INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Jan. 1991, p. 14&143
0020-7713/91/010140-04$02.00/0
Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 1
Clostridium xylanolyticum sp. nov. an Anaerobic Xylanolytic
Bacterium from Decayed Pinus patula Wood Chips
G. M. ROGERS1* AND A. A. W . BAECKER,
Department of Microbiology, University of the Witwatersrand, Johannesburg 2000, and Department of Microbiology,
University of Durban-Westville, Durban 4000,2 South Africa
An anaerobic, mesophilic, sporeforming, xylanolytic bacterium was isolated from decayed Pinus patula wood
chips. The cells of this organism were gram-negative rods, had peritrichous flagella, and formed terminal
round endospores which distended the cell wall. Xylan, cellobiose, fructose, glucose, maltose, mannose,
melezitose, raffinose, rhamnose, salicin, sucrose, and xylose were utilized. The optimum temperature and
optimum pH for growth were 35°C and 7.2, respectively. The DNA composition was 40 mol% guanine plus
cytosine. The name Clostridium xylanolyticum sp. nov. is proposed for this bacterium. The type strain is strain
ATCC 49623.
chips were obtained from a chipping plant in Richards Bay,
South Africa. The chips were collected in gas-tight containers and transported to our laboratory. The chips were cut
into small (1 .O-cm3)sections and immersed in liquid nitrogen
before being milled in a liquid nitrogen mill (model A-70; F.
Morat KG, Eissenbach, Federal Republic of Germany)
which had been swabbed with alcohol and flamed to ensure
sterility; 10-fold dilutions of each sample were prepared by
using anaerobic diluent (2). The dilutions were plated onto
BM containing 3% oat spelt xylan in the anaerobic cabinet
and incubated at 35°C for 48 h. Colonies which produced
clearing zones of xylan degradation were removed and
restreaked to obtain pure cultures. The isolate used in this
study was maintained by weekly transfers on slopes of BM
containing 0.3% cellobiose.
Morphology. Living and stained cells of the purified isolate
were examined by light microscopy. Flagella were examined
by electron microscopy. To do this, a colony grown on BM
containing 3% oat spelt xylan was suspended in physiological saline, and 1 drop of the resulting suspension was placed
onto a Formvar-coated copper grid and stained with 1.0%
(wthol) phosphotungstic acid (Sigma Chemical Co., St.
Louis, Mo.). Observations were made by using a JEOL
model lOOC transmission electron microscope. Cell wall
structure was also examined by using transmission electron
microscopy. For this procedure, cells were prepared as
described previously (6).
Biochemical reactions. Biochemical tests were performed
by using the standard procedures described in the Anaerobe
Laboratory Manual (8). Test results were read as soon as the
microorganisms exhibited good growth (optical density at
625 nm, 0.8) or 1 day after they achieved constant turbidity.
Temperature and pH studies. The temperature and pH
optima for growth were determined in BM broth. Three
tubes were inoculated and incubated for each temperature
and pH tested. Growth was measured by using a Bausch &
Lomb Spectronic 1001 spectrophotometer at 625 nm, and the
average values were calculated.
Fermentation end product analysis. Volatile fatty acids and
nonvolatile fatty acids were determined in cultures grown in
peptone-yeast extract-glucose broth (8). Ether extracts were
prepared and chromatographic analyses were performed as
described in Holdeman et al. (8).
DNA base composition. The guanine-plus-cytosine (G +C)
content was determined by using the melting point method of
Marmur and Doty (11). Salmon sperm DNA (Boehringer
The potential use of biological hydrolysis of the hemicellulose components of wood has attracted much interest in
the forestry and pulp and paper industries (9). After paper
manufacture the hemicellulose fraction is essentially a waste
product, but if it could be utilized efficiently by microorganisms, this fraction could become an important substrate for
the production of several products, such as alcohol. Many
xylanolytic organisms, mainly fungi and actinomycetes (1,
14-16), have been isolated from various environments. However, no economically feasible system for biopulping has
been developed (9). As a result of biopulping research in our
laboratories, an obligately anaerobic bacterium was isolated
from decayed Pinus patula wood chips. In this paper this
isolate is proposed as a new species, Clostridium xylanolyticum.
MATERIALS AND METHODS
Media. The basal medium (BM) used in this study contained 1.2% (wt/vol) Bacto-Agar (Difco), 230 ml of clarified
rumen fluid, 12.5 ml of mineral solution I (2), 12.5 ml of
mineral solution I1 (2), 0.25 g of yeast extract (Merck), 1.0 g
of peptone (Merck), 2.5 ml of a vitamin solution (3), 5.0 ml of
a volatile fatty acid mixture (4), 5.0 ml of hemin (0.01%,
wt/vol), 0.5 ml of Pfennig trace element solution (12), and
230 ml of distilled water. Sodium bicarbonate was used as a
buffering agent, indigocarmine (0.05%, wt/vol) was used as a
redox indicator, and 1.0 ml of cysteine monohydrochloride
(12.5%, wt/vol) and 1.0 ml of Na,S (12.5%, wt/vol) were
used as redox poisoning agents. The pH was adjusted to pH
6.8 with 1 N HC1 or 1 N NaOH as required. BM supplemented with 3% oat spelt xylan (Fluka Chemicals) was also
used, and for culture maintenance BM was supplemented
with 0.3% cellobiose (Merck).
Anaerobic culture methods. In this study an anaerobic
cabinet (Forma Scientific, Marietta, Ohio) was used to
culture organisms under strict anaerobic conditions. Cultures were maintained in 3-oz (ca. 90-ml) McCartney bottles
that had been purged with an 0,-free gas mixture containing
5.5% H,, 31.8% CO,, and 62.7% N, prior to use and sealed
with butyl rubber stoppers and screw caps. All cultures were
incubated at 35°C without agitation.
Isolation procedure. Samples of decayed P.patula wood
* Corresponding author
140
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VOL. 41, 1991
CLOSTRIDIUM XYLANOLYTICUM SP. NOV.
FIG. 1. Cells of C . xylanolyticurn grown for 24 h on BM supplemented with xylan. Terminal spores distending the cells are clearly
visible.
Mannheim) and DNA from Clostridium clostridiiforme DSM
933T (T = type strain) were used as the standards. The
DNAs were isolated by using the methods of Marmur (10)
and Meyer and Schleifer (13). The melting temperature (T,)
was determined by measuring the relative change in A,,
with a Varian Cary model 2200 spectrophotometer. The
G+C content was calculated by using the following equation
(11): T, = 69.3 + 0.41(G+C).
DNA-DNA hybridization. In the DNA-DNA hybridization
study DNA from our isolate was hybridized with DNA from
C . clostridiiforme. Genomic DNA was isolated by the
method of Marmur (10). The DNA was purified on a cesium
chloride gradient as described below. The DNA was suspended in 4.0 ml of TE buffer (10 mM Tris, 0.1 mM EDTA,
pH 7.5). The DNA concentration was adjusted to give a
concentration of 50 to 100 pglml. A 4.3-g portion of CsCl was
141
FIG. 2. Transmission electron micrograph of C. xylanolyticurn
cells, showing peritrichous flagella.
added to 4.0 ml of TE buffer, 200 pl of ethidium bromide
(Sigma) was added, and the mixture was centrifuged at
450,000 x g for 4 h at 15°C. The gradient was visualized with
UV light. The DNA band was removed by using a 15-gauge
needle and a 3.0-ml syringe, and the ethidium bromide was
extracted with CsCl-saturated isopropanol. The DNA was
dialyzed overnight against 2.0 liters of TE buffer. The DNA
was precipitated by adding 0.1 volume of 3 M sodium acetate
and 0.6 volume of isopropanol. DNAs from the isolate and
C. clostridiiforme were denatured by heating them at 95°C
for 5 min and then transferred to a nitrocellulose filter
(Amersham, Buckinghamshire, United Kingdom) by using a
Bio-Rad dot blot apparatus connected to a vacuum source.
The blots were washed with 100 pl of 20X SSC (1X SSC is
0.15 M NaCl plus 0.015 M sodium citrate) and fixed at 80°C
for 2 h in a vacuum oven. DNA from the isolate was labeled
with 32P by using a nick translation kit (Amersham). The
FIG. 3. Transmission electron micrograph showing the five-layer cell wall structure of C . xylanolyticurn.
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142
INT. J. SYST.BACTERIOL.
ROGERS AND BAECKER
n
I \
PH
FIG. 4. Effect of temperature on the growth of C . xylanolyticum.
blots were hybridized overnight submerged in a water bath
at 42°C in 30 ml of a solution containing lop1 of the probe,
500 ~1 of salmon sperm DNA, 50 p1 of 10 M NaOH, 300 pl
of 2 M Tris (pH 7.4), and 475 pl of 1 M HC1. After
hybridization the filter was rinsed in 0 . 2 ~SSC-0.02% sodium dodecyl sulfate for 5 min and then washed three times
(20 min each) at room temperature. Then the filter was
washed twice (20 min each) in 0.1X SSC-O.l% sodium
dodecyl sulfate under stringent conditions at 60°C. The
nitrocellulose filter was air dried and autoradiographed overnight at -70°C on X-ray film (Fuji Photo Film Co., Tokyo,
Japan). The densities of the autoradiographs were measured
by using an LKB model 2202 Ultroscan laser densitometer.
RESULTS AND DISCUSSION
The isolation procedures which we used yielded one
anaerobic xylanolytic isolate which was selected for identification on the basis of hydrolysis of xylan. This isolate is an
obligately anaerobic, sporeforming, rod-shaped bacterium
which does not reduce sulfate to hydrogen sulfide and
therefore is a member of the genus Clostridium Prazmowski
(5).
Clostridium xylanolyticum sp. nov. Clostridium xylanolyticum (xy.1an.o. ly’ ti. cum. Gr. n. xylanosum, xylan; Gr. adj.
lyticus, dissolving; N. L. adj. xylanolyticum, xylan dissolving). Cells are gram-negative, obligately anaerobic, and rod
shaped (length, 1.8 to 3.0 pm; width, 0.5 to 0.8 pm). A
single, terminal, round endospore is formed by each cell; the
spore distends the cell wall (Fig. 1).The cells are motile by
means of peritrichous flagella (Fig. 2). Thin sections reveal a
five-layer cell wall structure (Fig. 3).
Xylan is a good substrate for growth. Amygdalin, arabinose, glycogen, inositol, lactose, mannitol, ribose, sorbitol,
and trehalose are not fermented. Gelatin is not liquefied;
urease, indole, lecithinase, lipase, and catalase are not
produced; and nitrate is not reduced. The fermentation end
products from PYG (5) are formic, lactic, and acetic acids.
The optimum temperature for growth is 35°C (Fig. 4), and
the optimum pH for growth is 7.2 (Fig. 5 ) .
The DNA G+C content is 40 mol%. DNA-DNA hybridization results show that under stringent conditions (60°C)
the DNA of C. clostridiiforrne exhibits only 14% homology
FIG. 5. Effect of pH on the growth of C . xylanolyticurn.
with the DNA of our isolate. This suggests that although
there is a low level of homology between the two DNAs,
these organisms are not the same genetically.
Hydrolysis of starch and nonreduction of nitrate distinguish our isolate from C . clostridiiforme. When grown on
blood agar, our isolate is beta-hemolytic, whereas C. clostridiiforme is nonhemolytic. C. clostridiiforme is susceptible to
clindamycin, whereas our isolate is resistant. The G +C
content of C. clostridiiforme is 48 mol%, whereas the G+C
content of our isolate is 40 mol%. The position of the
endospores of C. clostridiiforme is central to subterminal,
whereas the position of the endospores of our isolate is
terminal. Our isolate also differs from Clostridium aerotolerans ATCC 43524T, which ferments lactose, ribose, and
trehalose. C. aerotolerans also utilizes xylan. The DNA
G + C content of C . aerotolerans is 40 mol%, which is the
same as the G + C content of our isolate. Another Clostridium strain which utilizes xylan as a growth source is Clostridium cellulovorans DSM 3052T. However, this organism
differs from our isolate in that it ferments lactose and does
not ferment melezitose, rhamnose, and xylose. The DNA
G + C content of C. cellulovorans is 26 mol%, which is
markedly different from the DNA base composition of our
isolate.
The type strain is strain ATCC 49623. This strain was
isolated from a P.patula chip pile which had been exposed
to environmental conditions for 10 months.
ACKNOWLEDGMENTS
We thank L. V. H. Moore, Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, who confirmed the results of our characterization studies.
We also acknowledge the financial assistance of project 135145
from the Commonwealth of Virginia for support of L. V . H. Moore.
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