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3173 Advances in Environmental Biology, 5(10): 3173-3178, 2011 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Isolation and characterization of some aquatic bacteria from Qurugöl Lake in Azerbaijan under aerobic conditions 1,2 Vahideh Tarhriz, 1Ghorbanali Nematzadeh, 3Fatemeh Mohammadzadeh, 1Ehtaram Rahimi, and 2Mohammad Saeid Hejazi 1 Rice and Citrus Research Institute (RCRI), Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran. 2 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 3 Faculty of Agriculture, Zanjan University, Zanjan, Iran. Vahideh Tarhriz, Fatemeh Mohammadzadeh, Mohammad Saeid Hejazi, Ghorbanali Nematzadeh, Ehtaram Rahimi: Isolation and characterization of some aquatic bacteria from Qurugöl Lake in Azerbaijan under aerobic conditions ABSTRACT Aquatic bacteria play crucial roles in environmental biology. They are a rich source of new genes; unfortunately most of them are left unknown and regarding the ones which have been recognized and known there is not enough and efficient information considering their applications. Considering the geographical conditions of Qurug l Lake that is mostly frozen during the cold seasons and also lack of published papers on the presence of aquatic bacteria of this lake, the present study aimed to investigate the bacterial community in the lake. In so doing, samples were taken from four different parts and at each part they were taken from three different depths 0.25, 1 and 8 meter, respectively. The taken samples were grown on special media including: modified marine agar and sea water medium (SWM). Biochemical tests consisted of determination of oxidase and catalase activity, gelatin liquefaction, ability to hydrolyze starch and casein, production of indole, H2S and nitrate reduction tests were carried out. Thirty two isolates recognized. Most of isolated strains were Gramnegative (21) and mostly were psychrophilic bacteria. According to the results obtained from the analysis of extra-cellular enzymes it was found that the isolated bacteria have high variety hydrolytic enzyme activity. In a way that few of the isolated bacteria could produce a mixture of the mentioned enzymes, but all in all Grampositive bacteria in comparison with Gram-negative bacteria tested showed more hydrolytic activities, also revealed that the capability of Gram-negative bacteria in production of lipase enzyme is more than Grampositive bacteria. On the basis of phylogenetic results, it is recommended that the isolates belong to Aeromonas, Alishewanella, Alteromonas, Jeotgalicoccus, Bacillus, Marinobacter, Marinococcus, Paracoccus, Pseudoalteromonas, Rheinheimera, Rodobacter, Prophyrobacter and Shewanella. Moreover, several members of the Alishewanella, Jeotgalicoccus, Bacillus, Marinococcus and Shewanella clades were recovered. In a way that Alishewanella clade was dominant. Key words: aquatic bacteria, hydrolytic enzymes, Qurug l Lake, 16S rRNA gene. Introduction Aquatic microorganisms have very important roles in food chains, biogeochemical cycling, decomposition and production industrial enzymes, yet they are largely uncharacterized due to their small size, the limited range of morphologies, and the difficulties in obtaining pure cultures that are representative of natural populations [16][10]. It is estimated that negligible percent of aquatic microorganisms can be recovered through standard tissue culturing technique. However, the action of recovery can be improved through applying molecular phylogenetic analysis such as 16s rRNA gene sequence [10]. Most of aquatic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases, pullulanases and other important industrial enzymes. For example Vibrio alginolyticu produces six proteases. One of these proteases, Pro A, is SDS resistant [5]. Also most of the metabolites produced by aquatic bacteria especially Cianobactéria have acute cytotoxic Corresponding Author Vahideh Tarhriz, Rice and Citrus Research Institute (RCRI), Faculty of Agriculture, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran. Email: [email protected]; Tel: +98 (411) 337 2256; Mob: +98 914 311 3708 Fax: +98 (411) 334 4798 3174 Adv. Environ. Biol., 5(10): 3173-3178, 2011 feature such as Apratoxin, private neurotoxic activity such as Kalkilatoxin and Antillatoxin or anti-cancer feature such as Dolastatin and Cutacia [17]. Qurugӧl Lake is a fresh water Lake and it is neglected to be studied for its potential interesting novel types of aquatic bacteria. Due to importance of aquatic bacteria, it is the aim of this paper to isolate and characterize of aquatic bacteria in Qurugӧl Lake by 16s rRNA gene, since according to our knowledge such studies have not been carried out up to now in this lake. Material and Methods Water Sample Collections: Samples from four different areas of the lake and at three different depths 0.25, 1 and 8 meter, respectively, were collected at the end of rainy seasons. Then the samples were transported to the RCRI (Rice and Citrus Research Institute) laboratory. Isolation and identification of aquaticbacteria were determined on SWM contained (per liter): Beef extract, 10.0 g; Peptone, 10.0 g; Agar, 20.0 g; Tap water, 250.0 ml; artificial sea water, 750.0 ml was tested, consist of NaCl, 28.13 g; KCl, 0.77 g; CaCl2 x 2 H2O, 1.6 g; MgCl2 x 6 H2O, 4.8 g; NaHCO3, 0.11 g; MgSO4 x 7 H2O, 3.5 g; and modified marine agar contained (per liter): peptone, 5.0 g; yeast extract, 1.0 g; Fe3+-citrate, 0.1 g; NaCl, (1.2, 2.4, 4.8, 9.75, 19.45 g); MgCl2 (dried), 5.9 g; Na2SO2, 3.24g; CaCl2, 1.8g; KCl ,0.55g; Na2O3, 0.16g; KBr, 0.08g; SrCl2, 34.0mg H3BO3, 22.0 mg; Na-silicate, 4.0 mg; NaF, 2.4 mg; (NH4)NO3, 1.6 mg; Na2HPO4, 8.0 mg, and pH was adjusted to 7.6 ± 0.2. 100 micro liters from the samples were inoculated onto plates. The cultures were incubated at 25°C for a couple of weeks. To obtain a pure culture, isolates were taken by marine broth medium and were stored at -70°C in marine broth supplemented with 30% (v/v) Glycerol. Gram staining was performed using the standard Gram staining method [8] and also Gram reaction was tested with KOH lysis test technique [9]. Growth of isolates was investigated at -5, 0, 5, 10, 15, 20, 25, 30, 37, 40, 45 and 50 °C in marine broth containing (per liter): Bacto peptone, 5.0 g; Bacto yeast extract, 1.0 g; Fe(III) citrate, 0.1 g; NaCl, 19.45 g; MgCl2 (dried), 5.9 g; Na2SO4, 3.24 g; CaCl2, 1.8 g; KCl, 0.55 g; Na2CO3, 0.16 g; KBr, 0.08 g; SrCl2, 34.0 mg; H3BO3, 22.0 mg; Na-silicate, 4.0 mg; NaF, 2.4 mg; (NH4)NO3, 1.6 mg; Na2HPO4, 8.0mg; with pH 7.2±0.5. In order to determine the optimal growth temperature, spectrophotometric absorbance of the samples was measured at 600 nm (OD: 600) after 48 hours. The pH tolerance was determined in marine broth with the pH set from 4 and 12 (using increments of 1 pH unit). Growth of isolates was tested at different NaCl concentrations in marine broth medium supplemented with 0 and 1-10% NaCl, at intervals of 0.5% (w/v) NaCl [23]. Biochemical tests consisted of determination of oxidase and catalase activity, gelatin liquefaction, ability to hydrolyze starch and casein, production of indole, H2S, nitrate reduction and motility tests were carried out according to [12]. Screening of strains for extracellular hydrolytic activities: In the next step the capability of the isolated bacteria was analyzed in producing extra-cellular amylase enzyme (Cowan et al., 1991), extra-cellular protease enzyme (Simankova et al., 1994), extracellular lipase enzyme (Simankova et al., 1994), extra-cellular nuclease enzyme (Onishi et al., 1983), extra-cellular inulinase enzyme (Allais et al., 1986), extracellular pectinase enzyme (Marcia et al., 1999), extra-cellular carboxymethyl cellulose enzyme (Simankova et al., 1994), extra-cellular pullulanase enzyme (Murray et al., 1994) and extra-cellular chitinase enzyme (Shaikh et al., 1993). All tests were carried out at optimum NaCl concentration of each isolate. The 16S rRNA gene sequences: For genotypic characterization, DNA was isolated from the isolates according to the method described by Corbin et al. (2001) with some modifications. For phylogenetic analysis based on the 16S rRNA gene sequence, the genes were amplified using PCR technique in the presence of forward 16F27 (5' -AGA GTT TGA TCT GGC TCA G- 3') and reverse 16R1488 (5' -TAC CTT GTT AGG ACT TCA CC- 3') primers [11]. The amplified fragments were purified using Roche kit (Germany) and then sequenced by utilizing forward and reverse and 3 other designed primers; H400 (5'-GGG TTG TAA AGC ACT TTC AG-3'), H550 (5'-CCA GTA ATT CCG ATT AAC GC-3') and H900 (5'-ACT CAA ATG AAT TGA CGG GG3') used for PCR amplification by Macrogen company (Korea). The 16S rRNA gene reads were assembled using Chromas pro software and aligned using the multiple sequence alignment program CLUSTAL X (version 1.83) (Thompson et al., 1997). Phylogenetic trees were constructed using neighbor-joining, method in MEGA version 4 software package [21]. 3175 Adv. Environ. Biol., 5(10): 3173-3178, 2011 Table 1: Comparison of the characteristics of isolated bacteria based on phenotypic features, Taxa: the some aquatic isolates in each genus. Table 2. Closest known bacteria to various isolates Qurug l Lake based on 16S rRNA gene sequence. Fig. 1: The comparison of production of extra-cellular enzymes in Gram-negative and Gram-positive bacteria existing in Qurug l Lake. 3176 Adv. Environ. Biol., 5(10): 3173-3178, 2011 Fig. 2: Phylogenetic analysis of the isolates was members of thirteen genera of the domain Bacteria based on a partial sequence of the 16S ribosomal RNA gene. The sequence alignment was performed using the CLUSTAL-X program and the tree was generated using the NJ method in MEGA 4 software. Results and Discussion We could separate thirty two isolates from twelve water samples collected from Qurug l Lake, most of which were Gram-negative (21 isolates). Colony pigmentation from these samples was red to white, most of which were cream. All isolates were catalase positive and some of which were able to grow in the absence of NaCl (Table 2). Based on the results obtained from the analysis of extra-cellular enzymes it was found that the isolated bacteria have high variety hydrolytic enzyme activity. In a way that few of the isolated bacteria could produce a mixture of the mentioned enzymes, but Gram-positive bacteria in comparison with Gram-negative bacteria tested showed more hydrolytic activities. The findings also revealed that the capability of Gram-negative bacteria in production of lipase enzyme is more than Grampositive bacteria (Fig. 1). The comparison of isolated bacteria based on variety and production of hydrolytic enzymes shows that Gram-positive bacilli are at the top of the list. Most of the isolated bacteria in this research have had the capability to produce DNase enzyme. The dominant enzyme in Gram-negative was lipase and Gram-positive was DNase. In order to find out their phylogenetic position, the 16S rRNA gene sequence of each strain was analyzed, and a phylogenetic tree was constructed (Fig. 2). We were able to sequence the isolates successfully and the results showed that the collections have been put into at least thirteen bacterial genera. The bacterial isolates were mostly ascribable to members of Aeromonas, Alishewanella, Alteromonas, Jeotgalicoccus, Bacillus, Marinobacter, Marinococcus, Paracoccus, Pseudoalteromonas, Rheinheimera, Rodobacter, Prophyrobacter and Shewanella. Moreover, several members of the Alishewanella, Jeotgalicoccus, Bacillus, Marinococcus and Shewanella clades were recovered. In a way that Alishewanella clade was dominant. Discussion: In the last few years, increased attention has been given to aquatic bacteria, especially producing hydrolytic enzymes bacteria. Several studies have been conducted on their biotechnological applications as well as their ecology and phylogeny [18]. There are some studies about aquatic bacterial 3177 Adv. Environ. Biol., 5(10): 3173-3178, 2011 communities of several lakes by amplification and sequencing of 16S rRNA gene such as study of aquatic bacteria communities in the Adirondack Mountains lakes by [10]. The present study tries to make an attempt to study the isolation and characterization of some aquatic bacteria from Qurugöl Lake under aerobic condition. This study, as far as we know, provides the first publication on the diversity of aquatic bacteria in Qurugöl Lake in Azerbijan-Iran. Even though from morphological point of view most of the separated isolates are cold environment favoring kind due to their ability to grow in less than five centigrade, the best temperature for the growth of the isolates was determined as between 25 to 30 ºC (Table 1). Furthermore the isolates were able to grow in a wide range of pH, even though the ideal conditions for the growth of majority of them is determined in pH=6.5-7. Throughout the course of this work, we isolated thirty two strains. Most of the strains grow in the absence of NaCl. Phylogenetic analysis indicated that all isolates were members of thirteen genera of the domain bacteria, in five of which were dominant; Alishewanella, Bacillus, Jeotgalicoccus, Marinococcus and Shewanella. The collection contains only one member from each of the Alteromonas, Pesodoalteromonas, Porphyrobacter and Rhodobacter(Table 2). Six strains which were isolated on marine medium belonged to Alishewanella genus, most of which had high level of 16S rRNA gene similarity with Alishewanella aestuarii (>99%) (Table 1), but Similarities between strain RCRI4 (=RCRI16) (=BCCM/ LMG 26473T = JCM 17275T) and closely related type strains, achieved using the EzTaxon (Chun et al., 2007), were 98% with Alishewanella agri BLO6T. So the GenBank/EMBL accession number for the 16S rRNA gene sequences of strain RCRI4 (=RCRI16) is GQ505294. These strains are Gram-negative, rod shaped, nonmotile with 1.5-2.0 μm long, and 0.9 μm width, the biochemical tests indicated that the strain RCRI4 is capable to produce amylase, lipase, DNase, inulinase, pectinase, but is not capable to produce other hydrolytic enzymes. The phylogenetic tree showed that strains RCRI7 (=RCRI8) and RCRI18 (= JCM 17276T) fall within the radiation of a cluster composed of Shewanella species ≥99.1%. 16S rRNA gene sequence similarity with Shewanella xiamenensis, Shewanella putrefaciens and Shewanella oneidensis (Fig. 2; Table 2). They could produce lipase and amylas, but they were not able to produce other hydrolytic enzymes. The GenBank/EMBL accession number for the 16S rRNA gene sequences of strain RCRI7 (=RCRI8) is GQ988720. Also the closest genus with strain RCRI19 (=BCCM/ LMG 25773T =JCM 17277T) is genus Rhodobacter spp. Phylogenic tree clustered this strain with Rhodobacter blasticus(95.3%). The strain is Gram-negative, rod shaped and motile with 1.3-3.0 μm length, and 0.9 μm widths. The strains grow in the absence of NaCl and also in the presence of 0.5 and 1 percent NaCl, but not in 1.5% NaCl in marine agar. The strain RCRI19 was able to produce only lipase and amylase. The 16S rRNA gene sequences of strain RCRI19 was scored HQ392507 accession number in the GenBank/EMBL. Conclusion: Among the culture collection strains tested for hydrolytic enzyme activities most of them were negative for the production of the enzymes, only two genera Bacillus and Marinococcus showed the highest variety of production of hydrolytic enzyme activities (Table 1), both of them belong to Grampositive bacteria. Since dominant bacteria (work in progress) were Gram-negative, so this may be influence the results. In comparison with Gramnegative species, the Gram-positive bacteria tested showed more hydrolytic activities, particularly DNase enzyme production. Between the Grampositive, representatives of the genus Bacillus was predominant. The genus Bacillus is well known as an enzyme-producer and many industrial processes use species belonging to this genus for commercial production of enzymes (Sanchez-porro et al., 2003). Finally xylanase producers are limited to representatives of the genus Bacillus. In exhaustive comparison with moderately halophilic bacteria, it was sound fresh water bacteria with less tolerant NaCl, were very poor to produce hydrolytic enzyme activities, in the other hands halophile and halotolerant bacteria were higher variety producers of hydrolytic enzymes. Furthermore, the potential use of these microorganisms in degrading aromatic compounds as underlined earlier (Dyksterhouse et al., 1995), prompts us to screen our collection of aquatic bacteria for their ability (work in progress). Besides, it is reported that marine bacteria Marinobacter, jeotgalicoccus and Bacillus strains, are able to degrade organic pollutants [7]. Study of our collection for enzyme production of these bacteria to degrade environmental pollutants, is another step in our interests. 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