INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1987, p. 102-105 Vol. 37, No. 2 0020-7713/87/020102-04$02.OO/O Copyright 0 1987, International Union of Microbiological Societies

Clostridium aerotolerans sp. nov. a Xylanolytic Bacterium from Corn Stover and from the Rumina of Sheep Fed Corn Stover N. 0. VAN GYLSWYK" AND J. J. T. K. VAN DER TOORN Anaerobic Microbiology Division, Laboratory for Molecular and Cell Biology, Onderstepoort 01 10, South Africa

Two strains of sporeforming, xylan-digesting, rod-shaped bacteria were isolated from a lo-* dilution of rumen ingesta taken from two sheep fed a corn stover ration. To determine the source of these strains, we examined the corn stover and isolated two strains of similar bacteria from it. The four strains were remarkably tolerant to oxygen although they did not grow in liquid medium that was shaken while exposed to air. They fermented a wide variety of carbon sources and produced formic, acetic, and lactic acids, ethanol, carbon dioxide, and hydrogen from xylan. The deoxyribonucleic acid base composition was 40 mol% guanine plus cytosine. The name proposed for these strains is Clostridiurn aerotolerans; the type strain is strain XSA62 (ATCC 43524).

Two strains of sporeforming bacteria were found among a in which glucose (5 g/liter) replaced xylan. Spores were large number of bacteria isolated from colonies that pro- stained with malachite green (10). Motility was determined duced clearing zones in xylan (3%) agar medium inoculated after cells were grown overnight on maintenance slants. with a loh8 dilution of rumen ingesta from sheep fed corn Growth in basal medium containing cellobiose (5 g/liter) stover diets (14) as briefly described by van der Toorn and from which reducing agent (cysteine and sulfide) had been vim Gylswyk (15). To discover the origin of these bacteria, deleted was used to determine whether a low redox potential we examined the feed (corn stover) and isolated two strains was required. Tolerance to oxygen was assessed with 5-ml of bacteria with similar characteristics. In this paper, the amounts of basal medium in 50-ml Erlenmeyer flasks characteristics of the four strains are described; it is pro- (loosely stoppered with sterile aluminium caps), from which posed that they can be included in a new species, Clostrid- carbonate and reducing agent had been omitted. Resazurin iurn aerotolerans. (1 mg/liter) replaced indigo carmine (5 mg/liter) in the me- dium which contained clarified rumen fluid (40%), yeast MATERIALS AND METHODS extract (5 ghiter), and glucose (5 g/liter). For each 5 ml of medium, the inoculum was 0.5 ml of the water of syneresis Strains. Strains X8A62 and X4B63 were isolated from the from maintenance slants incubated overnight. In all cases, rumina of two different sheep adapted to corn stover fed ad inocula were examined microscopically for the presence of lib and supplemented with a protein-mineral mixture (14). an abundance of healthy cells. The mixture contained casein (46%), fish meal (41%), and Fermentation acids and alcohols were determined after minerals plus trace elements (12.5%). The corn stover diet growth in basal medium containing 20 g of xylan per liter. was fed (calculated on a daily basis) so that crude protein For acetylmethylcarbinol production, basal medium was formed 13.8% of total intake of solids. Strain X2 was isolated supplemented with cellobiose (5 g/liter) and glucose (10 from 2 g of corn stover (from the same batch as that fed to g/liter), and indigo carmine was deleted. the sheep) added to 200 ml of sterile, anaerobic diluent The medium used for the gelatin liquefaction test con- containing inorganic salts. This mixture was treated with a tained cellobiose (2 g/liter) and 120 g of gelatin per liter. Waring blender run for 3 min at half speed followed by Casein digestion was tested with a similar medium in which treatment with an Ultra-Turrax homogenizer (Janke and gelatin was replaced by 10 g of casein (Koch-Light Labora- Kunkel, Staufen i Br., Federal Republic of Germany) oper- tories Ltd., Colnbrook, England) per liter. Hydrogen sulfide ating at 20,000 rpm for 30 s. Strain X9 was isolated from a production was assessed in medium containing 5 g of separate sample of 4 g of stover treated in the same way. cellobiose and 10 g of peptone (tryptic digest of casein; E. Culture techniques and media. The techniques used and Merck AG, Darmstadt, Federal Republic of Germany) per the composition of basal medium and maintenance medium liter, and sulfide was deleted. Production of indole and were the same as those described before (16) except that a-methylindole was tested in medium containing 5 g of Na2C03(4 g/liter) replaced NaHC03 in these two media as cellobiose and 10 g of Casitone (Difco) per liter. Indigo well as in other media and that the maintenance medium carmine was deleted. For ability to reduce nitrate, cellobiose (used in preparing maintenance slants) contained 5 g of xylan (5 ghiter) and KN03 (1 g/liter, added in filter-sterilized (catalogue no. 95590; Fluka AG Buchs, Switzerland) per solution) were included in basal medium without a reducing liter. Soluble carbohydrate energy sources were included in agent and indigo carmine. Lipase activity was tested in basal sterile media in separate filter-sterilized or (for glucose) medium plus cellobiose (10 g/liter), agar (15 g/liter), and heat-sterilized solutions. tributyrin (Merck; ca. 10 g/liter). The presence or absence of The strains were isolated from basal medium (in roll catalase was assessed with maintenance slants containing 5 g bottles) containing xylan (30 g/liter) and agar (20 g/liter). of cellobiose per liter as the sole added energy source. For Colony morphology was examined after growth in this determining urease activity, cell suspensions were incubated medium but to which was added 5 g of yeast extract (Difco with basal medium to which was added filter-sterilized urea Laboratories, Detroit, Mich.) per liter and also in a medium solution to give a final concentration of 20 g of urea per liter. Basal medium containing 1.4 g of sodium DL-lactate served * Corresponding author. to test for ability to utilize lactate.

102 VOL. 37. 1987 CLOSTRIDIUM AEROTOLERANS SP. NOV. 103

Yeast extract (Difco) and peptone (Difco) at 5 g/liter each caps) free of added reducing agent but exposed to air. In this were incorporated in the following media: (i) medium to test medium growth is observed within 7 or 8 h after inoculation for ability to grow in the absence of carbohydrate in which and can be seen as a turbid layer on the bottom of the flasks clarified rumen fluid was used; and (ii) medium to test for where the medium is reduced with respect to resazurin. ability to grow in the absence of rumen fluid, carbonate, and About 15 min after gentle shaking of the flasks to suspend the C02 (C02 was replaced by N2 in the gas phase), which bacteria, the resazurin is almost completely reduced. On contained 5 g of cellobiose per liter. The effects of peptone standing, the upper layer of medium again becomes at least (10 g/liter) and yeast extract (5 g/liter) as growth stimulants partly oxidized with respect to resazurin, and after incuba- were examined with the inclusion of either or both in media tion for a further period of 16 to 17 h, growth appears as a containing 5 g of cellobiose per liter. For guanine-plus- dense layer on the bottom of the flasks with only the cytosine (G+C) determination, cells were grown in basal uppermost layer of medium showing some color due to medium supplemented with glucose and Difco yeast extract oxidized resazurin. At this stage vigorous shaking is required (5 g of each per liter). to dislodge the bacteria from the bottom of the flask where Media in which lowering of the pH served as a criterion of most growth has occurred. Incubation in a shaking water growth contained 0.1 X the usual concentration of carbonate, bath of inoculated medium prepared in the same way does and the gas phase was COrNTH* in a ratio of approxi- not produce any growth, and the medium is not reduced with mately 10:85:5. Also included were a medium for testing respect to resazurin even after 3 days. tolerance to different temperatures which contained Cells from 16-h cultures on agar slopes containing either cellobiose (5 g/liter), media for testing ability to ferment cellobiose or xylan are gram negative. Very little, if any, different carbohydrate energy sources (5 g of each per liter), stain is adsorbed if crystal violet is the only stain used, and a medium for assessing the types of gases produced Vegetative cells are straight rods often with somewhat during the fermentation of xylan (5 g/liter). In the last case, pointed ends (Fig. 1A and B). They usually occur singly. the CO2 content of the gas phase had to be low to facilitate Their width varies from 0.4 to 0.6 pm, and their length is the determination of C02produced during fermentation. The about 1.5 to 3.0 pm and sometimes more. Cells are motile by incubation temperature was 38 to 39°C except where indi- peritrichous flagella (Fig. 1B). Spores stain green with mal- cated, and the initial pH of the media varied from 6.6 to 6.9. achite green. They are terminal and distend the cells where Identification tests. Formic, acetic, D- and L-lactic and they are formed (Fig. 1C). They are slightly oval and succinic acids, and ethanol were determined by enzymatic measure about 0.6 by 0.8 pm. methods (I). Gas-liquid chromatographic techniques were After incubation for 3 days on films of glucose (0.5%) agar employed to determine other alcohols, volatile acids, and medium in roll bottles, surface colonies are round with gases, Tests for gelatin liquefaction, casein digestion, cata- smooth or almost smooth edges. They are convex and lase, amylase, urease, nitrate reduction, and production of appear translucent at the edges to semitranslucent and light acetylmethylcarbinol, indole, and methylindole were done brown at the center. Colonies are up to 2.5 mm wide. by the method of Holdeman et al. (3). Hydrogen sulfide Submerged colonies are lens shaped and appear brown when production was tested with lead acetate-impregnated paper viewed from the side. Surface colonies in xylan (3%) agar strips (2), and lipase activity was determined by the forma- medium in roll bottles appear similar to those that develop tion of clearing zones in agar medium containing tributyrin on glucose medium and are surrounded by a clear zone (1 to (5). Utilization of lactate was assessed by comparing the 3 cm in diameter) due to the dissolution of xylan. In addition, concentration of lactate (determined enzymically) in inocu- there are large spreading colonies with lobate edges and thin lated and uninoculated media after incubation. translucent growth. Dissolution of xylan extends to several To determine the G+C content, cells grown for 16 h were millimeters beyond the outer periphery of these colonies. harvested by centrifugation and lysed with sodium dodecyl Submerged, lens-shaped colonies give rise to clearing zones sulfate (7). Deoxyribonucleic acid (DNA) was isolated and of 1 cm or more in diameter. purified (8), and the G+C content was estimated from the The four strains are saccharoclastic and show little, if any, ratio of absorbance at 245 nm to absorbance at 270 nm (13). growth in media containing peptone and yeast extract but DNAs from Clostridiurn perfringens, Escherichia coli, without added carbohydrate. Peptone and yeast extract can Micrococcus luteus (‘ ‘Micrococcus lysodeikticus”), calf thy- replace rumen fluid in medium containing carbohydrate. mus, and salmon sperm (all from Sigma Chemical Co., St. Media containing rumen fluid as the sole source of growth Louis, Mo.) served as calibration standards. factors support moderate growth. For two strains tested (X8A62 and X2) absorbance at 578 nm (1-cm path length) of RESULTS AND DISCUSSION medium containing clarified rumen fluid and cellobiose in- creases by about 0.3 U in 16 h. If this medium is supple- The four strains studied are motile, straight rods that mented with peptone (10 g/liter), the absorbance increases to produce single, terminal endospores which distend the cells; about 0.6 U over the same period. If the medium is further they do not grow in well-aerated liquid culture and do not supplemented with yeast extract (5 g/liter), the absorbance reduce sulfate to hydrogen sulfide. They are therefore mem- reached after 16 h is about 0.9 U. The last value is also bers of the genus Clostridium Prazmowski (12). Their char- obtained when the medium is enriched with only the yeast acteristics do not conform with those of any previously extract (without the peptone). Incubation for longer periods described species. does not yield appreciably higher absorbance readings. A Clostridium aerotolerans sp. nov. Clostridium aerotolerans medium containing (apart from minerals and Na2C03)only (ae.ro.to’ 1e.rans Gr. masc. n. aer, air, gas; L. v. tolerare, to glucose (5 ghiter), yeast extract (5 ghiter), cysteine endure, to put up with; L. neut. part. adj. aerotolerans, hydrochloride . H20 (0.125 g/liter), and Na2S 9H20(0.125 air-enduring). Cells grow under strictly anaerobic conditions g/liter) gives a maximum absorbance of about 1.8 U. This and also in agar or liquid medium free of added reducing medium was used to assess the approximate optimum agent. They also grow in a shallow layer of liquid medium (5 growth temperature of strain X8A62. The maximum absor- ml in 50-ml Erlenmeyer flasks loosely covered with sterile bance (between 1.4 and 1.8 U) is attained after about 24 h at 104 VAN GYLSWYK AND VAN DER TOORN INT. J. SYST.BACTERIOL.

FIG. 1. Cells of C. aerotolerans X8A62T grown for 17 h on a cellobiose agar slant. Bar, 10 km. (B) Transmission electron micrograph of cells and flagella of strain X8A62T grown for 17 h on an agar slant containing xylan and yeast extract. Bar, 1 p,m. (C) Scanning electron micrograph of sporulating cells of strain X8A62T grown for 20 h on an agar slant containing glucose and cellobiose. Bar, 1 pm.

38T, 30 h at 30"C, and 60 h at 22°C. After 60 h at 15"C, aerotolerant sporeformers that will not grow in media in absorbance is about 0.1 U. Growth does not occur at 45°C. which equilibration with air is maintained (3,6, 11). It can be A wide range of carbohydrate energy sources is fermented distinguished from C. tertium, which produces butyric acid (Table 1) with the final pH not falling below 5.2. Xylan is but not ethanol, ferments mannitol, and does not ferment fermented to formic, acetic, and lactic acids, ethanol, hydro- arabinose (3); C. carnis, which produces butyric acid but not gen, and carbon dioxide (Table 2). Acetylmethylcarbinol is ethanol and does not ferment arabinose, melibiose, raffinose, not produced. or xylose (3); C. dururn, which does not ferment arabinose, None of the four strains liquefies gelatin or digests casein, cellobiose, lactose, melibiose, starch, or xylose (3, 11); C. and no H2S is formed from peptone or from sulfate reduc- histolyticum, which digests gelatin and ferments no carbo- tion. Neither indole nor a-methylindole is produced. There hydrates tested (3); and Sporolactobacillus inulinus, which is no indication that the bacteria possess urease, catalase, or produces only lactic acid and ferments mannitol and inulin enzymes for reducing nitrate. Some lipase activity was (6). exhibited by all four strains. C. aerotolerans does not appear to be an aerotolerant The G + C content of the DNA is about 40 mol% for all four variation of mesophilic clostridia producing formic, acetic, strains. All nine values fell between 39 and 41 mol%. The and lactic acids, ethanol, and hydrogen and having a G+C type strain is X8A62 (ATCC 43524), isolated from the rumen content of 40 to 41 mol% for DNA. It can be distinguished of a sheep. from C. cellulolyticum, which differs in Gram reaction and C. aerotolerans differs from other Clostridium spp. and cell dimensions and ferments cellulose but not lactose,

TABLE 1. Acid production from different carbon energy TABLE 2. End products of xylan fermentation by sources by C. aerotolerans isolated from corn stover C. aerotolerans in a medium containing rumen fluid" and from the rumina of sheep fed corn stoveP Amtb produced by strain: Reactionb of strain: End product Carbon X8A62T X4B63 x2 x9 energy source ~ X8A62T X4B63 x2 x9 Formic acid 0.8' 0.7 0.7 0.9 L-A.rabinose + W + + Acetic acid 1.7 1.5 2.1 2.4 Dextrin W W W W L-Lactic acid 0.4 0.1 0.3 0.1 Lactose + W + + D-Lactic acid 0.3 0.1 0.3 0.1 L-Rhamnose + + + W Ethanol 2.2 1.9 1.7 1.9 Ribose + + W - Trehalose + - W - Hydrogen 10.4 12.6 20.2 10.3 ~~ Carbon dioxide' 6.2 7.8 9.5 8.0 ' All strains ferment cellobiose, fructose, galactose, glucose, maltose, mannose, melibiose, pectin, raffinose, salicin, starch (no residual starch was a None of the strains produce or utilize significant amounts of propionic, detected after incubation), sucrose, xylan, and xylose, the pH being lowered butyric, isobutyric, 2-methylbutyric, valeric, isovaleric, n-caproic, or succinic by 0.8 or more. Esculin is fermented weakly by all four strains. None of the acids nor did they produce significant amounts of alcohols other than ethanol. strains ferments cellulose (ball-milled filter paper [Whatman no. l]),glycerol, Values are millimoles per 100 ml of medium for the acids and ethanol and inositol, inulin, mannitol, or sorbitol or utilizes DL-lactic acid (residual lactic represent differences between inoculated and uninoculated medium; the acid was determined). values for Hz and COz are given as percent (voVvol) in the gas phase. + ,pH lowered by 0.8 or more; w, pH lowered by between 0.3 and 0.8; -, ' Poorly buffered medium was used with 10% C02 in the gas phase to pH unchanged or lowered by less than 0.3. facilitate the determination of C02 produced. VOL. 37, 1987 CLOSTRIDIUM AEROTOLERANS SP. NOV. 105 maltose, raffinose, rhamnose, salicin, or sucrose (9); and C. Coprococcus,twelve new species, and emended descriptions of nexile, which is nonmotile and does not ferment arabinose, four previously described species of bacteria from human feces. cellobiose, maltose, mannose, or rhamnose (4). Int. J. Syst. Bacteriol. 24:260-277. Corn stover has a high xylan content (about 30%) which is 5. Holding, A. J., and J. G. Collee. 1971. Routine biochemical 32%) (14). tests. Methods Microbiol. 6A:1-32. only slightly exceeded by that of cellulose (about 6. Kitahara, K. 1974. Genus Sporolactobacillus, p. 550-551. In Soil is probably the normal habitat of C. aerotolerans, which R. E. Buchanan and N. E. Gibbons (ed.), Bergey’s manual of could contribute to the degradation of the xylan-containing determinative bacteriology, 8th ed. The Williams & Wilkins components of corn plant and other plant residues in soil, Co., Baltimore. particularly in the presence of growth factors that could be 7. Marmur, J. 1961. A procedure for the isolation of deoxyribo- supplied by manure. The ease with which this bacterium nucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. creates an anaerobic environment may aid it to adapt swiftly 8. Meyer, S. A., and K. H. Schleifer. 1975. Rapid procedure for the to changing conditions such as may occur in cultivated soils. approximate determination of the deoxyribonucleic acid base Its ability to reach high numbers in the rumen may, at least composition of micrococci, staphylococci, and other bacteria. Int. J. Syst. Bacteriol. 25383-385. partly, be related to the fact that it grows best at a temper- 9. Petitdemange, E., F. Caillet, J. Giallo, and C. Gaudin. 1984. ature close to that of the rumen. Clostridium cellulolyticurn sp. nov., a cellulolytic, mesophilic species from decayed grass. Int. J. Syst. Bacteriol. 34:155-159. 10. Schaeffer, A. B., and McD. Fulton. 1933. A simplified method of ACKNOWLEDGMENTS staining endospores. Science 77:194. 11. Smith, L. D. S., and E. P. Cato. 1974. Clostridium durum sp. We thank the Director of the Veterinary Research Institute, nov., the predominant organism in a sediment core from the Onderstepoort, for facilities which made this work possible, and Black Sea. Can. J. Microbiol. 20:1393-1397. also the following workers of this institute who generously gave of 12. Smith, L. D. S., and G. Hobbs. 1974. Genus Clostridium, p. their time: A. M. Spickett (scanning electron microscopy), T. Phil- 551-572. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey’s lips (transmission electron microscopy), and J. J. Paulsen (light manual of determinative bacteriology, 8th ed. The Williams & photography). We are also indebted to Denise N. Barry of our Wilkins Co., Baltimore. laboratory for expert technical assistance. 13. Ulitzur, S. 1972. Rapid determination of DNA base composition by ultraviolet spectroscopy. Biochim. Biophys. Acta 272:l-11. LITERATURE CITED 14. van der Linden, Y., N. 0. van Gylswyk, and H. M. Schwartz. Anonymous. 1980. Methods of enzymatic food analysis, Boehr- 1984. Influence of supplementation of corn stover with corn inger-Mannheim, Mannheim, Federal Republic of Germany. grain on the fibrolytic bacteria in the rumen of sheep and their Conn, H. J. 1951. Routine tests for the descriptive chart. relations to the intake and digestion of fiber. J. Anim. Sci. Morphological and biochemical. Leaflet V. In Committee of 59:772-783. Bacteriological Technic of the Society of American Bacteriolo- 15. van der Toorn, J. J. T. K., and N. 0. van Gylswyk. 1985. gists (ed.), Manual of methods for pure culture study of bacte- Xylan-digesting bacteria from the rumen of sheep fed maize ria, 12th ed. Biotech Publications, New York. straw diets. J. Gen. Microbiol. 131:2601-2607. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977. 16. van Gylswyk, N. O., and J. J. T. K. van der Toorn. 1985. Anaerobe laboratory manual, 4th ed. Virginia Polytechnic In- Eubacterium uniforme sp. nov. and Eubacterium xylanophilum stitute and State University, Blacksburg. sp. nov., fiber-digesting bacteria from the rumina of sheep fed Holdeman, L. V., and W. E. C. Moore. 1974. New genus, corn stover. Int. J. Syst. Bacteriol. 35323-326.