INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1990, p. 297-301 Vol. 40, No. 3 0020-7713/9O/030297-05$02.OO/O Copyright 0 1990, International Union of Microbiological Societies Amphibacillus xylanus gen. nov. , sp. nov. , a Facultatively Anaerobic Sporeforming Xylan-Digesting Bacterium Which Lacks Cytochrome, Quinone, and Catalase YOUICHI NIIMURA,l* ENKI KOH,l FUJITOSHI YANAGIDA,l KEN-ICHIRO SUZUKI,, KAZUO KOMAGATA,3 AND MICHIO KOZAKI' Tokyo University of Agriculture, Setagaya-ku, Tokyo 1.56,' Japan Collection of Microorganisms, RIKEN, Wako-shi, Saitama 351 -01 ,2 and Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113,3 Japan Three strains of gram-positive, facultatively anaerobic, sporeforming, rod-shaped bacteria were isolated from composts of manure with grass and rice straw. These organisms grew well in an alkaline medium and digested xylan both in strictly anaerobic cultures when titanium(III) citrate was used as a reducing agent and in aerobic cultures with shaking. The cells contained meso-diaminopimelic acid, and their cellular fatty acids consisted of iso- and anteiso-branched acids and considerable amounts of straight-chain acids. The DNA base composition of these strains ranged from 36 to 38 mol% guanine plus cytosine. Cytochromes, isoprenoid quinones, and catalase activity were not detected. DNA-DNA homology determinations did not show relatedness to strains of representative species of the genera Bacillus, Clostridium, and Sporolactobacillus. Considering the uniqueness of the characteristics, the sequence of the 5s rRNA, and the unique metabolic pathways, we propose Amphibacillus xylanus gen. nov., sp. nov., for these strains. The type strain is strain EpOl (= JCM 7361). Previously, Niimura et al. reported the isolation from an 10.0 with a 10% Na,CO, solution. Cultivation was carried alkaline compost of manure with grass and rice straw of a out at 393°C. For aerobic cultivation, a cotton-plugged facultatively anaerobic, sporeforming, xylan-digesting bac- culture flask containing medium (20% of the volume of the terium (strain EpOIT [T = type strain]) that lacks cy- flask) was shaken vigorously. For anaerobic cultivation, 0.13 tochromes, quinones, and catalase (10). This strain grew mM titanium(II1) citrate (19) was added to medium prepared well in xylan medium and digested xylan both under strictly anaerobically (10). The E, of the medium was decreased by anaerobic conditions and under aerobic conditions with adding titanium(II1) citrate, which produced values less than shaking. -300 mV (pH 10.0). Sporulation was determined by using In this work, we included two new isolates similar to strain cells cultivated under aerobic or anaerobic conditions by EpOIT in a taxonomic study. Our data revealed that the three phase-contrast microscopy and by heating culture broth at strains are members of an independent taxon that is different 80°C for 10 min. Utilization of sugars was determined by from members of the genera Bacillus, Clostridium, and measuring the turbidity and pH of the broth after 4 days of Sporolactobacillus with respect to phenotypic and chemo- incubation in glucose medium (see above) in which glucose taxonomic characteristics. Consequently, we propose a new was replaced with other carbohydrates and also in basal genus, Amphibacillus, and a new species, Amphibacillus medium (3). Turbidity was determined by using naked eye xylanus, for these organisms. observations, and pH was determined with a pH meter (Horiba Seisakusho, Tokyo, Japan), Nitrate reduction, H,S MATERIALS AND METHODS production, indole production, citrate utilization, and hy- drolysis of gelatin were determined as described by Holde- Strains and isolation. We examined three strains of a man et al. (3) and Komagata (6); the pH of the media for all facultatively anaerobic xylan-utilizing bacterium, strains of these tests was adjusted to 10.0 with a 10% Na,CO, EpOIT, SinIV, and 15abb. Strains SinIV and 15abb were solution. newly isolated from different composts of manure with grass and rice straw by using the same enrichment culture tech- nique that was used previously (10). TABLE 1. Effects of oxygen on the growth of A. xylanus strains Identification methods. Cell morphology, Gram reaction, in glucose medium motility, and flagella were determined by using cells grown CL-~ (h-9" Cell yield on glucose medium which contained (per liter) 10 g of Strain Anaerobic Aerobic Anaerobic Aerobic glucose, 1 g of K,HPO,, 2 g of NH,NO,, 200 mg of growth' growthd growth growth MgS04.7H,0, 5 mg of MnSO4.7H,O, 5 mg of ~ ~~ ~~ ~~ FeSO, - 7H,O, 100 mg of CaC1, . 2H20, 3 g of yeast extract EpOIT 0.82 0.70 0.65 0.72 (Difco Laboratories, Detroit, Mich.), 300 mg of Polypeptone SinIV 0.68 0.97 0.73 0.78 (Daigo Eiyo Chemical Co., Tokyo, Japan), and 1 mg of 15abb 0.56 0.26 0.58 0.43 resazurin. All of the media used for cultivation and biochem- " kmanwas measured at pH 9.5 (start). ical and physiological characterizations were adjusted to pH Cell yield was measured at pH 10.0 (start). Strictly anaerobic cultures in which titanium (111) citrate was used as a reducing agent. * Corresponding author. Aerobic cultures with shaking. 297 298 NIIMURA ET AL. INT.J. SYST.BACTERIOL. TABLE 2. Products of xylan fermentation by A. xylanus strainsa Concn of the following products Strain Culture (mmol/liter): Ethanol Acetic acid Formic acid EpOIT Anaerobicb 22 22 46 Aerobic" 35 SinIV Anaerobic 26 25 57 Aerobic 22 15abb Anaerobic 28 32 67 Aerobic 35 a The medium contained 1%xylan as a carbon source. StrictIy anaerobic culture in which titanium (111) citrate was used as a reducing agent. Aerobic culture with shaking. Quinone systems were determined as described by Koma- gata and Suzuki (7). Amino acids in the cell wall peptidoglycan were analyzed with a model 835 automatic amino acid analyzer (Hitachi, Ltd., Tokyo, Japan) (7). The isomers of diaminopimelic acid were determined by thin-layer chromatography, using the modified method (7) of Staneck and Roberts (14). Cellular fatty acid composition was determined by gas FIG. 1. Phase-contrast photomicrograph of A. xylanus EpOIT chromatography as described by Suzuki and Komagata (15). cells grown on glucose agar at 39.5"C. Bar * = 5 pm. The arrow indicates an endospore. DNA base composition and DNA-DNA hybridization. DNA base composition was determined by the thermal melting point method as described previously (10). The levels of The fermentation products from xylan were analyzed by DNA-DNA relatedness with representatives of the genera gas chromatography and high-performance liquid chroma- Bacillus, Clostridium, and Sporolactobacillus were deter- tography (10). The xylan was prepared from oat spelt (Ald- mined by using the sodium dodecyl sulfate membrane filter rich Chemical Co., Milwaukee, Wis.). method (4, 5). Strains of the following species were used in Cytochrome systems were determined by spectropho- the homology experiments: Bacillus alcalophilus, Bacillus tometry as described previously (10). cereus, Bacillus lentus, Bacillus megaterium, Bacillus sub- FIG. 2. Electron micrograph of A. xylanus 15abb. The cell was negatively stained with phosphotungstic acid. Bar = 0.5 pm. VOL. 40, 1990 AMPHIBACILLUS XYLANUS GEN. NOV., SP. NOV. 299 TABLE 3. Cellular fatty acid compositions of A. xylanus strains Fatty acid composition (% of total) Strain Culture Straight-chain acids Iso-branched acids Anteiso-branched acids c14:0 c16:0 c14:o c16:0 c15:o c17:0 c15:o c17:0 EpOIT Anaerobic" 11 27 8 17 10 1 19 5 Aerobicb 11 20 11 19 10 1 21 4 SinIV Anaerobic 12 24 7 11 10 1 28 5 Aerobic 8 14 6 7 12 1 38 4 15abb Anaerobic 10 36 6 15 8 1 15 5 Aerobic 5 21 9 22 10 1 22 6 a Strictly anaerobic culture in which titanium(II1) citrate was used as a reducing agent. Aerobic culture with shaking. tilis, Clostridiurn arninovalericum, Clostridiurn butyricum, These results suggest that our isolates differ from mem- Clostridium mangenotii, Clostridium thermocellurn, and bers of the genera Bacillus, Clostridium, and Sporolactoba- Sporolactobacillus inulinus (see Table 4 for strain designa- cillus (Table 5). Furthermore, these three isolates were not tions). identified as any previously described species by using the data in references 2 and 13. Thus, we propose a new genus RESULTS AND DISCUSSION and new species for these isolates, Amphibacillus xylanus. Description of Amphibacillus gen. nov. Amphibacillus Strains EpOIT, SinIV, and 15abb grew well under anaer- (Am. phi. ba. cil' lus. Gr. pref. amphi, both sides or double; obic and aerobic conditions (Table 1) and formed heat- L. dim. n. bacillus, a small rod; M. L. masc. n. Amphiba- resistant spores under both aerobic and anaerobic conditions cillus, rod capable of both aerobic and anaerobic growth). (Fig. 1). Spore formation under both conditions differenti- Cells are rods that are 0.3 to 0.5 km in diameter and 0.9 to ated these organisms from species of Bacillus, Clostridium, 1.9 pm long. Gram positive in the very early stages of growth and Sporolactobacillus (13). Strains SinIV and 15abb were and loosely gram positive in stationary growth phase. Heat- motile by means of peritrichous flagella (Fig. 2), and strain resistant, oval endospores are formed, but the sporangia are EpOIT was nonmotile. These three strains produced ethanol, rapidly lysed and the spores are liberated. Spores are formed acetic acid, and formic acid from xylan under anaerobic under both aerobic and anaerobic conditions. Grows well conditions and acetic acid under aerobic conditions (Table both in well-aerated cultures and in aerobic cultures (Eh, 2). -370 mV; pH 10) when titanium(II1) citrate is used as a Catalase and oxidase were not produced, and the isolates reducing agent. Does not grow in nutrient broth. Chemoor- had neither cytochromes a, 6, c, or d nor any respiratory ganotrophic. Produces ethanol, acetic acid, and formic acid isoprenoid quinones, as reported previously (10).