International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1681–1685 DOI: 10.1099/ijs.0.02169-0 Alicyclobacillus acidiphilus sp. nov., a novel NOTE thermo-acidophilic, ω-alicyclic fatty acid- containing bacterium isolated from acidic beverages 1 R&D Laboratory, KIRIN Hiroshige Matsubara,1 Keiichi Goto,2 Terumi Matsumura,1 Beverage Corporation, 2 1 1 Kurami, Samukawa, Koza, Kaoru Mochida, Masaharu Iwaki, Motohiro Niwa Kanagawa 253-0101, Japan and Kazuhide Yamasato3 2 Food Functions R&D Section, Food Research Laboratories, Mitsui Norin Author for correspondence: Hiroshige Matsubara. Tel: j81 467 75 6166. Fax: j81 467 75 8158. Co., Ltd, Miyabara, Fujieda, e-mail: HiroshigeIMatubara!beverage.co.jp Shizuoka 426-0133, Japan 3 Department of A novel thermo-acidophilic bacterium was isolated from an acidic beverage Fermentation Science, Faculty of Applied that had the odour of guaiacol. The cells are aerobic, Gram-positive, spore- Bioscience, Tokyo forming rods. The organism, strain TA-67T, grows at temperatures from 20 to University of Agriculture, 55 SC (optimum, 50 SC) and at pH values from 25to55 (optimum, pH 30). It Sakuragaoka, Setagaya, Tokyo 156-8502, Japan possesses ω-cyclohexane fatty acid as a major cellular fatty acid. The GMC content of the DNA is 541 mol%. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strain TA-67T constituted a distinct lineage in the Alicyclobacillus cluster, with Alicyclobacillus acidoterrestris as the closest neighbour (966% homology). Phenotypically, it is similar to, but can be distinguished from, ω-cyclohexane fatty acid-possessing alicyclobacilli (A. acidoterrestris, Alicyclobacillus acidocaldarius, Alicyclobacillus hesperidum and ‘Alicyclobacillus mali’) by the morphology of spores and sporangia, by the growth response to different temperatures, and by the profiles for acid production from carbon sources. It is the alicyclobacillus that produces guaiacol, a causative substance for an ‘off’ flavour of orange juice. On the basis of the phenotypic and phylogenetic evidence, it is concluded that strain TA-67T represents a new species of the genus Alicyclobacillus, for which the name Alicyclobacillus acidiphilus is proposed. The type strain is TA-67T (l DSM 14558T l IAM 14935T l NRIC 6496T). Keywords: Alicyclobacillus acidiphilus sp. nov., ω-cyclohexane fatty acid, thermo- acidophile The genus Alicyclobacillus is characterized by endo- 1987b), were reclassified in a new genus, i.e. Alicyclo- spore-formation, a thermo-acidophilic nature, and the bacillus (Wisotzkey et al., 1992). Recently, Alicyclo- possession of ω-alicyclic fatty acid as a major cellular bacillus hesperidum (Albuquerque et al., 2000), ‘Ali- fatty acid (Hippchen et al., 1981; Poralla & Konig, cyclobacillus mali’ and ‘Alicyclobacillus acidocaldarius 1983). Those organisms had been assigned to the genus subsp. rittmannii’ (Nicolaus et al., 1998), which possess Bacillus for two decades. In 1992, on the basis of ω-cyclohexane fatty acid, and Alicyclobacillus her- phylogenetic distinctness revealed by comparative 16S barius (Goto et al., 2002), which possesses ω-cyclohep- rRNA gene sequence analysis and the possession of ω- tane fatty acid, have been reported. In the preceding alicyclic fatty acid, three thermo-acidophilic species, studies, strains of the genus Alicyclobacillus had been Bacillus acidocaldarius (Darland & Brock, 1971; Rosa isolated from geothermal sources, and, subsequently, et al., 1971), Bacillus acidoterrestris (Deinhard et al., also from non-geothermal soils. The isolation of 1987a), Bacillus cycloheptanicus (Deinhard et al., thermo-acidophiles from spoiled apple juice was re- ................................................................................................................................................. ported by Cerny et al. (1984); these were later The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene recognized as Alicyclobacillus acidoterrestris. Since sequence of Alicyclobacillus acidiphilus sp. nov. TA-67T is AB076660. then, the occurrence of this species, which is the 02169 # 2002 IUMS Printed in Great Britain 1681 H. Matsubara and others causative organism of an ‘off’ flavour, in spoiled fruit carbon compounds) by 0n4 or more were scored as juices, has been reported in the US and Japan (Jensen, positive. Growth responses to temperature and pH 1999; Pettipher et al., 1997; Splittstoesser et al., 1994; were examined by measuring the turbidity (at 578 nm) Yamazaki et al., 1996). In the hot summer of 1994, an of cultures. The pH range and the optimum pH for outbreak of spoilage of fruit-juice products caused by growth were determined at optimum temperature for thermo-acidophiles occurred in Europe. A microbio- each strain. For guaiacol measurement, culture super- logical survey of orange trees and garden soil con- natants were filtered through Sep-Pak Plus tC18 ducted in Brazil, a raw-material-exporting country, (Waters) and a dehydration column. GC\MS was showed that the primary niche of these micro-organ- performed on a 5973 mass spectrometer (Hewlett isms was possibly soil (Eguchi et al., 1999). Alicyclo- Packard) in conjuction with a 6890 gas chromatograph bacilli are now generally considered to inhabit soil, (Hewlett Packard) equipped with an HP-5MS capillary and to contaminate, and occasionally grow in, fruit column (30 mi0n25 mm; Hewlett Packard). juices. Recently, they have been focused on as target Quinones were extracted from freeze-dried cells organisms in quality control in the production of (200 mg) with a chloroform\methanol mixture (2:1) acidic beverages. and examined by HPLC with a Crest Pack (C18T-5; We isolated a new thermo-acidophilic, ω-cyclohexane Japan Spectroscopic Co.) according to the method fatty acid-possessing, spore-forming bacterium, strain described by Tamaoka et al. (1983). Cell lipids were TA-67T, from an ‘off’-flavoured acidic beverage as an prepared from about 1 g wet cells, according to the organism capable of producing guaiacol. Phylogenetic Bligh–Dyer method (Bligh & Dyer, 1959). Cells were analysis based on 16S rRNA gene sequences and broken using a Waring blender with glass beads characterization of phenotypic features revealed that (80–100 mesh), and the lipids were extracted with a the organism should constitute a new species in the chloroform\methanol mixture (2:1). The extracted genus Alicyclobacillus, for which the name Alicyclo- lipids were hydrolysed at 60 mC for 5 min with meth- bacillus acidiphilus is proposed. anol containing 7% boron trifluoride (Metcalfe et al., T 1996). GC\MS was performed on a 5971 mass spec- Strain TA-67 was isolated from an ‘off’-flavoured, trometer (Hewlett Packard) in conjuction with a 5890 acidic beverage. Diluted samples were spread on agar −" gas chromatograph (Hewlett Packard) equipped with plates of YSG medium containing the following (l ): a DB-23 capillary column (0n25 mmi30 m; J&B 2 g yeast extract (Difco), 2 g soluble starch (Merck), Scientific). The column temperature was increased by −" 1g-glucose and 15 g agar, pH 3n7, adjusted with 1 M 3 mCmin from 130 to 210 mC. Identification of the H#SO%; the plates were incubated at 45 mC for 3 days. T methyl esters was performed by comparing mass A. acidocaldarius ATCC 27009 and A. acidoterrestris spectra and retention times with fatty acid methyl ester DSM 3923, obtained from the American Type Culture standards (Larodan). Collection (ATCC; Manassas, VA) and Deutsche Sammlung von Mikroorganismen und Zellkulturen For DNA preparation, about 1 g wet cells grown (DSMZ; Braunschweig, Germany), respectively, were aerobically for 3 days was resuspended in TE buffer T (10 mM Tris, 1 mM EDTA, pH 8 0). Then, lysozyme used as reference strains. Strain TA-67 and the " n (final concentration 20 mg ml− ), achromopeptidase reference strains were cultivated in B. acidocaldarius −" −" medium (BAM medium) at 45 mC unless otherwise (20 mg ml ; Sigma), RNase A (50 µgml ) and NaCl stated (Deinhard et al., 1987a). For solid medium, (10 mM) were added and the mixture incubated at 1n5% of agar was added. For cellular fatty acid 37 mC for 1 h. Total lysis was achieved by adding 10% analysis, strains were cultivated in a semi-synthetic SDS (final concentration 1%). Proteins were digested basal medium for 3 days (Cerny et al., 1984). by incubation with Proteinase K (Boehringer Mann- heim) at 65 mC for 2 h. The residual proteins were For young cells, morphology was examined by phase- removed by either phenol or hexadecyltrimethylam- contrast microscopy. Motility was observed micro- monium bromide treatment, depending on the strain. scopically for young cells and by the prick test with For DNA–DNA hybridization experiments, DNAs BAM medium containing 0n05% gellan gum. The were further purified by equilibrium centrifugation in other biochemical tests were carried out according to CsCl-ethidium bromide gradients. Hybridization was Bergey’s Manual of Systematic Bacteriology (Claus & carried out by using the photobiotin-microplate Berkeley, 1986) and Deinhard et al. (1987a) with BAM method described by Ezaki et al. (1989). For measure- medium as the basal medium. Acid formation from ment of the GjC contents (mol%), the purified carbon compounds was determined using the method DNAs were hydrolyzed into nucleosides and analyzed described by Deinhard et al. (1987a) with the API 50 by HPLC using a C18T-5 Crest Pack. The 16S rRNA CH kit (bioMe! rieux). When acidification was am- genes were amplified by the PCR technique in com- biguous, strains were cultivated in BAM basal salts