Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 07 Dec 2018 09:52:40 Fusibacter tunisiensis sp. nov., isolated from an anaerobic reactor used to treat olive-mill wastewater Wajdi Ben Hania, 1,2 Belkis Fraj, 1,2 Anne Postec, 1 Khaled Fadhlaoui, 1 Moktar Hamdi, 2 Bernard Ollivier 1 and Marie-Laure Fardeau 1 Correspondence Marie-Laure Fardeau marie-laure.fardeau@univ-amu.fr 1 Laboratoire de Microbiologie M10, UMR 2359, Aix-Marseille Univ., Case 925, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France 2 Laboratoire d’Ecologie et de Technologie Microbienne, Institut National des Sciences Applique ´es et de Technologie, Centre Urbain Nord, BP 676, 1080 Tunis, Tunisia Strain BELH1 T , a novel mesophilic, anaerobic, halotolerant, rod-shaped bacterium, was isolated from a Tunisian wastewater digester. The cells of the strain are motile, measure 0.5¾2–5 mm, and occur singly or in pairs. The strain reduced thiosulfate and elemental sulfur (but not sulfate or sulfite) into sulfide. It grew at 15–40 6C (optimum 30 6C), pH 5.8–8.4 (optimum 7) and with 0– 10 % (w/v) NaCl (optimum 3.0 %). The genomic DNA G+C content of strain BELH1 T was 38.2 mol% and the strain’s predominant cellular fatty acids were C 14 : 0 , a summed feature that contained iso-C 17 : 1 and/or anteiso-C 17 : 1 B, and C 16 : 0 . Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain was most closely related to Fusibacter paucivorans (94.8 % sequence similarity). Based on phenotypic, phylogenetic and taxonomic characteristics, strain BELH1 T represents a novel species of the genus Fusibacter, for which the name Fusibacter tunisiensis sp. nov. is proposed. The type strain is BELH1 T (5DSM 24436 T 5JCM 17481 T ). The genus Fusibacter, first proposed by Ravot et al. (1999), belongs to the phylum Firmicutes, order Clostridiales. To date, the genus contains only one recognized species, Fusibacter paucivorans, an anaerobic heterotrophic and thiosulfate-reducing bacterium isolated from a sample of reservoir water collected from an offshore oil-producing well in Congo, Central Africa (Ravot et al., 1999). In the present study, a polyphasic approach was used for the taxonomic characterization of a novel mesophilic and halotolerant bacterium isolated from a digester used to treat wastewater from a Tunisian olive mill. The novel strain, which was able to use thiosulfate as a terminal electron acceptor, possessed phenotypic and phylogenetic traits that allowed its assignment as a novel species of the genus Fusibacter. Samples were collected in Tunisia, from the sludge of a 2-litre anaerobic sequencing batch reactor, which was run at 35 u C and pH 7.5, with a flow rate of 100 ml day 21 , and fed with olive-mill wastewater (100 ml l 21 ) and phospho- gypsum (10 g l 21 ). The samples were collected under anaerobic conditions and transported to the research labo- ratory at ambient temperature. Strict anaerobic procedures were followed for the microbial isolation and culture (Hungate, 1969). The basal medium used for the isolation contained (l 21 ): 1.0 g NH 4 Cl, 0.3 g K 2 HPO 4 , 0.3 g KH 2 PO 4 , 0.1 g KCl, 0.1 g CaCl 2 . 2H 2 O, 23 g NaCl, 1.0 g yeast extract (Difco), 1.0 g tryptone (Panreac Quimica), 0.5 g cysteine-HCl, 1.6 g Na 2 S 2 O 3 and 1 ml Widdel trace element solution (Widdel & Pfennig, 1982). The medium was adjusted to pH 7 with 10 M KOH solution before being boiled and cooled to room temperature under a stream of oxygen- free N 2 gas. Aliquots of 5 ml were dispensed into Hungate tubes, degassed under N 2 /CO 2 (80 : 20, v/v) and then sterilized by autoclaving at 120 uC for 20 min. Before inoculation with 0.5 ml sample, the basal medium in each tube was supplemented with 0.1 ml 10 % (w/v) NaHCO 3 , 0.1 ml 2 % (w/v) Na 2 S . 9H 2 O, 0.1 ml 15 % (w/v) MgCl 2 . 6H 2 O and 0.1 ml 1 M glucose, all from sterile stock solutions. Inoculated tubes were incubated at 30 u C. Cultures were purified by repeated use of the Hungate roll-tube method (Hungate, 1969) and medium solidified with 1.6 % (w/v) agar, before transfer of each isolate back into liquid medium. The pH, temperature and NaCl concentration ranges for growth were determined using basal medium supplemen- ted with 20 mM glucose. Initial pH was varied, from 5.8 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BELH1 T is FR851323. International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1365–1368 DOI 10.1099/ijs.0.034603-0 034603 G 2012 IUMS Printed in Great Britain 1365