J. Gen. Appl. Microbiol., 48, 269–279 (2002) Full Paper Paraliobacillus ryukyuensis gen. nov., sp. nov., a new Gram-positive, slightly halophilic, extremely halotolerant, facultative anaerobe isolated from a decomposing marine alga Morio Ishikawa,* Shihomi Ishizaki, Yasushi Yamamoto, and Kazuhide Yamasato Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156–8502, Japan (Received August 12, 2002; Accepted September 24, 2002) A slightly halophilic, extremely halotolerant, alkaliphilic, and facultatively anaerobic rod bac- terium was isolated from a decomposing marine alga collected in Okinawa, Japan. The isolate, designated O15-7T, was Gram-positive, endospore-forming, catalase-positive, menaquinone-7- possessing bacterium that is motile by peritrichous flagella. The isolate was an inhabitant of ma- rine environments; the optimum NaCl concentration for growth was 0.75–3.0% (w/v) with a range of 0–22.0%, and the optimum pH was 7.0–8.5 with a range of 5.5–9.5. Catalase was produced in aerobic cultivation but not in anaerobic cultivation. Carbohydrate, sugar alcohol or a related car- bon compound was required for growth. In aerobic cultivation, the isolate produced pyruvate, acetate and CO2 from glucose, and in anaerobic cultivation, it produced lactate, formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1 for the last three products. No gas was produced anaerobically. Lactate yield per consumed glucose was markedly affected by the pH of the fermentation medium: 51% at pH 6.5 and 8% at pH 9.0. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Phylogenetically, the isolate occupied an independent lineage within the group composed of the halophilic/halotolerant/alkaliphilic and/or alkalitolerant species in Bacillus rRNA group 1 with the highest 16S rRNA gene sequence similarity of 95.2% to the genus Gracilibacillus. For this isolate, Paraliobacillus ryukyuensis gen. nov., sp. nov. was proposed. mol%), has been deposited in the DSMZ, IAM, NBRC, and 35.6؍The type strain, O15-7T (G؉C .(NRIC 0520T؍NBRC 10001T؍IAM 15001T؍NRIC (DSM 15140T Key Words——alkaliphile; extremely halotolerant; facultative anaerobe; halophile; lactic acid producing bacterium; marine bacteria; Paraliobacillus ryukyuensis gen. nov., sp. nov. Introduction have halophilic/halotolerant/alkaliphilic and/or alkalitol- erant properties compose a considerably large phylo- Isolation and taxonomic and phylogenetic studies genetic group within rRNA 1 group of Ash et al. (1991) performed in this decade have revealed that most in the phyletic assemblage of bacteria classically de- Gram-positive, aerobic spore-forming bacteria which fined as the genus Bacillus (Ash et al., 1991; Garabito et al., 1997; Heyndrickx et al., 1999; Lu et al., 2001; Wainø et al., 1999; Zhilina et al., 2001). They have * Address reprint requests to: Dr. Morio Ishikawa, Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo been isolated mostly from saline or hypersaline envi- University of Agriculture, 1–1 Sakuragaoka 1-chome, Setagaya- ronments such as saltern, salt lake, soda lake and ku, Tokyo 156–8502, Japan. seawater, and several genera including Halobacillus, E-mail: [email protected] Gracilibacillus, Oceanobacillus, Amphibacillus and 270 ISHIKAWA et al. Vol. 48 Marinococcus have been described (Hao et al., 1984; (Wako Pure Chemical Industries), 5 g; soy sauce, 50 Lu et al., 2001; Spring et al., 1996; Wainø et al., 1999; ml; K2HPO4, 10 g; NaCl, 60/170 g; sodium thioglyco- Zhilina et al., 2001). late, 1 g; salt solution (MgSO4 ·7H2O, 40 mg; MnSO4 · Ϫ1 Littoral environments harbor a large number of vari- 4H2O, 2 mg; and FeSO4 ·7H2O, 2 mgml ), 5 ml; and ous kinds of marine plants and animals. Decomposing cycloheximide, 10 mg. The medium was adjusted to dead organisms and intestinal contents of living organ- pH 7.5 and autoclaved at 110°C for 10 min. A small isms in these environments are rich sources of organic piece of a decomposing alga was placed in 7% NaCl nutrients that support life for heterotrophic microorgan- GYPFSK isolation broth and incubated for 21 days for isms. These environments provide favorable niches for the first enrichment followed by the second enrichment facultatively anaerobic bacteria, which generally re- in 18% NaCl GYPFSK isolation broth for 15 days at quire complex nutrients for growth. Those bacteria are 25°C in static culture. Enrichment culture was pour- thought to have characteristic properties that reflect plated with 12% NaCl GYPFSK isolation agar (2.0% Ϫ1 the physicochemical nature of seawater [total salt con- agar) supplemented with 5 g L CaCO3. centration: 3.2–3.8% (w/v), pH 8.2–8.3 (surface)]. Media and growth conditions. For the cultivation These environments are considered to be good and taxonomic characterization, 2.5% NaCl GYPF sources of those bacteria in reference to the diversity broth (pH 8.5) was used as the basal medium, which of microorganisms. While Gram-positive, aerobic bac- was composed of the following (per 1,000 ml): glu- teria have been isolated from coastal seawater and cose, 10 g; yeast extract, 5 g; Polypepton, 5 g; Extract sediments (Bonde, 1981; Hao et al., 1984; Rüger and Bonito, 5 g; K2HPO4, 1 g; NaCl, 25 g; sodium thioglyco- Richter, 1979; Schlesner et al., 2001; Ventosa et al., late, 1 g; and salt solution, 5 ml. The medium was ster- 1989), Gram-positive, facultatively anaerobic bacteria ilized by filtration. In some experiments, a 2.5% NaCl from littoral environments have been much less stud- GYPFK broth was used, which was prepared by in- ied taxonomically. creasing the concentration of K2HPO4 in a 2.5% NaCl In the isolation and taxonomic studies of Gram-posi- GYPF broth to 1%. The media without sodium thiogly- tive bacteria that inhabit marine environments and pro- colate were used for aerobic cultivation. Cultivation duce lactic acid, a facultatively anaerobic rod-shaped was performed at 30°C. bacterium belonging to the halophilic/halotolerant/alka- Morphological, physiological and biochemical fea- liphilic and/or alkalitolerant group (henceforth referred tures. Gram staining was conducted using crystal vi- to as HA group in this paper) was isolated from a de- olet-phenol solution (Merck) following the procedure as composing alga. In the present paper, we describe the described elsewhere (Gerhardt et al., 1994). Flagella- taxonomic characterization of the isolate, for which the tion was studied for cells grown on 2.5% NaCl GYPFK name Paraliobacillus ryukyuensis gen. nov., sp. nov. agar at 20°C for 2 days using the staining method of has been proposed. Nishizawa and Sugawara as described (Alumni Asso- ciation of The Institute of Medical Sciences, The Uni- Materials and Methods versity of Tokyo, 1988). Spore formation was observed microscopically for cultures grown at 37°C on yeast Isolation of strain. Experiments to isolate lactic extract salts agar (pH 7.5) which had the following acid-producing bacteria were conducted using marine composition (per 1,000 ml): yeast extract, 5 g; NaCl, materials (living or decomposing algae, fish, sponges, 20 g; MgSO4, 5 g; CaCl2, 2 g; K2HPO4, 0.1 g; salt solu- and shellfish) collected from Okinawa Prefecture, a tion, 10 ml; and agar, 15 g and Marine agar (Difco). For subtropical area of Japan, in September 1998. Strain the catalase test, the evolution of bubbles upon addi- T O15-7 was obtained from a decomposing alga, taken tion of 3% H2O2 solution to aerobically or anaerobically at a foreshore site near the Oujima islet adjacent to the grown cells was observed under a stereoscopic micro- main island of Okinawa, by enrichment culture using scope. The utilization of carbohydrates and related 7% and 18% (for a subsequent second enrichment) compounds was observed by incubating in 2.5% NaCl NaCl GYPFSK isolation broths. The compositions of GYPF broth without glucose as the basal medium, to these media were as follows (per 1,000 ml): glucose, which substrates were added at 1.0% (w/v). The test 10 g; yeast extract, 5 g; Polypepton (Wako Pure Chem- media were sterilized by filtration except for starch and ical Industries, Osaka, Japan), 5 g; Extract Bonito inulin-added media which were sterilized by autoclav- 2002 Paraliobacillus ryukyuensis gen. nov., sp. nov. 271 ing at 121°C for 15 min. The utilization of ethanol and from glucose were analyzed by HPLC. organic acids other than gluconic acid was examined Chemotaxonomic characteristics and DNA base in aerobic incubation with shaking. Growth was evalu- composition. For the analysis of isoprenoid quinones, ated based on both the amount of acid produced and cells grown aerobically or anaerobically were freeze- the turbidity estimated based on absorbance at dried and subjected to extraction of the lipid fraction as 660 nm. Other biochemical tests were conducted fol- described earlier (Yamada, 1987; Yamada and Ku- lowing the methods described previously (Gerhardt et raishi, 1982). Molecular species was determined by al., 1994; Okada et al., 1992), with modifications as HPLC (Yamada, 1987). The presence of meso-di- follows: supplementation with 2.5% NaCl and adjust- aminopimelic acid in cell-wall peptidoglycan was deter- ment of pH to 8.5, or the use of 2.5% NaCl GYPF mined by TLC as described elsewhere (Hasegawa et broth without glucose as the basal medium. al., 1983; Okada et al., 1992). DNA was extracted and Growth characteristics in relation to NaCl concentra- purified as previously described (Marmur, 1961; Saito tion, pH and temperature. The optimum NaCl con- and Miura, 1963). DNA base composition was deter- centration, pH and temperature and their ranges for mined by reverse-phase HPLC (Tamaoka and Koma- bacterial growth were investigated with 2.5% NaCl gata, 1984). The isomeric form of lactate produced GYPF broth as the basal medium in static culture. For was determined enzymatically using D- and L-lactic pH studies, Good’s buffers (MES, MOPS, HEPES, acid dehydrogenases (R-Biopharm, Darmstadt, Ger- EPPS, CHES, and CAPSO) were added at 100–200 many).