growth on agar slopes
containing 5 g of cellobiose per liter and 5 g of xylan per liter,
respectively. The ability to ferment different carbon and
energy sources (5 g/liter) was tested in poorly buffered
medium (6). The fermentation products were determined in
medium containing ball-milled filter paper (10 g/liter) for
cellulolytic strains and xylan (20 ghiter) for strain X4B54.
Four strains (strains 2, ST [T = type strain], C3E34, and
X4B54) were grown in basal medium containing 5 g of
cellobiose per liter to determine the isomers of lactic acid
produced. To determine guanine-plus-cytosine contents,
cells were grown in basal medium (6) containing 5 g of
cellobiose per liter.
Determinations of fermentation products by gas-liquid
chromatography or enzymic methods and of the base com-
DISCUSSION
The most obvious differences among strains are found in
the types of fermentation products (Table 3). At least two
groups may be distinguished, those strains that produce
n-valeric acid together with D-lactic acid (including strain
X4B54) and those strains that do not produce valeric acid but
produce L-lactic acid with or without the D-lactic acid. A
possible third group, represented by strains B348 and 273
(Table 3), is practically homofermentative ,producing mainly
lactic acid. Unfortunately, the latter strains, as well as other
strains described by Prins et al. (3), are no longer available
for comparison (R. A. Prins, personal communication).
To the general description of the species given by Moore
and Holdeman (in press) may be added that strain X4B54 is
not capable of fermenting cellulose and that the guanine275
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INT. J . SYST.BACTERIOL.
TABLE 1. Origins of E. cellulosolvens strains isolated from the
rumina of sheep fed corn stover plus a protein-mineral mixture
without or with different amounts of corn grits supplement"
Amt of supplement
(g/kg of stover)
C3A38, C2A64, C3E39, X4B54
C3E34, C6E34
C9C54, C7E54
C6C38
C14D56
C8E58
None
90
120
150
210
360
a The composition of the rations is described in greater detail by van der
Linden et al. (4).
All strains except strain X4B54 were isolated from clearing zones produced in cellulose agar inoculated with a lo-' dilution of rumen ingesta. Strain
X4B54 was isolated from a clearing zone produced in 3% xylan agar medium
inoculated with a
dilution of ingesta. The final two digits of each strain
designation indicate the animal from which the strain was isolated.
plus-cytosine content of the deoxyribonucleic acid lies between 49 and 51 md% for two strains with different patterns
of fermentation end products.
Neotype strain. We suggest that strain 6 of van Gylswyk
and Hoffman (5) be designated the neotype strain of E .
cellulosolvens because it represents the most common type
of strain isolated. This strain has been deposited in the
American Type Culture Collection, and its characteristics
are as given below.
Cells are obligately anaerobic and motile with a number
(two to at least nine) of peritrichously arranged flagella per
cell. The cells move in a tumbling fashion. In young cultures
the cells are mostly gram positive and occur in chains. In
older cultures more gram-negative cells are found, but few
chains are present. The cells then occur singly or in pairs.
The cells have rounded ends but are often pointed when they
are joined to other cells. The shape varies from coccoid to
rod shaped, with dimensions varying from 0.5 to 1.0 pm
wide by 0.5 to 3 pm long.
Surface colonies on 0.5% cellobiose-40% rumen fluid agar
slopes are circular, convex, entire, and about 1 mm in
diameter after incubation for 18 h at 38 to 39°C. Surface
colonies within the clearings produced in films of cellulose
agar after 2 weeks are usually entire, flat, and translucent;
they often extend to the outer limit of the zofie of clearing.
Strain 6* does not grow at 22°C and grows poorly at 45°C.
It grows well at 37 to 39°C. Carbon dioxide is not required
for growth, and Trypticase plus yeast extract can replace
rumen fluid as a source of growth factors. Only poor growth
is obtained when peptone is supplied as the sole energy
source.
Gelatin is not digested, nitrate is not reduced, and catalase, indole, hydrogen sulfide, acetylmethylcarbinol, and
TABLE 2. Acid production from different carbohydrate energy sources by E. cellulosolvens strains
Carbohydrate
energy
source
Data from
Bryant et al.
for type
strain B348"
Data from van Gylswyk and
Hoffmanb
Data from this study
Eight strains
Strain
C3A38
Strain
C3E34
Strain
X4B54
Eight strains
72n
:ypse
Data from b i n s et a].'
Five strains
Strain
273
Strain
SW7-1
Arabinose
Am ygdalin
Cellobiose
Cellulose
Dextrin
Esculin
Fructose
Galactose
Glucose
Glycerol
Gum arabic
Inositol
Inulin
Lactate
Lactose
Maltose
Mannitol
Mannose
Melezitose
Pectin
Raffinose
Rhamnose
Ribose
Salicin
Sorbitol
Starch
Sucrose
Trehalose
Xylan
Xylose
See reference 1.
See reference 5.
See reference 3.
+ , pH lowered by 0.6 U or more; w, pH lowered by between 0.2 and 0.6 U; -, pH unchanged or lowered by less than 0.2 U; v, variable (i.e., may or may
not be fermented). The numbers in parentheses indicate the numbers of strains in cases where reactions were not identical for all strains.
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VOL. 36, 1986
EUBACTERIUM CELLULOSOLVENS NEOTYPE STRAIN
277
TABLE 3. Fermentation end products of E. cellulosolvens strains
Amt produced or utilized
mmoVlOO ml of medium
Data from this study
Formic acid
Acetic acid
Propionic acid
Butyric acid
n-Valeric acid
D-Lactic acid
L-Lactic acid
Total lactic acid
Succinic acid
Hydrogen
Carbon dioxide
1 T-m-
tor type
strain B348"
Eight strainsb
Strain
C3A38b
Strain
C3E34b
Strain
X4B54c
0.26
0.42
0
0
0
1.16 k 0.29
-1.49 k 0.34
-0.67 k 0.09
2.28 k 0.32
0.49 k 0.07
3.72 k 0.48
0
0.85
-1.82
-0.80
2.18
0.52
0.13
0
1.03
-1.84
-0.87
2.34
-0.02
3.03
0.55
0.44
0
0.11
0.21
0.04
0
0
6.42
0
0
0
0.12
0
Tr
Data from b i n s et al.
Data from van Gylswyh
0
-
Eight strains
NeotvDestrain 6
1.28 ? 0.21
-1.11 2 0.55
-0.67 k 0.09
1.87 2 0.38
0.47 0.09
1.12
-0.83
-0.55
1.85
0.46
0.55
4.85
*
4.37
k
0
Small amt
~
, I
Small amt
_.
Five strains
0.49
-0.06
0.04
0.43
2
0.12
k
0.03
* 0.02
* 0.04
0
0.34 -+ 0.09
0.39 k 0.03
0
0.33 +- 0.09
0.37 k 0.12
Strain
0
-0.10
-0.15
0.03
0
0
1.46
0.01
0.004
0.004
" See reference 1. The energy substrate used was 1%cellobiose.
f
The energy substrate used was 1% cellulose.
The energy substrate used was 2% xylan.
See reference 5 . The energy substrate used was 1.2% cellulose.
See reference 3. The energy substrate used was 0.5% glucose.
Mean f standard deviation.
ethanol are not produced. Acid is produced from cellobiose,
cellulose, esculin, galactose, glucose, inulin, lactose,
maltose, pectin, raffinose, salicin, and sucrose. Weak acid is
produced from fructose. Acid is not produced from
arabinose, dextrin, glycerol, inositol, mannitol, mannose,
rhamnose, sorbitol, starch, trehalose, xylan, or xylose, and
lactic acid is not fermented. Strain 6Tproduces formic acid,
butyric acid, n-valeric acid, and lactic acid. Only the D
isomer of lactic acid is produced. Acetic and propionic acids
are utilized (when present in the medium).
The guanine-plus-cytosine content of strain 6T is approximately 50 mol%.
LITERATURE CITED
1. Bryant, M. P., N. Small, C. Bouma, and I. M. Robinson. 1958.
The characteristics of ruminal anaerobic cellulolytic cocci and
Cillobacterium cellulosolvens n. sp.. J. Bacteriol. 76529-537.
2. Holdeman, L. V., and W. E. C. Moore (ed.). 1972. Anaerobe
laboratory manual. Anaerobe Laboratory, Virginia Polytechnic
Institute and State University, Blacksburg.
3. Prins, R. A., F. van Vugt, R. E. Hungate, and C. J. A, H. V. van
Vorstenbosch. 1972. A comparison of strains of Eubacterium
cellulosolvens from the rumen. Antonie van Leeuwenhoek J.
Microbiol. Serol. 38:153-161.
4. van der Linden, Y., N. 0. van Gylswyk, and H. M. Schwartz.
1984. Influence of supplementation of corn stover with corn grain
on the fibrolytic bacteria in the rumen of sheep and their relation
to the intake and digestion of fiber. J. Anim. Sci. 59:772-783.
5. van Gylswyk, N. O., and J. P. L. Hoffman. 1970. Characteristics
of cellulolytic cillobacteria from the rumens of sheep fed teff
(Eragrostis teJ) hay diets. J. Gen. Microbiol. 60:381-386.
6. van Gylswyk, N. O., and J. J. T. K. van der Toorn. 1985.
Eubacterium uniforme sp. nov. and Eubacterium xylanophilum
sp. nov., fiber-digesting bacteria from the rumina of sheep fed
corn stover. Int. J. Syst. Bacteriol. 35323-326.
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