INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1990, p. 268-272 0020-7713/90/030268-05$02.OO/O Copyright 0 1990, International Union of Microbiological Societies
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Clostridium aldrichii s p . nov., a Cellulolytic Mesophile Inhabiting a Wood-Fermenting Anaerobic Digester? JUNCHANG C. YANG,l* DAVID P. CHYNOWETH,l DONNA S. WILLIAMS,, AND ANMING L12$ Bioprocess Engineering Research Laboratory, Agricultural Engineering Department,' and Microbiology and Cell Science Department,2 University of Florida, Gainesville, Florida 32611 An anaerobic, mesophilic, spore-forming, cellulolytic bacterium was repeatedly isolated from a woodfermenting anaerobic digester. Cells of this organism were gram-positive rods, motile with a bundle of polar flagella, and formed subterminal oblong spores. The colonies in agar had an irregular shape with many platelike structures and were greyish white. Cellulose, xylan, and cellobiose served as substrates for growth. Acetate, propionate, butyrate, isobutyrate, isovalerate, lactate, succinate, H,, and CO, were products of cellobiose fermentation. The optimal temperature and pH for growth were 35°C and 7, respectively. The DNA composition was 40 mol% G+C. The name Clostridium aldn'chii sp. nov. is proposed. The type strain is P-1 (= OGI 112, = ATCC 49358).
10 ml; resazurin (0.5 mg/ml), 1ml; L-cysteine hydrochloride, 0.5 g; Na,S . 9H20, 0.2 g. The agar concentration was 1.5% (wthol). The pH was adjusted to 7.0 with NaHCO,. The medium was prepared and dispensed with continuous flushing of the gas mixture mentioned above. Medium was dispensed into serum tubes (1)(18 by 150 mm; Bellco Glass, Inc., catalog no. 2048-00150) or serum bottles, according to need, stoppered with butyl rubber stoppers (Bellco, catalog no. 2048-11800), sealed with aluminum seals (Wheaton Industries; 20-mm tear-off, catalog no. 224193), and sterilized at 121°C for 20 min. The cellulose slurry was prepared as follows: 30 g of Whatman no. 1 filter paper was ground with 1,000 ml of distilled water for 72 h in a 5-liter alumina grinding jar with pebbles (20 to 30 mm in diameter). The cellulose slurry was added (50 ml/liter) to the basic medium to make the cellulose medium or cellulose agar medium (CAM). The amount of the cellulose slurry added was varied according to the purpose of the culture. The cellulose concentration in the medium was typically in the range of 0.2 to 0.4% (wthol). A Whatman no. 1 filter paper strip (50 by 5 mm) was submerged in the 4.5-ml basal medium tube as filter paper strip medium (CSM) to determine the highest dilution of the mixed population exhibiting cellulolytic activity. Other substrates, including cellobiose, pectin, xylan, and glucose, were added to the basal medium at a final concentration of 1% (wthol). The media and methods of Holdeman et al. (6) were used for biochemical tests and fermentation product analysis. Isolation, purification, and enumeration. Serial dilutions of sample (0.5 ml) were inoculated in triplicate into CAM (4.5 ml per tube) for roll tubes and CSM for cellulolytic activity detection. The tubes were incubated at 35°C until colonies surrounded by clear zones appeared in CAM or until the submerged filter paper strips were digested in the CSM. Cellulolytic colonies in the highest-dilution roll tube were picked and suspended in cellulose medium, which was used to inoculate CAM roll tubes. The tubes were incubated at 35°C until cellulolytic colonies appeared again. This transfer procedure was repeated several times until a pure culture was obtained. To confirm the purity, the isolate was transferred to cellobiose or glucose agar medium in roll tubes and incubated at 35°C. After ascertaining that only one morphological type of colony appeared, a single colony was picked and transferred back to CAM roll tubes to check its cellu-
Woody biomass generally was not considered to be biodegradable under anaerobic conditions until Zeikus and Ward (22) showed that a methane fermentation occurs in the heartwood of live trees and Chynoweth and co-workers (4, 9) showed that the carbohydrates of poplar and other hardwoods are degraded to methane by a domestic sludge digester inoculum without pretreatment other than reduction of particle size to the millimeter range. These observations have stimulated research to determine the organisms and interactions involved with the possible goal of improving the extent and rate of these depolymerization reactions. Sleat and co-workers (18, 19) have isolated two new cellulolytic, Clostridium species from wood methane fermentation. Our laboratory has begun to isolate predominant cellulolytic pectinolytic, and xylanolytic bacteria from poplar-fed wood digesters. The first of these, a new cellulolytic Clostridium species, has been repeatedly isolated from high dilutions of the samples over a period of 2 years and is described below. The name Clostridium aldrichii is proposed for this organism. MATERIALS AND METHODS Samples and treatments. Strict anaerobic methods (7) were used for sample collection, medium preparation, inoculation, and identification. Samples were collected from the effluent of a continuously stirred tank, bench-scale, mesophilic (35°C)) poplar-fed digester. The effluent was blended for less than 1 min in a homogenizer under oxygen-free N,-CO, (80:20). The treated sample was diluted in basal medium up to lo-'' in order to exceed the highest dilution with cellulolytic activity. The first sample was collected 65 days after the digester was started. Culture media. The basal medium contained (per liter): NH,C1,2 g; KCl, 0.5 g; K,HP04,1.65 g; MgSO4 7H,O, 0.5 g; Trypticase (BBL Microbiology Systems), 0.5 g; yeast extract (BBL), 0.5 g; clarified rumen fluid, 200 ml; clarified woody-biomass digester sludge, 100 ml; mineral solution "aCl(6 g), (NH4),S04 (6 g), CaCl, (0.6 g), H,O (1,000 mOl, 150 ml; trace mineral solution (l),10 ml; vitamin solution (l), *
* Corresponding author. t Florida Agricultural Experiment Station Journal Series no. R-00436. $ Present address: Chengdu Institute of Biology, Academia Sinica, Chengdu, Sichuan, People's Republic of China. 268
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FIG. 1. (A) Ultrathin section of P-1cells, showing gram-positive-typecell wall structures (an enlarged cross section is shown at bottom right). (B) Ultrathin section showing the round particle between two cells.
lolytic activity. The colony and cell morphologies from the secondary growth of the clear-zoned colonies in CAM were compared and were identical to those of the previous isolate (8). Numbers of cellulolytic bacteria were estimated from the reciprocal of the highest dilution of the inoculated samples in which the filter paper strip was digested in CSM and in which clear-zoned colonies appeared in CAM. Morphological studies and microscopy. Morphological observations of the cells from 24- to 48-h growth broth cultures and single colonies were made by both light microscopy (Nikon Labophot equipped with phase optics, epifluorescence, and an automatic photomicrographic Nikon FX-35A camera with UFX-I1 control) and electron microscopy (Philips EM-301 transmission electron microscope). Gram stains, the KOH test (S), and the ~danine-4-nitroanilide(LANA) test (2) were used to complement the Gram reaction. Medium without Trypticase, yeast extract, clarified rumen fluid, and digester sludge but with the vitamin solution and cellobiose was used to induce spore formation. A fresh culture (1 to 2 days) in cellobiose medium was used for electron microscopic observation of flagella. To prepare specimens for electron microscopy, glutaraldehyde was injected into the fresh broth culture to a final concentration of approximately 1.3%. Cells were immediately centrifuged, and the supernatant was replaced with 1% glutaraldehyde in 0.025 M sodium cacodylate buffer (pH 7.2). Cells were postfixed in buffered 1% osmium tetroxide followed by 1% aqueous uranyl acetate, dehydrated in a graded ethanol to acetone series, and embedded in Spurr low-viscosity resin. Sections were cut with a diamond knife on an LKB Ultrotome I11 and poststained with uranyl acetate and lead citrate. For observation of flagella, cells were gently pipetted from the fresh culture and placed on Formvar-coated 200-mesh copper grids which had been precoated with 0.1% bacitracin to improve surface wettability. Cells were allowed to settle for 3 to 5 min, and the excess medium was drawn off. The grids were washed with water to remove soluble medium components and shadowed at a 30" angle with a carbon-platinum source in an electron gun using
a Balzers model MED 010 vacuum evaporator. Grids were examined by transmission electron microscopy, Physiological and biochemical identifications. Basal medium with cellobiose was used for the measurements of optimal growth temperature and pH. Cell density was determined by measurement of optical densities of cultures with a spectrophotometer (Perkin-Elmer model 35) at 610 nm. Optimal temperature and pH measurements were determined twice, and each measurement was made in triplicate. Nutrient requirements were tested in the medium with cellobiose as the only carbon and energy source and with different combinations of yeast extract, Trypticase, clarified rumen fluid, digester sludge, and vitamin solution. Twoday-old cultures grown on cellobiose basal agar slants were washed with the salt medium to serve as inocula for these tests. The fermentation products were analyzed after incubation for 2 days in PY basal medium (6) with cellobiose as the substrate. Biochemical tests were performed in PY basal medium with carbohydrates, enzymes, and other substrates. Fermentation product analyses and biochemical tests were done in the laboratory of W.E. C. and L. V. H. Moore, Virginia Polytechnic Institute and State University, Blacksburg. Analysis of DNA base composition. Resuspended cells from a centrifuged pellet were treated with 5% (voVvol) Triton X-100, and DNA was extracted and purified by the method of Marmur (13). DNA base compositions were determined from the buoyant density of DNA in CsCl gradients by the method of Preston and Boone (16). DNAs from Micrococcus lysodeikticus and Escherichia coli were used as standards. The mol% G+C of the DNA was calculated by the method of Schildkraut et al. (17).
RESULTS AND DISCUSSION The isolate was rod shaped, gram positive, motile, formed endospores, and obligately anaerobic but was unable to reduce sulfate to sulfide. These differential characters indicate that the isolate can be placed in the genus Clostridium (3). It was unlike previously described mesophilic, cellu-
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FIG. 2. Cell morphology of strain P-1 visualized by light microscopy. (A) Vegetative cells after 24 to 48 h of growth in basal medium with cellobiose as the substrate; a round particle (arrow) can be seen between two cells (B) Spores of strain P-1 (arrow).
lolytic clostridia in growth substrates, fermentative products, morphology, and immunospecificity . Therefore, we propose a new species, Clostridium aldrichii, as follows. Clostridium aldrichii sp. nov. Clostridium aldrichii (Al. dri’chii, N.L. gen. n., of Aldrich, named for Henry C. Aldrich, a professor of the University of Florida, Gaines-
ville, for his contributions to ultrastructural research on the strictly anaerobic bacteria) cells are easily decolorized in Gram stains, but KOH and LANA tests are negative. Cell wall structure is that of gram-positive bacterium (Fig. 1A). Cells are 0.5 to 1.0 Fm by 3 to 5 Fm, straight or slightly curved with rounded ends (Fig. 2). Pleomorphism is evident,
FIG. 3. Carbon-platinum-shadowed whole-cell preparation; bundled flagella can be seen at both ends of the paired cells.
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FIG. 4. Colonial morphology of strain P-1 in CAM with clear zones around each colony. Arrow indicates the surface colony.
with some cells up to 8 to 12 Fm in length; occasionally, some cells are swollen at both attached ends of paired cells. Cells are arranged mostly in pairs but some are single, or in short chains (Fig. 2). Round particles are sometimes seen between two connected cells (Fig. 1B and 2A). Young cells of strain P-1 are motile, with a bundle of flagella at one end of the cell (Fig. 3). Oblong spores occur subterminally and are formed after 2 weeks in a medium containing salts, trace vitamins, and cellobiose as the only carbon source at 35°C. Mature spores are 0.5 to 1.0 Fm by 1 to 2 pm (Fig. 2B). Spores are viable after being heated at 80°C for 10 min. Morphologically identical colonies appear in CAM 4 to 6 days after transfer of strain P-1 from medium to medium and within 24 h in the medium with cellobiose as the substrate. The surface colonies are round, smooth, entire, soft, jellylike, semitransparent or greyish white, and 1 to 2 mm in diameter. Clear zones caused by cellulolytic activity around colonies are usually 4 to 6 mm in diameter, and the widths of the clear zones from the edge of the colonies are 2 to 3 mm. In roll tubes, surface colonies often flow down along the agar surface to form elongated colonies; the clear zones subsequently appear around the elongated growth. Subsurface colonies are irregular in shape, with many platelike structures in different dimensions; they are greyish white and more opaque than surface colonies (Fig. 4). This distinctive colony morphology and associated clear zone facilitate recognition of colonies of strain P-1. Strain P-1 is a strict anaerobe. The optimal temperature for growth is 35”C, with a range between 20 and 45°C.The optimal pH for growth is 7, and the range is 6.2 to 7.8. P-1 does not require rumen fluid, digester sludge, Trypticase, yeast extract, and added trace vitamins for growth but grows more slowly without these additions. Cellulose, xylan, and cellobiose serve as growth substrates, and esculin is hydrolyzed. Amygdalin, arabinose, erythritol, fructose, glucose, glycogen, inositol, lactose, maltose, mannitol, mannose, melezitose, melibiose, pectin, raffinose, rhamnose, ribose, salicin, sorbitol, starch, sucrose, trehalose, and xylose are not utilized. Gelatin is not liquified, and milk is not clotted. Indole and H,S are not produced. The isolate does not reduce nitrate to nitrite, and the lecithinase A test is negative. The fermentation products from cellobiose are acetic, propionic, isobutyric, butyric, isovaleric, lactic, and SUCcinic acids, hydrogen, and carbon dioxide.
DNA base composition. The G+C content of strain P-1 DNA is 40 mol%. The type strain of C. afdrichii is strain P-1 (= OGI 112, = ATCC 49358 [OGI, Collection of Methanogenic Archaeobacteria of Oregon Graduate Institute of Science and Technology]). It was isolated in 1986 from a 3-month-old, mesophilic , poplar wood-fed continuously stirred anaerobic digester. It has been considered a predominant cellulolytic bacterium, since it inhabited (at lo6 to 10’) this digester for at least 2 years. Enumeration of strain P-1.Cellulolytic activities in CSM (digestion of the immersed part of the filter paper strip; Fig. 5 ) and CAM (colonies with clear zones; Fig. 4) correspondingly appear in the same or nearly the same dilution in isolations, and the highest dilutions showing positive celluto lo-’. lolytic activity fall in the range of Distinguishing characteristics. Clostridium aldrichii P-1 is considered a new species on the basis of the following evidence. Most mesophilic, cellulolytic Cfostridium species which have been reported (10-12, 14, 15, 18, 19) grow and produce acids in glucose; strain P-1 does not produce acids from glucose. No immunological cross-reactions were observed between the polyclonal antibody of C. afdrichii P-1 and 14 other known bacterial species (including 11 cellu-
FIG. 5. Filter paper strips in CSM digested in dilutions up to lo-’ of the sample inocula. Higher dilutions (right three tubes) did not digest filter paper strips.
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YANG ET AL. TABLE 1. Comparison of C. aldrichii and C . polysaccharolyticum C . polysaccharolyticum
C . aldrichii
Characteristic"
Rods (0.5 to 1 pm by 3 to 5 pm) Oblong (0.5 to 1 pm by 1 to 2 pm); subterminal
Cell morphology Spores
Polar bundles -
Flagella Acids from glucose Gelatin hydrolysis Esculin hydrolysis
+
Rods (0.8 pm by 3 to 6 pm) OvaVspherical (1.2 by 0.8 pm); terminalhbterminal Peritrichous -
+
Acid production from: Cellobiose Xylan Starch Maltose L-Arabinose Xylose Fermentation products from cellobiose: Formate Acetate Propionate Butyrate Isobutyrate Lactate Mol% G+C Source of isolates
40 Wood digester
42 Sheep rumen
a Methods used for biochemical tests and fermentative product detections were the same (6) under different laboratory conditions, except DNA base ratio measurements. Symbols: +, positive reaction; -, negative reaction; w, weak reaction.
lolytic species) by using an enzyme-linked immunosorbent assay technique (an immunological study of C. aldrichii P-1 will be reported elsewhere). Only one reported cellulolytic, mesophilic Clostridiurn species, Clostridiurn polysaccharolyticurn (20, 21), does not produce acids from glucose. The important characteristics of the two species are compared in Table 1. Although the DNA base ratios are similar, the arrangement of the flagella, acid production from different carbohydrates, and the differences in fermentation products from cellobiose are the primary bases for separating them into different species. ACKNOWLEDGMENTS
We gratefully acknowledge L. V. H. and W. E. C. Moore of the Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, for biochemical tests and fermentative product analyses. This work was supported by the Gas Research Institute, New York State Energy Development Authority, and New York Gas Group under contract no. 5086-226-1199. LITERATURE CITED 1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296.
2. Carlone, G. M., M. J. Valadez, and M. J. Pickett. 1983. Methods for distinguishing gram-positive from gram-negative bacteria. J. Clin. Microbiol. 16:1157-1159. 3. Cato, E. P., W. L. George, and S. M. Finegold. 1986. Genus Clostridium Prazmowski 1880, 23*=, p. 1141-1200. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 4. Chynoweth, D. P., and D. E. Jerger. 1985. Anaerobic digestion of woody Biomass. Dev. Ind. Microbiol. 265235-246. 5 . Halebian, S., B. Harris, S. M. Finegold, and R. D. Rolfe. 1981. Rapid method that aids in distinguishing gram-positive from gram-negative anaerobic bacteria. J. Clin. Microbiol. 13:444448. 6. 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. 7. Hungate, R. E. 1969. A roll tube method for cultivation of strict anaerobes, p. 117-132. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 3B. Academic Press, Inc., New Y ork . 8. Hungate, R. E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14:149. 9. Jerger, D. E., M. Novil, and D. P. Chynoweth. 1982. Bioconversion of woody biomass as a renewable source of energy. Biotechnol. Bioeng. Symp. 12:233-248. 10. Kelly, W. J., R. V. Asmundson, and D. H. Hoperon. 1987. Isolation and characterization of a strictly anaerobic, cellulolytic spore former: Clostridium chartatabidum sp. nov. Arch. Microbiol. 147:169-173. 11. Leschine, S. B., and E. Canale-Parola. 1983. Mesophilic cellulolytic clostridia from freshwater environments. Appl. Environ. Microbiol. 46:72&737. 12. Madden, R. H., M. J. Bryder, and N. J. Poole. 1982. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium papyrosolvens sp. nov. Int. J. Syst. Bacteriol. 32:87-91. 13. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. 14. Palop, M. L., S. Valles, F. Pinaga, and A. Flors. 1989. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int. J. Syst. Bacteriol. 39:68-71. 15. Petitdemange, E., F. Caillet, J. Giallo, and C. Gaudin. 1984. Clostridium cellulolyticum sp. nov., a cellulolytic, mesophilic species from decayed grass. Int. J. Syst. Bacteriol. 34:155-159. 16. Preston, J. F., and D. R. Boone. 1972. Analytical determination of the buoyant density of DNA in acrylamide gels after preparative CsCl gradient centrifugation. FEBS Lett. 37:321-324. 17. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J. Mol. Biol. 4:430-443. 18. Sleat, R., and R. A. Mah. 1985. Clostridium populeti sp. nov., a cellulolytic species from a woody-biomass digester. Int. J. Syst. Bacteriol. 35160-163. 19. Sleat, R., R. A. Mah, and R. Robinson. 1984. Isolation and characterization of an anaerobic, cellulolytic Bacterium, Clostridium cellulovorans sp. nov. Appl. Environ. Microbiol. 48: 88-93. 20. van Gylswyk, N. 0. 1980. Fusobacterium polysaccharolyticum sp. nov., a gram negative rod from the rumen that produces butyrate and ferments cellulose and starch. J. Gen. Microbiol. 116:157-163. 21. van Gylswyk, N. O., E. J. Morris, and H. J. Els. 1980. Sporulation and cell wall structure of Clostridium polysaccharolyticum comb. nov. (formerly Fusobacterium polysaccharolyticum). J. Gen. Microbiol. 121:491493. 22. Zeikus, J. G., and J. C. Ward. 1974. Methane formation in living trees: a microbial origin. Science 184:1181-1183.
Vol. 40, No. 3
Clostridium aldrichii s p . nov., a Cellulolytic Mesophile Inhabiting a Wood-Fermenting Anaerobic Digester? JUNCHANG C. YANG,l* DAVID P. CHYNOWETH,l DONNA S. WILLIAMS,, AND ANMING L12$ Bioprocess Engineering Research Laboratory, Agricultural Engineering Department,' and Microbiology and Cell Science Department,2 University of Florida, Gainesville, Florida 32611 An anaerobic, mesophilic, spore-forming, cellulolytic bacterium was repeatedly isolated from a woodfermenting anaerobic digester. Cells of this organism were gram-positive rods, motile with a bundle of polar flagella, and formed subterminal oblong spores. The colonies in agar had an irregular shape with many platelike structures and were greyish white. Cellulose, xylan, and cellobiose served as substrates for growth. Acetate, propionate, butyrate, isobutyrate, isovalerate, lactate, succinate, H,, and CO, were products of cellobiose fermentation. The optimal temperature and pH for growth were 35°C and 7, respectively. The DNA composition was 40 mol% G+C. The name Clostridium aldn'chii sp. nov. is proposed. The type strain is P-1 (= OGI 112, = ATCC 49358).
10 ml; resazurin (0.5 mg/ml), 1ml; L-cysteine hydrochloride, 0.5 g; Na,S . 9H20, 0.2 g. The agar concentration was 1.5% (wthol). The pH was adjusted to 7.0 with NaHCO,. The medium was prepared and dispensed with continuous flushing of the gas mixture mentioned above. Medium was dispensed into serum tubes (1)(18 by 150 mm; Bellco Glass, Inc., catalog no. 2048-00150) or serum bottles, according to need, stoppered with butyl rubber stoppers (Bellco, catalog no. 2048-11800), sealed with aluminum seals (Wheaton Industries; 20-mm tear-off, catalog no. 224193), and sterilized at 121°C for 20 min. The cellulose slurry was prepared as follows: 30 g of Whatman no. 1 filter paper was ground with 1,000 ml of distilled water for 72 h in a 5-liter alumina grinding jar with pebbles (20 to 30 mm in diameter). The cellulose slurry was added (50 ml/liter) to the basic medium to make the cellulose medium or cellulose agar medium (CAM). The amount of the cellulose slurry added was varied according to the purpose of the culture. The cellulose concentration in the medium was typically in the range of 0.2 to 0.4% (wthol). A Whatman no. 1 filter paper strip (50 by 5 mm) was submerged in the 4.5-ml basal medium tube as filter paper strip medium (CSM) to determine the highest dilution of the mixed population exhibiting cellulolytic activity. Other substrates, including cellobiose, pectin, xylan, and glucose, were added to the basal medium at a final concentration of 1% (wthol). The media and methods of Holdeman et al. (6) were used for biochemical tests and fermentation product analysis. Isolation, purification, and enumeration. Serial dilutions of sample (0.5 ml) were inoculated in triplicate into CAM (4.5 ml per tube) for roll tubes and CSM for cellulolytic activity detection. The tubes were incubated at 35°C until colonies surrounded by clear zones appeared in CAM or until the submerged filter paper strips were digested in the CSM. Cellulolytic colonies in the highest-dilution roll tube were picked and suspended in cellulose medium, which was used to inoculate CAM roll tubes. The tubes were incubated at 35°C until cellulolytic colonies appeared again. This transfer procedure was repeated several times until a pure culture was obtained. To confirm the purity, the isolate was transferred to cellobiose or glucose agar medium in roll tubes and incubated at 35°C. After ascertaining that only one morphological type of colony appeared, a single colony was picked and transferred back to CAM roll tubes to check its cellu-
Woody biomass generally was not considered to be biodegradable under anaerobic conditions until Zeikus and Ward (22) showed that a methane fermentation occurs in the heartwood of live trees and Chynoweth and co-workers (4, 9) showed that the carbohydrates of poplar and other hardwoods are degraded to methane by a domestic sludge digester inoculum without pretreatment other than reduction of particle size to the millimeter range. These observations have stimulated research to determine the organisms and interactions involved with the possible goal of improving the extent and rate of these depolymerization reactions. Sleat and co-workers (18, 19) have isolated two new cellulolytic, Clostridium species from wood methane fermentation. Our laboratory has begun to isolate predominant cellulolytic pectinolytic, and xylanolytic bacteria from poplar-fed wood digesters. The first of these, a new cellulolytic Clostridium species, has been repeatedly isolated from high dilutions of the samples over a period of 2 years and is described below. The name Clostridium aldrichii is proposed for this organism. MATERIALS AND METHODS Samples and treatments. Strict anaerobic methods (7) were used for sample collection, medium preparation, inoculation, and identification. Samples were collected from the effluent of a continuously stirred tank, bench-scale, mesophilic (35°C)) poplar-fed digester. The effluent was blended for less than 1 min in a homogenizer under oxygen-free N,-CO, (80:20). The treated sample was diluted in basal medium up to lo-'' in order to exceed the highest dilution with cellulolytic activity. The first sample was collected 65 days after the digester was started. Culture media. The basal medium contained (per liter): NH,C1,2 g; KCl, 0.5 g; K,HP04,1.65 g; MgSO4 7H,O, 0.5 g; Trypticase (BBL Microbiology Systems), 0.5 g; yeast extract (BBL), 0.5 g; clarified rumen fluid, 200 ml; clarified woody-biomass digester sludge, 100 ml; mineral solution "aCl(6 g), (NH4),S04 (6 g), CaCl, (0.6 g), H,O (1,000 mOl, 150 ml; trace mineral solution (l),10 ml; vitamin solution (l), *
* Corresponding author. t Florida Agricultural Experiment Station Journal Series no. R-00436. $ Present address: Chengdu Institute of Biology, Academia Sinica, Chengdu, Sichuan, People's Republic of China. 268
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FIG. 1. (A) Ultrathin section of P-1cells, showing gram-positive-typecell wall structures (an enlarged cross section is shown at bottom right). (B) Ultrathin section showing the round particle between two cells.
lolytic activity. The colony and cell morphologies from the secondary growth of the clear-zoned colonies in CAM were compared and were identical to those of the previous isolate (8). Numbers of cellulolytic bacteria were estimated from the reciprocal of the highest dilution of the inoculated samples in which the filter paper strip was digested in CSM and in which clear-zoned colonies appeared in CAM. Morphological studies and microscopy. Morphological observations of the cells from 24- to 48-h growth broth cultures and single colonies were made by both light microscopy (Nikon Labophot equipped with phase optics, epifluorescence, and an automatic photomicrographic Nikon FX-35A camera with UFX-I1 control) and electron microscopy (Philips EM-301 transmission electron microscope). Gram stains, the KOH test (S), and the ~danine-4-nitroanilide(LANA) test (2) were used to complement the Gram reaction. Medium without Trypticase, yeast extract, clarified rumen fluid, and digester sludge but with the vitamin solution and cellobiose was used to induce spore formation. A fresh culture (1 to 2 days) in cellobiose medium was used for electron microscopic observation of flagella. To prepare specimens for electron microscopy, glutaraldehyde was injected into the fresh broth culture to a final concentration of approximately 1.3%. Cells were immediately centrifuged, and the supernatant was replaced with 1% glutaraldehyde in 0.025 M sodium cacodylate buffer (pH 7.2). Cells were postfixed in buffered 1% osmium tetroxide followed by 1% aqueous uranyl acetate, dehydrated in a graded ethanol to acetone series, and embedded in Spurr low-viscosity resin. Sections were cut with a diamond knife on an LKB Ultrotome I11 and poststained with uranyl acetate and lead citrate. For observation of flagella, cells were gently pipetted from the fresh culture and placed on Formvar-coated 200-mesh copper grids which had been precoated with 0.1% bacitracin to improve surface wettability. Cells were allowed to settle for 3 to 5 min, and the excess medium was drawn off. The grids were washed with water to remove soluble medium components and shadowed at a 30" angle with a carbon-platinum source in an electron gun using
a Balzers model MED 010 vacuum evaporator. Grids were examined by transmission electron microscopy, Physiological and biochemical identifications. Basal medium with cellobiose was used for the measurements of optimal growth temperature and pH. Cell density was determined by measurement of optical densities of cultures with a spectrophotometer (Perkin-Elmer model 35) at 610 nm. Optimal temperature and pH measurements were determined twice, and each measurement was made in triplicate. Nutrient requirements were tested in the medium with cellobiose as the only carbon and energy source and with different combinations of yeast extract, Trypticase, clarified rumen fluid, digester sludge, and vitamin solution. Twoday-old cultures grown on cellobiose basal agar slants were washed with the salt medium to serve as inocula for these tests. The fermentation products were analyzed after incubation for 2 days in PY basal medium (6) with cellobiose as the substrate. Biochemical tests were performed in PY basal medium with carbohydrates, enzymes, and other substrates. Fermentation product analyses and biochemical tests were done in the laboratory of W.E. C. and L. V. H. Moore, Virginia Polytechnic Institute and State University, Blacksburg. Analysis of DNA base composition. Resuspended cells from a centrifuged pellet were treated with 5% (voVvol) Triton X-100, and DNA was extracted and purified by the method of Marmur (13). DNA base compositions were determined from the buoyant density of DNA in CsCl gradients by the method of Preston and Boone (16). DNAs from Micrococcus lysodeikticus and Escherichia coli were used as standards. The mol% G+C of the DNA was calculated by the method of Schildkraut et al. (17).
RESULTS AND DISCUSSION The isolate was rod shaped, gram positive, motile, formed endospores, and obligately anaerobic but was unable to reduce sulfate to sulfide. These differential characters indicate that the isolate can be placed in the genus Clostridium (3). It was unlike previously described mesophilic, cellu-
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FIG. 2. Cell morphology of strain P-1 visualized by light microscopy. (A) Vegetative cells after 24 to 48 h of growth in basal medium with cellobiose as the substrate; a round particle (arrow) can be seen between two cells (B) Spores of strain P-1 (arrow).
lolytic clostridia in growth substrates, fermentative products, morphology, and immunospecificity . Therefore, we propose a new species, Clostridium aldrichii, as follows. Clostridium aldrichii sp. nov. Clostridium aldrichii (Al. dri’chii, N.L. gen. n., of Aldrich, named for Henry C. Aldrich, a professor of the University of Florida, Gaines-
ville, for his contributions to ultrastructural research on the strictly anaerobic bacteria) cells are easily decolorized in Gram stains, but KOH and LANA tests are negative. Cell wall structure is that of gram-positive bacterium (Fig. 1A). Cells are 0.5 to 1.0 Fm by 3 to 5 Fm, straight or slightly curved with rounded ends (Fig. 2). Pleomorphism is evident,
FIG. 3. Carbon-platinum-shadowed whole-cell preparation; bundled flagella can be seen at both ends of the paired cells.
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FIG. 4. Colonial morphology of strain P-1 in CAM with clear zones around each colony. Arrow indicates the surface colony.
with some cells up to 8 to 12 Fm in length; occasionally, some cells are swollen at both attached ends of paired cells. Cells are arranged mostly in pairs but some are single, or in short chains (Fig. 2). Round particles are sometimes seen between two connected cells (Fig. 1B and 2A). Young cells of strain P-1 are motile, with a bundle of flagella at one end of the cell (Fig. 3). Oblong spores occur subterminally and are formed after 2 weeks in a medium containing salts, trace vitamins, and cellobiose as the only carbon source at 35°C. Mature spores are 0.5 to 1.0 Fm by 1 to 2 pm (Fig. 2B). Spores are viable after being heated at 80°C for 10 min. Morphologically identical colonies appear in CAM 4 to 6 days after transfer of strain P-1 from medium to medium and within 24 h in the medium with cellobiose as the substrate. The surface colonies are round, smooth, entire, soft, jellylike, semitransparent or greyish white, and 1 to 2 mm in diameter. Clear zones caused by cellulolytic activity around colonies are usually 4 to 6 mm in diameter, and the widths of the clear zones from the edge of the colonies are 2 to 3 mm. In roll tubes, surface colonies often flow down along the agar surface to form elongated colonies; the clear zones subsequently appear around the elongated growth. Subsurface colonies are irregular in shape, with many platelike structures in different dimensions; they are greyish white and more opaque than surface colonies (Fig. 4). This distinctive colony morphology and associated clear zone facilitate recognition of colonies of strain P-1. Strain P-1 is a strict anaerobe. The optimal temperature for growth is 35”C, with a range between 20 and 45°C.The optimal pH for growth is 7, and the range is 6.2 to 7.8. P-1 does not require rumen fluid, digester sludge, Trypticase, yeast extract, and added trace vitamins for growth but grows more slowly without these additions. Cellulose, xylan, and cellobiose serve as growth substrates, and esculin is hydrolyzed. Amygdalin, arabinose, erythritol, fructose, glucose, glycogen, inositol, lactose, maltose, mannitol, mannose, melezitose, melibiose, pectin, raffinose, rhamnose, ribose, salicin, sorbitol, starch, sucrose, trehalose, and xylose are not utilized. Gelatin is not liquified, and milk is not clotted. Indole and H,S are not produced. The isolate does not reduce nitrate to nitrite, and the lecithinase A test is negative. The fermentation products from cellobiose are acetic, propionic, isobutyric, butyric, isovaleric, lactic, and SUCcinic acids, hydrogen, and carbon dioxide.
DNA base composition. The G+C content of strain P-1 DNA is 40 mol%. The type strain of C. afdrichii is strain P-1 (= OGI 112, = ATCC 49358 [OGI, Collection of Methanogenic Archaeobacteria of Oregon Graduate Institute of Science and Technology]). It was isolated in 1986 from a 3-month-old, mesophilic , poplar wood-fed continuously stirred anaerobic digester. It has been considered a predominant cellulolytic bacterium, since it inhabited (at lo6 to 10’) this digester for at least 2 years. Enumeration of strain P-1.Cellulolytic activities in CSM (digestion of the immersed part of the filter paper strip; Fig. 5 ) and CAM (colonies with clear zones; Fig. 4) correspondingly appear in the same or nearly the same dilution in isolations, and the highest dilutions showing positive celluto lo-’. lolytic activity fall in the range of Distinguishing characteristics. Clostridium aldrichii P-1 is considered a new species on the basis of the following evidence. Most mesophilic, cellulolytic Cfostridium species which have been reported (10-12, 14, 15, 18, 19) grow and produce acids in glucose; strain P-1 does not produce acids from glucose. No immunological cross-reactions were observed between the polyclonal antibody of C. afdrichii P-1 and 14 other known bacterial species (including 11 cellu-
FIG. 5. Filter paper strips in CSM digested in dilutions up to lo-’ of the sample inocula. Higher dilutions (right three tubes) did not digest filter paper strips.
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YANG ET AL. TABLE 1. Comparison of C. aldrichii and C . polysaccharolyticum C . polysaccharolyticum
C . aldrichii
Characteristic"
Rods (0.5 to 1 pm by 3 to 5 pm) Oblong (0.5 to 1 pm by 1 to 2 pm); subterminal
Cell morphology Spores
Polar bundles -
Flagella Acids from glucose Gelatin hydrolysis Esculin hydrolysis
+
Rods (0.8 pm by 3 to 6 pm) OvaVspherical (1.2 by 0.8 pm); terminalhbterminal Peritrichous -
+
Acid production from: Cellobiose Xylan Starch Maltose L-Arabinose Xylose Fermentation products from cellobiose: Formate Acetate Propionate Butyrate Isobutyrate Lactate Mol% G+C Source of isolates
40 Wood digester
42 Sheep rumen
a Methods used for biochemical tests and fermentative product detections were the same (6) under different laboratory conditions, except DNA base ratio measurements. Symbols: +, positive reaction; -, negative reaction; w, weak reaction.
lolytic species) by using an enzyme-linked immunosorbent assay technique (an immunological study of C. aldrichii P-1 will be reported elsewhere). Only one reported cellulolytic, mesophilic Clostridiurn species, Clostridiurn polysaccharolyticurn (20, 21), does not produce acids from glucose. The important characteristics of the two species are compared in Table 1. Although the DNA base ratios are similar, the arrangement of the flagella, acid production from different carbohydrates, and the differences in fermentation products from cellobiose are the primary bases for separating them into different species. ACKNOWLEDGMENTS
We gratefully acknowledge L. V. H. and W. E. C. Moore of the Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, for biochemical tests and fermentative product analyses. This work was supported by the Gas Research Institute, New York State Energy Development Authority, and New York Gas Group under contract no. 5086-226-1199. LITERATURE CITED 1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296.
2. Carlone, G. M., M. J. Valadez, and M. J. Pickett. 1983. Methods for distinguishing gram-positive from gram-negative bacteria. J. Clin. Microbiol. 16:1157-1159. 3. Cato, E. P., W. L. George, and S. M. Finegold. 1986. Genus Clostridium Prazmowski 1880, 23*=, p. 1141-1200. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 4. Chynoweth, D. P., and D. E. Jerger. 1985. Anaerobic digestion of woody Biomass. Dev. Ind. Microbiol. 265235-246. 5 . Halebian, S., B. Harris, S. M. Finegold, and R. D. Rolfe. 1981. Rapid method that aids in distinguishing gram-positive from gram-negative anaerobic bacteria. J. Clin. Microbiol. 13:444448. 6. 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. 7. Hungate, R. E. 1969. A roll tube method for cultivation of strict anaerobes, p. 117-132. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 3B. Academic Press, Inc., New Y ork . 8. Hungate, R. E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14:149. 9. Jerger, D. E., M. Novil, and D. P. Chynoweth. 1982. Bioconversion of woody biomass as a renewable source of energy. Biotechnol. Bioeng. Symp. 12:233-248. 10. Kelly, W. J., R. V. Asmundson, and D. H. Hoperon. 1987. Isolation and characterization of a strictly anaerobic, cellulolytic spore former: Clostridium chartatabidum sp. nov. Arch. Microbiol. 147:169-173. 11. Leschine, S. B., and E. Canale-Parola. 1983. Mesophilic cellulolytic clostridia from freshwater environments. Appl. Environ. Microbiol. 46:72&737. 12. Madden, R. H., M. J. Bryder, and N. J. Poole. 1982. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium papyrosolvens sp. nov. Int. J. Syst. Bacteriol. 32:87-91. 13. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. 14. Palop, M. L., S. Valles, F. Pinaga, and A. Flors. 1989. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int. J. Syst. Bacteriol. 39:68-71. 15. Petitdemange, E., F. Caillet, J. Giallo, and C. Gaudin. 1984. Clostridium cellulolyticum sp. nov., a cellulolytic, mesophilic species from decayed grass. Int. J. Syst. Bacteriol. 34:155-159. 16. Preston, J. F., and D. R. Boone. 1972. Analytical determination of the buoyant density of DNA in acrylamide gels after preparative CsCl gradient centrifugation. FEBS Lett. 37:321-324. 17. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J. Mol. Biol. 4:430-443. 18. Sleat, R., and R. A. Mah. 1985. Clostridium populeti sp. nov., a cellulolytic species from a woody-biomass digester. Int. J. Syst. Bacteriol. 35160-163. 19. Sleat, R., R. A. Mah, and R. Robinson. 1984. Isolation and characterization of an anaerobic, cellulolytic Bacterium, Clostridium cellulovorans sp. nov. Appl. Environ. Microbiol. 48: 88-93. 20. van Gylswyk, N. 0. 1980. Fusobacterium polysaccharolyticum sp. nov., a gram negative rod from the rumen that produces butyrate and ferments cellulose and starch. J. Gen. Microbiol. 116:157-163. 21. van Gylswyk, N. O., E. J. Morris, and H. J. Els. 1980. Sporulation and cell wall structure of Clostridium polysaccharolyticum comb. nov. (formerly Fusobacterium polysaccharolyticum). J. Gen. Microbiol. 121:491493. 22. Zeikus, J. G., and J. C. Ward. 1974. Methane formation in living trees: a microbial origin. Science 184:1181-1183.
