Orenia Salinaria Sp. Nov., a Fermentative Bacterium Isolated from Anaerobic Sediments of Mediterranean Salterns

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 721–729 Printed in Great Britain Orenia salinaria sp. nov., a fermentative bacterium isolated from anaerobic sediments of Mediterranean salterns Sophie Moune! ,1,4 Claire Eatock,2 Robert Matheron,3 John C. Willison,4 Agne' s Hirschler,3 Rodney Herbert2 and Pierre Caumette1 Author for correspondence: Pierre Caumette. Tel: j33559923146.Fax:j33559808311. e-mail: pierre.caumette!univ-pau.fr 1 Laboratoire d’Ecologie A diverse range of fermentative bacteria have been isolated from the Mole! culaire-Microbiologie, commercial salterns of Salin-de-Giraud (Camargue, France). One of these IBEAS, Universite! de Pau, T 64000 Pau, France isolates, strain SG 3902 , has many of the morphological and physiological characteristics of the genus Orenia, as was confirmed by a phylogenetic study 2 Department of Biological Sciences, University of based on 16S rRNA gene sequencing. The closest species is Orenia marismortui, Dundee, Dundee with a similarity of only 951%. However, strain SG 3902T, unlike O. DD1 4HN, UK marismortui, does not ferment mannose, glycogen or starch. The GMC contents 3 Microbiologie IMEP, of the DNA also differ significantly, being 296 mol% for O. marismortui and Faculte! des Sciences et 337 mol% for strain SG 3902T. On the basis of these physiological and genetic Techniques St Je! ro# me, T 13397 Marseille, Cedex 20, differences, it is proposed that strain SG 3902 should be considered as a France representative of a new species belonging to the genus Orenia, under the T 4 Laboratoire de Biochimie name Orenia salinaria sp. nov. The type strain is SG 3902 (l ATCC 700911). et Biophysique des Syste' mes Inte! gre! s, DBMS, CEA Grenoble, 38054 Grenoble, Keywords: halophilic fermentative bacteria, salterns, Haloanaerobiales, hypersaline Cedex 9, France environments, Orenia INTRODUCTION previously by Caumette et al. (1994). Most of the fermentative bacterial isolates belong to the family Several halophilic, obligately anaerobic, fermentative Halobacteroidaceae. One of these isolates has been bacteria belonging to the domain Bacteria have been described as a new species with the name Haloanaero- isolated from sediments of hypersaline environments. bacter salinarius, strain SG 3903T (Moune! et al., 1999). Most of these bacteria were isolated from athalasso- Among the other strains isolated, strain SG 3902T is haline environments such as the Dead Sea, inland salt phylogenetically related to the genus Orenia according lakes or subterranean waters in oilfields (Oren, 1992; to 16S rDNA similarities. This genus is currently Ollivier et al., 1994; Rainey et al., 1995; Ravot et al., represented by a single species, Orenia marismortui, 1997). Few of them have been isolated from thalasso- isolated from the Dead Sea (Oren et al., 1987; Rainey haline environments such as solar salterns (Liaw & et al., 1995). Isolate SG 3902T showed sufficient Mah, 1992; Zhilina et al., 1991, 1992; Simankova et physiological and genetic differences from the species al., 1993). O. marismortui to be considered as a representative of a new member of the genus Orenia. Thus, strain SG During ecological investigations in solar salterns of 3902T is described here as a new species of the genus, the French Mediterranean Coast (Salin-de-Giraud, under the name Orenia salinaria sp. nov. Camargue, Rhone Delta), we isolated several strains of fermentative halophilic bacteria from the sediments of hypersaline lagoons with total salinities ranging from 13 to 34%. These bacteria co-exist with halophilic, METHODS phototrophic and sulfate-reducing bacteria described Source of strains. Strain SG 3902T was isolated from the sediment of hypersaline ponds (20–34% total salinity) in the ................................................................................................................................................. Salin-de-Giraud salterns (Camargue, France). The sulfide- The GenBank/EMBL/DDBJ accession number for the 16S rRNA sequence of rich black sediment was covered by a thin layer of gypsum; strain SG 3902T is Y18485. at the highest salinities, a deposit of halite was present. 01185 # 2000 IUMS 721 S. Moune! and others Table 1. Substrates fermented by Orenia salinaria strain Flagella were observed by transmission electron microscopy SG 3902T grown with 10% (w/v) NaCl under optimal with a JEOL 1200 ES electron microscope after negative conditions staining with 1% (v\v) tungstic acid neutralized to pH 7n2. ................................................................................................................................................. The ultrastructure of the cells was studied by transmission The following substrates were tested but not utilized: 0n1% electron microscopy after fixation of a cell pellet with osmic starch or glycogen, 6 mM arabinose, xylose, rhamnose, acid and ultrathin sectioning of the cells according to the lactose, raffinose, dextrose, arginine or glycerol. j, Substrate method of Glazer et al. (1971). utilized; k, substrate not utilized. Physiological tests. The ability to produce endospores was checked by growth in liquid medium supplemented Substrate (6 mM) Utilization with 1 mM glucose and 0n18 mM MnSO% or 0n5 g yeast extract\soil extract for spore induction, after exposure of the Glucose j cells to a temperature of 80 mC for 20 min. Fructose j Utilization of carbon sources and electron donors was tested Galactose k in triplicate in basal liquid medium amended with substrates Mannose k at the concentrations given in Table 1. Growth tests for Trehalose j utilizable substrates, optimum concentrations of NaCl and Sucrose j MgCl#, optimum pH, optimum temperature and sulfide Maltose j tolerance were assessed in completely filled 25 ml screw-cap tubes, as described by Caumette et al. (1988). The glucose Cellobiose j fermentation test was carried out in synthetic medium Glucosamine k lacking sodium bicarbonate and buffered with 0n4 M Tris\ N-Acetylglucosamine k HCl. The medium was prepared under an N# gas phase. Mannitol j Pyruvate k For aerobic growth tests, the basal growth medium without sodium bicarbonate and sulfide was buffered with 0 4M Lactate k n Tris\HCl and supplemented with glucose as the substrate. Glutamate k Growth was checked in test tubes open to the air and Glycine-betaine k plugged with a cotton-wool stopper. Antibiotic- and anti- Trimethylamine k bacterial susceptibility were tested in completely filled screw- cap tubes with the growth medium used for maintenance of strains, supplemented with the following substances: aniso- " " mycin (40 µgml− ), chloramphenicol (20 µgml− ), tetra- −" −" Media, isolation and culture conditions. The basal synthetic cycline (40 µgml ), erythromycin (40 µgml ), kanamycin −" −" medium used for the growth of strain SG 3902T contained (40 µgml ) and sodium taurocholate (50 µgml ), accord- the following (per litre distilled water): NaCl, 150 g; ing to the method described by Oren (1990). Growth was MgCl#;6H#O, 15 g; KCl, 3 g; NH%Cl, 0n5g; KH#PO%, measured by following the increase in optical density of the 0n35 g; CaCl#;2H#O, 0n05 g; yeast extract, 0n1g;0n1%(w\v) cultures at 450 nm (Spectronic 20; Bausch and Lomb) over resazurin solution, 1 ml; trace-element solution SL12 a period of 10 d. (Overmann et al., 1992), 1 ml; selenite\tungstate solution −" −" The utilization of nitrogen sources (ammonia, nitrate, N#, (Na#SeO$;5H#O, 6 mg l ;Na#WO%;2H#O, 8 mg l ; −" cysteine) was checked by growth in liquid medium lacking NaOH, 0n4gl ), 1 ml; NaHCO$,2g;Na#S;9H#O, 0n5g; organic or mineral nitrogen compounds, buffered with " vitamin V7 solution (Pfennig et al., 1981), 1 ml; pH 7n2–7n4. MOPS (3 g l− ) and prepared under an argon gas phase with The medium was prepared under a gas mixture (N#\CO#, an adequate nitrogen source. The growth was checked 90:10) according to the method of Pfennig et al. (1981). through five consecutive transfers. Prior to inoculation, the medium was supplemented with Vitamin requirements were determined in 60 ml serum organic substrates as carbon and energy sources (see Table 1 bottles by means of growth tests involving five consecutive for substrate utilization). transfers in synthetic medium lacking vitamins and yeast Pure cultures of strain SG 3902T were obtained by repeated extract. Catalase was tested by adding a few drops of 3% passage through a deep agar dilution series (Pfennig & (v\v) H#O# to a cell pellet on a microscope slide. Oxidase Tru$ per, 1981) in Hungate tubes, with N#\CO# (90:10) in the was checked by using the oxidase kit (bioMe! rieux). The gas phase and glucose as the organic substrate. The tubes utilization of sulfate, sulfite, thiosulfate or nitrate was tested were incubated at 30 mC in the dark. The purity of the in Postgate’s Medium B (Postgate, 1984), modified by sulfate cultures was checked microscopically and by inoculation omission, in 60 ml serum bottles, with glucose as the electron into different media specific for aerobic bacteria and sulfate- donor. After growth, the utilization of sulfur compounds reducing bacteria. was determined by means of sulfide production, as revealed by the formation of a black precipitate of FeS in the liquid Pure strains were grown in liquid cultures under a gas phase culture; nitrate reduction was determined by nitrite pro- (N#\CO#, 90:10) in 60 ml serum bottles stoppered with butyl duction, as revealed by the formation of a red colour rubber stoppers, using the Hungate anaerobic technique. following the addition of Griess reagent. The basal medium was

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