Vol. 36, No. 2
INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1986, p. 314-316
0020-7713/86/020314-03$02.OOIO
Copyright 0 1986, International Union of Microbiological Societies
Acetivibrio cellulosolvens Is a Synonym for Acetivibrio
cellulolyticus: Emendation of the Genus Acetivibrio?
WILLIAM D. MURRAY
Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada K I A OR6
Acetivibrio cellulosolvens BAST was compared with Acetivibrio cellulolyticus CD2T and was found to be
identical in all morphological and biochemical characteristics. The description of the genus Acetivibrio is
emended as originally described by I. M. Robinson and A. E. Ritchie (Int. J. Syst. Bacteriol. 31:333-338, 1981)
to include anaerobic, gram-negative, straight to slightly curved rods that produce mainly acetic acid, ethanol,
H2 and C 0 2 and that are motile by means of a single flagellum or multiple flagella. It is proposed that A .
cellulosolvens BAST be correctly classified as A . cellulolyticus.
Khan et al. (4) reported the isolation of Acetivibrio
cellulosolvens BAST, a new species of cellulolytic anaerobe.
This microbe was described as a nonmotile straight rod
capable of hydrolyzing and growing on esculin and therefore
different from Acetivibrio cellulolyticus CD2T (6). The description of the genus Acetivibrio (6, 9) was emended by
these authors (4) to include nonmotile strains. However,
when strain BAST was grown in this laboratory in the
cellulose-basal medium of Patel et al. (7), the cells were
found to be straight to slightly curved and showed tumbling
motility in hanging-drop preparations. The inconsistency of
these observations with the published description (4)
prompted a taxonomic comparison of A . cellulosolvens
BAST with A . cellulolyticus CD2T.
MATERIALS AND METHODS
Microorganisms and media. A . cellulosolvens BAST
(NRCC 2936) and A . cellulolyticus CD2T (NRCC 2248) were
obtained from the National Research Council of Canada
Culture Collection. Also compared was a stock strain of A .
cellulolyticus which had been maintained in this laboratory
for 3 years on cellulose-basal medium. This stock strain will
be referred to as strain LS (laboratory strain).
The following two types of media were used in this study:
a synthetic basal medium and a peptone-yeast extract (PY)
broth. The composition of and procedure for preparation of
synthetic basal medium were as described by Patel et al. (7).
The three Acetivibrio strains were maintained in this medium, which contained approximately 0.5% (wthol) cellulose in the form of small squares of four-ply facial tissue. The
PY broth, which was used for biochemical tests, was prepared as described by Holdeman et al. (3).
Electron micrographs. For electron microscopy, strains
BAST, CD2T and LS were grown for 48 h at 35°C without
shaking in cellulose-basal medium. After dilution in distilled
water, the cells were placed in Formvar films and dialyzed
overnight. The films were placed on carbon-coated grids,
shadowed with Pd-Au, and examined with a Siemens electron microscope, model 101.
Biochemical tests. The procedures of Holdeman et al. (3)
were used for biochemical characterization. In substrate
t Issued as National Research Council of Canada paper no.
25336.
utilization tests, the desired substrates were added to both
PY broth and synthetic basal medium at concentrations of
0.5 to 1.0% (wthol) (3). When a test required the addition of
a fermentable substrate (e.g., semisolid motility medium),
0.5% cellobiose was used. The test media were inoculated
with 0.05 ml of 72-h-old cultures grown in cellulose-basal
medium and were incubated at 35°C. Test results were
determined after 72 h and 7 days. Negative tests were held
for 4 weeks. Metabolic products, from cellulose-basal medium, were quantified by gas chromatography. Gas volume
was measured with a gas manometer, and the gas composition (N2, C02, and H2) was determined by gas chromatography by the method of van Huyssteen (10). Alcohols and
volatile acids were assayed by the method of Ackman (1).
Lactic acid was determined enzymatically (5).
Khan et al. (4) reported the same pH and temperature
growth ranges for strain BAST that Patel et al. (7) described
for CD2T. The reported DNA base compositions for these
two strains were also similar. Accordingly, these determinations were not repeated in the present study.
RESULTS
Surface colonies of strains BAST, CD2T, and LS on
cellulose agar were visible after 7 days and showed identical
colony morphology. Colonies were 1 to 2 mm in diameter,
round, raised, and cream colored. After 3 weeks, the colonies were slightly larger and had undulate margins. Immediately around and below the colonies were clear zones where
the cellulose had been digested.
Cells of the three strains grown for 48 h in cellulose-basal
medium showed identical morphology (Fig. 1). Single cells
were straight to slightly curved rods, 0.4 to 0.6 pm wide by
2 to 6 km long, Cells occurred singly, in pairs, and occasionally in short chains. All three strains stained gram negative.
This Gram stain reaction was verified by the dissolution of
the cell wall and cytoplasmic membrane with 3% KOH (2).
All three strains were motile. Hanging-drop preparations
of cells grown for 48 h in cellulose-basal medium without
shaking showed tumbling motility. Motility was also indicated in cellobiose-semisolid medium (SIM medium containing 0.5% cellobiose; Difco). However, flagella were not seen
in electron micrographs.
In PY broth, only cellobiose, cellulose, and salicin supported the growth of all three strains. Growth on these
substrates was much heavier in synthetic basal medium, and
3 14
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SYNONYM OF A . CELLULOLYTICUS
VOL. 36, 1986
315
FIG. 1. Electron micrographs of strains BAST, CD2T, and LS grown for 48 h in cellulose-basal medium. Bar = 1 bm.
strains BAST, CD2T, and LS were also able to grow in
esculin-basal medium but not in PY broth-esculin medium.
Upon addition of ferric ammonium citrate to the three
esculin containing culture broths, all gave positive reactions
for esculin hydrolysis. Strain BAST was also able to ferment
xylose in cellulose-basal medium but not in PY broth,
whereas CD2T and LS could not ferment xylose in either
medium. However, the ability to ferment xylose was acquired by CD2T and LS after 2 months of successive
biweekly subcultures in cellobiose-basal medium. This ability was not acquired by parallel cultures carried in
microcrystalline (Avicel) cellulose-basal medium.
No growth occurred in either PY broth or synthetic basal
medium supplemented with adonitol, amygdalin, arabinose,
dulcitol, erythritol, fructose, galactose, glucose, glycerol,
glycogen, hippurate, inositol, inulin, lactate, lactose,
maltose, mannitol, mannose, melezitose, melibiose, pyruvate, raffinose, ribose, sorbitol, sorbose, starch, sucrose, or
trehalose. Tests for acetylmethylcarbinol, indole, ammonia,
urease, gelatinase, and catalase production were negative.
Sulfate and nitrate were not reduced.
When strains BAST, CD2T, and LS were grown in 1%
Solka Floc (ball-milled cellulose) at 35°C for 5 days with
rotary shaking, they produced the same metabolites: C 0 2 ,
H2, acetic acid, and ethanol, at approximately the same
concentrations. When these cultures were grown without
shaking, there was a shift in metabolite production towards
greater ethanol production with trace quantities of lactic acid
and a concomitant decrease in acetic acid and H2 production
(Table 1).
DISCUSSION
Patel et al. (7) carried out substrate utilization tests in PY
broth and found that A . cellulolyticus CD2T could utilize
only cellobiose, cellulose, and salicin. In the present study,
these were also the only substrates in PY broth utilized by
strains CD2T, LS, and BAST. Patel et al. (7) indicated that
PY broth was not the ideal growth medium for CD2T. It
appeared that PY broth lacked some of the vital nutrients
present in synthetic basal medium, because this strain could
only be successfully cultivated in cellobiose-PY broth for
three successive transfers. The delicate balance between
growth and no growth in PY broth was further demonstrated
by the ability of A . cellulolyticus CD2T to grow in cellobiosePY broth under either an 80% N2-20% C 0 2 or a 100% N2
headspace but in salicin-PY broth only under an 80%
N2-20% C02 headspace (7). It appears, therefore, that the
ability of strains BAST, CD2T, and LS to grow in and
hydrolyze esculin was caused by the presence of additional
required nutrients in esculin-basal medium.
The only nutritional difference noted among strains BAST,
CD2T, and LS was the ability of BAST to utilize xylose. The
ability of strain BAST to utilize xylose was not reported by
Khan et al. (4). It was previously noted in this laboratory
(unpublished data) that the ability to utilize xylose was
acquired by strain CD2T after it had been maintained in
cellobiose-basal medium for 2 months. In the present study,
both CD2T and LS developed the ability to utilize xylose
after repeated transfers in cellobiose-basal medium but not
in microcrystalline cellulose-basal medium. The cellobiose
may have been contaminated by small amounts of xylose,
which may have induced the enzymes necessary for xylose
assimilation. Similarly, A . cellulolyticus CD2T enzyme inTABLE 1. Metabolic products of cellulose fermentation"
Amt (mmol/liter) of product formed in cellulose
fermentation by:
Product
CO?
H?
Acetic acid
Ethanol
Lactic acid
Shaken
Unshaken
BAST
CD2T
LS
BAST
CD2T
LS
36.0
68.2
18.4
3.0
35.5
67.0
17.6
2.5
36.1
68.4
18.2
3.1
30.7
44.1
12.5
9.7
trb
31.2
43.9
12.2
9.1
tr
31.5
44.6
12.5
9.4
tr
Cellulose-basal medium contained 1% Solka Floc (ball-milled cellulose)
and was incubated at 35°C for 5 days.
Tr, Trace amounts.
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316
MURRAY
INT.
duction for glucose fermentation was accomplished by Patel
and MacKenzie (8) in 1%(wthol) glucose-basal medium.
Patel et al. (7) reported that strain CD2T produced COz,
H2, and acetic acid as the major metabolites and trace
amounts of ethanol; lactic acid was not detected. Strain
BAST was reported to produce C 0 2 , HZ,acetic acid,
ethanol, and trace amounts of lactic acid (4). The results
from the present study show that all three strains produced
the same products and that the level of these metabolites
could be shifted in favor of reduced products by stationary
incubat ion.
Strain CD2T was reported to possess a single flagellum
located one-third of the distance from the end of the cell (7).
Although flagella were not seen in electron micrographs in
the present study, all three strains were found to be motile.
Flagella may have been lost during handling. Failure to
observe flagella in electron micrographs of strain BAS was
used as evidence by Khan et al. (4) to state that BAS was
nonmotile. Although visual observations of attached flagella
indicate that a microorganism is motile, the absence of
flagella does not prove that a microorganism is nonmotile.
This investigation has shown that strain BAST is morphologically and biochemically identical to strains CD2= and LS
and that A . cellulosolvens is therefore a synonym for A .
cellulolyticus. Since strain BAST is motile, the emendation
by Khan et al. (4) to include nonmotile strains in the
description of the genus Acetivibrio is nullified. It is proposed that A . cellulosolvens BAST be correctly classified as
A . cellulolyticus. It is further proposed that the description
of the genus Acetivibrio, which contains the two species A .
cellulolyticus and A . ethanolgignens (6), be emended as
originally stated by Robinson and Ritchie (9) to include
anaerobic, gram-negative, straight to slightly curved rods
and C 0 2 and
that produce mainly acetic acid, ethanol, HZ,
that are motile by means of a single flagellum or multiple
flagella.
J. SYST. BACTERIOL.
ACKNOWLEDGMENT
I thank G. B. Patel of the National Research Council of Canada for
helpful discussion and comments.
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3. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977.
Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg.
4. Khan, A. W., E. Meek, L. C. Sowden, and J. R. Colvin. 1984.
Emendation of the genus Acetivibrio and description of
Acetivibrio cellulosolvens sp. nov., a nonmotile cellulolytic
mesophile. Int. J. Syst. Bacteriol. 34:419-422.
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Isolation and characterization of an anaerobic, cellulolytic microorganism, Acetivibrio cellulolyticus gen. nov., sp. nov. Int.
J. Syst. Bacteriol. 30:179-185.
8. Patel, G. B., and C. R. MacKenzie. 1982. Metabolism of
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9. Robinson, I. M., and A. E. Ritchie. 1981. Emendation of
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