J. Gen. Appl. Microbiol., 52, 63–72 (2006) Full Paper Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt Hanan Ghozlan,* Hisham Deif, Rania Abu Kandil, and Soraya Sabry Department of Botany, Faculty of Science, University of Alexandria, Moharrem Bey, Egypt (Received January 19, 2005; Accepted November 24, 2005) Screening bacteria from different saline environments in Alexandria. Egypt, lead to the isolation of 76 Gram-negative and 14 Gram-positive moderately halophilic bacteria. The isolates were characterized taxonomically for a total of 155 features. These results were analyzed by numerical techniques using simple matching coefficient (SSM) and the clustering was achieved by the un- weighed pair-group method of association (UPGMA). At 75% similarity level the Gram-negative bacteria were clustered in 7 phena in addition to one single isolate, whereas 4 phena repre- sented the Gram-positive. Based on phenotypic characteristics, it is suggested that the Gram- negative bacteria belong to the genera Pseudoalteromonas, Flavobacterium, Chromohalobacter, Halomonas and Salegentibacter, in addition to the non-identified single isolate. The Gram-posi- tive bacteria are proposed to belong to the genera Halobacillus, Salinicoccus, Staphylococcus and Tetragenococcus. This study provides the first publication on the biodiversity of moderately halophilic bacteria in saline environments in Alexandria, Egypt. Key Words——moderate halophiles; numerical taxonomy; saline environments Introduction physiological adaptation to highly saline concentra- tions and their ecology (Martinez-Canovas et al., 2004; Moderately halophilic bacteria are microorganisms Tokunaga et al., 2004; Ventosa et al., 1998a, b). that can grow optimally in media containing between Hypersaline environments in Egypt are neglected 3% and 15% (w/v) salt (Lichfield, 2002). Because of hunting grounds for potential interesting new types of the wide range of salinity in which this group of mi- moderately halophilic bcteria. It was thus aimed in this croorganisms can grow optimally, they are widely dis- paper to explore the biodiversity of this group of bacte- tributed in different saline habitats such as hypersaline ria in different saline habitats in Egypt since to our lakes, desert and saline soils, saltern ponds, salt knowledge such study has not been carried out. mines, salted foods and others (Lichfield and Gillevet, 2002; Ventosa et al., 1998a, b). Materials and Methods The aspects that attracted the interest of re- searchers recently were mainly those related to their Study area and analysis of samples. Water and soil samples were collected from three sites represent- ing different saline environments. These are the * Address reprint requests to: Dr. Hanan A. Ghozlan, Depart- coasted salt marshes and the solar salterns (40 km ment of Botany & Microbiology, Faculty of Science, University of Alexandria, Moharrem Bey, Egypt. west of Alexandria) and the salt lakes (21 km west of Tel: 0020–12–2491034 Fax: 0020–3–4240959 Alexandria). E-mail: [email protected] Samples collected in sterile bottles were sent to the 64 GHOZLAN et al. Vol. 52 laboratory, and examined within 2 to 3 h of collection. Halophilic medium (HM) adopted after Ventosa et al. (1982) was used for the isolation and contained in g%: NaCl, 17.8; MgSO4 ·H2O, 0.1; CaCl2 ·2H2O, 0.036; KCl, 0.2; NaHCO3, 0.006; NaBr, 0.023; FeCl3 ·6H2O, traces; bacto-peptone, 0.5; yeast extract, 1.0; glucose, 0.1; agar, 2.0. Agar plates were inoculated with 1 ml of water or soil suspension samples or an appropriate di- lution of the sample and incubated at 30°C for up to 7 days. Samples were analyzed for salinity and major cation (Mg2ϩ, Naϩ, Kϩ) contents according to Ramos-Cor- menzana (1989). Isolates representing different mor- photypes were selected and successively streaked on HM to ensure purity. Selected strains were kept on agar slants of maintenance halophilic media containing 10% (w/v) total marine salts; supplemented with bacto- Fig. 1. Simplified dendrogram showing the clustering of peptone, 0.5; yeast extract, 1.0; glucose, 0.1 and agar, strains into 7 phena based on SSM coefficient and UPGMA 2%. analysis, for 76 moderately halophilic Gram-negative bacteria. Characterization and numerical analysis. The se- * No.ϭNumber of strains. # Ph.ϭPhenon. lected strains were phenotypically characterized as previously described (Ventosa et al., 1982). All tests total isolates were Gram-negative, whereas the Gram- were carried out in cultures incubated at 30°C for up to positive (14 isolates) formed only 15.5% of the total. 7 days. A total of 155 characteristics were used in the Characteristics of the Gram-negative isolates are listed numerical analysis. Positive and negative results were in Table 1. Analysis of 155 characteristics using a SSM coded as 1 and 0, respectively. Strain similarities were coefficient with UPGMA clustering yielded the dendo- estimated using the simple matching coefficient (SSM) gram in Fig. 1. Data show that the majority of the (Sokal and Michenu, 1985) and the clustering was strains were grouped at the 75% similarity level into achieved by the unweighted pair-group method of as- seven phena. Only one strain, isolated from the salt sociation (UPGMA) (Sneath and Sokal, 1973). The lakes, formed a single separate cluster. The estimated test error was estimated by examining 10 strains in du- error was less than 3%. plicate (Sneath and Johnson, 1972). The computations Phenon A. The 6 strains grouped in phenon A were performed by using the SYSTAT-PC program V.7 possess several features that are common to mem- (Wilkinson et al., 1992) on an IBM computer. bers of genus Pseudoalteromonas. It is thus proposed that the strains are different species of this genus. The Results and Discussion data in Table 1 show that all of the strains are long motile rods. They require up to 20% NaCl for growth Increased attention has been given in the last few and tolerate up to 30% salinity. Colonies are round years to moderately halophilic bacteria. Several stud- convex and produce brown pigments. They utilize a ies have been conducted on their ecology, taxonomy wide range of compounds as a carbon source. and phylogeny as well as their biotechnological appli- Phenon B. The orange pigmented bacteria com- cations (Ramos-Cormenzana, 1991; Sanchez-Porro et prising phenon B (six strains) show phenotypic fea- al., 2003; Ventosa et al., 1998a, b). tures closely related to members of genus Flavobac- In the present study, a total of 90 strains chosen to terium. Flavobacterium is placed in the Flexibacer- represent different morphotypes were purified and Bacteroides-Flavobacterium phylum a (Woese et al., characterized with 155 tests. 1990). Flavobacterium is the type genus of the family Flavobacteriaceae which also encompasses several Gram-negative isolates other genera. The majority (76 isolates) representing 84.5% of the Data show that the strains are long rods with gliding 2006 Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt 65 Table 1. Frequencies of positive characteristics found in the seven phena (expressed as a percentage of the total scored by each group for the given test). ABCDEFG 664 62723 3 Salt concentration 10% 100 100 100 100 100 100 100 20% 100 100 100 100 100 100 100 30% 17 0 25 17 47 0 0 40% 0 0 25 17 67 0 0 Growth at pH 5 83 83 0 0 100 96 0 pH 9 83 67 100 100 85 57 100 Temperature 10°C 17 83 25 100 93 48 100 Temperature 40°C 0 0 0 0 81 52 0 Biochemical tests Oxidase 100 100 75 100 91 84 100 Methyl red 50 0 0 0 0 0 17 Voges-Proskauer 50 0 0 0 0 0 0 H2S production 83 50 75 83 93 43 0 Nitrate reductase 0 0 25 0 85 74 0 Nitrite reductase 0 0 0 0 85 74 0 Utilization of organic compounds as sole source of carbon and energy Arabinose 0 83 100 50 85 96 100 Mannose 100 100 0 100 85 100 100 Raffinose 17 0 0 17 74 48 100 Rhamnose 83 33 0 17 85 43 100 Ribose 83 100 0 67 85 100 100 Xylose 0 83 100 67 85 100 100 Trehalose 83 100 75 100 100 96 0 Fructose 67 17 50 0 7 52 0 Galactose 67 100 50 67 100 100 100 Sucrose 0 100 100 83 96 100 100 Lactose 0 0 0 0 74 26 100 Maltose 100 100 100 100 100 83 100 Cellobiose 0 17 0 17 100 74 100 Glycerol 33 83 0 100 93 48 100 Mannitol 100 83 75 100 96 96 100 Lactate 33 83 25 100 78 48 100 Utilization of amino acids as sole source of carbon, nitrogen and energy L-Alanine 83 0 0 100 100 100 100 L-Phenylalanine 100 67 0 100 93 91 100 L-Arginine 100 0 100 100 100 100 100 L-Asparagine 33 67 25 100 93 48 100 L-Tyrosine 0 0 0 33 81 70 67 L-Glutamine 33 83 25 100 93 48 100 L-Glutamic acid 0 0 0 0 0 4 0 L-Cystine 0 0 0 33 0 9 33 L-Cysteine 0 0 0 33 11 13 33 L-Proline 33 100 100 100 100 100 100 L-Histidine 0 50 0 33 74 100 67 66 GHOZLAN et al. Vol. 52 Table 1. (Continued). ABCDEFG 664 62723 3 L-Isoleucine 83 17 0 33 37 78 67 L-Leucine 0 0 0 83 100 91 100 L-Lysine 100 17 100 100 100 100 100 L-Ornithine 0 33 75 33 100 100 0 L-Serine 0 100 0 33 100 100 33 L-Tryptophan 0 83 0 100 89 100 33 L-Methionine 0 17 0 100 81 100 100 L-Threonine 0 0 100 0 33 0 0 L-Valine 100 17 0 100 85 100 100 Acid production from Glucose 100 100 75 50 100 91 100 Galactose 0 100 0 0 0 87 0 Maltose 100 17 0 0 0 4 0 Lactose 0 67 0 0 0 4 0 Sucrose 100 33 75 67 11 17 0 Mannitol 67 0 0 0 4 0 0 Degradation of Alginate 100 50 75 100 44 91 100 Aesculin 0 100 0 0 93 4 0 Chitin 100 0 0 0 0 0 0 Cellulose 50 50 0 17 0 13 100 Starch 0 50 0 0 0 4 100 Gelatine 67 100 50 0 7 0 33 Casein 100 0 0 0 0 0 100 DNA 100 33 0 33 4 30 33 Tween 20 33 0 0 17 26 13 0 Tween 40 0 67 0 0 67 22 0 Tween 80 50 0 0 50 15 9 100 Antibiotic sensitivity Penicillin G (10 units) 83 33 100 83 7 26 100 Polymyxin B (300 units) 100 83 50 67 93 70 67 Tetracycline (30 mg) 33 33 0 17 11 26 100 Ampicillin (10 mg) 83 100 100 100 30 96 100 Cephalothine (30 mg) 67 0 100 83 15 9 67 Chloramphenicol (30 mg) 83 83 100 100 100 96 100 Novobiocin (30 mg) 83 67 100 100 33 52 100 motility, forming orange round convex colonies.