Int. J. Aquat. Biol. (2017) 5(4): 268-274OI: ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2017 Iranian Society of Ichthyology Original Article Caspian Sea’s Navicula salinicola Hustedt 1939 and effect of the prolonged culture on its fatty acid profile Ehsan Etesami1, Farkhondeh Saba1, Mostafa Noroozi*2,1Mohammad Ali Amoozegar1, 3, Gholamreza Bakhshi Khaniki4, Seyed Abolhassan Shahzadeh Fazeli1, 5 1Microorganisms Bank, Iranian Biological Resource Centre (IBRC), Tehran, Iran. 2Department of Biotechnology, Faculty of Biological Sciences, ALzahra University, Tehran, Iran. 3Department of Microbiology, Faculty of Biology and Centre of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran. 4Department of Biotechnology, Faculty of Sciences, Payam-e Noor Tehran-Shargh University, Tehran, Iran. 5Departments of Molecular and Cellular Biology, Faculty of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran. Article history: Received 3 June 2017 Accepted 24 August 2017 Available online 2 5 August 2017 Keywords: Algae Omega3 Eicosapentaenoic acid Docosahexaenoic acid Abstract: Diatoms are a potent source of polyunsaturated fatty acids. This study was conducted for screening a Naviculoid diatom strains from the southern Caspian Sea with analyzing its lipid production and accumulation potentials. The isolate was identified as Navicula salinicola strain IBRC-M 5083 based on micro-morphological characterization and analysis of 18S rRNA genomic region. Navicula salinicola were cultured in the f/2 medium under both normal and prolonged culture (21 days) conditions. Total lipid percentages of this strain were found to be 31.83% under normal condition and 43.72±1.4% in prolonged culture respectively on the basis of their dry cell weight (DCW). Also, the oil droplets were detected in 21 days’ cells as shown by Sudan Black B staining experiments. Furthermore, the main fatty acids were found by Gas Chromatography analyses of this strain under prolonged condition to be Eicosapentaenoic acid (25.58%TFA). Such oil accumulation capabilities seem to be promising for performing further studies on this strain as a source of Omega- 3 in aquafeed, pharmaceutical and biofuel industries. Introduction Microalgae have many applications in different fields such as pharmaceutical, food industries, aquafeed, phytoremediation, sewage treatment and biofuel. There are several microalgae species which are able to accumulate lipids such as diatoms having a high potential of lipid production (Dunahay et al., 1998; Hildebrand et al., 2012). Therefore, they are potent and proper candidates for many biotechnological applications like biofuels (Merz and Main, 2014). Among them, the marine diatoms have been investigated more than others (Griffiths and Harrison, 2009) except the freshwater genus Navicula (Sheehan et al., 1998; Graham et al., 2012). Lipid in diatoms could be accumulated as intracellular droplets included TAGs (Triacylglycerol) (Yoneda et al., 2016; *Corresponding author: Mostafa Noroozi DOI: https://doi.org/10.22034/ijab.v5i4.271 E-mail address: noroozi.mostafa@alzahra.ac.ir Yongmanitchai and Ward, 1991). The 16 or 18 carbon TAGs could be used as biodiesel sources because of its chemical similarity with aliphatic hydrocarbons in petrol (Yongmanitchai and Ward, 1991; Sivakumar et al., 2010). Furthermore, over 18 carbons such as C20:5ω3 (EPA) and C22:6ω3 (DHA) have also been found in diatoms which could be valuable sources for Omega3 (Brett and Muller‐Navarra, 1997). Strong evidence of beneficial properties of Omega3 for human nutrition led to increase of interest in omega3 as nutritional supplements and aquafeed (Merz and Main, 2014). Previous studies about Navicula sp. showed its proper level of C16:0, C16:1, C17:01, C18:0 (Matsumoto et al., 2010; Thajuddin et al., 2015), but many environmental variations such as nutrient 269 Int. J. Aquat. Biol. (2017) 5(4): 268-274 deficiency can change its fatty acid composition (Lison, 1934; Mekhalfi et al., 2010). For instance, under nitrogen and silicon deficiencies in N. salinicola, the lipid content was induced up to 58% and 22-49%, respectively (Nagle and Lemke, 1990). Nonetheless, prior to any lipid analysis, the target cells should be identified. Identification of diatoms is done using morphological characters such as the siliceous wall (frustules) ornamentations and proto- plast features (Preisig and Andersen, 2005). For precise morphological identification of diatoms, it would be better to observe the frustules by Scanning Electron Microscope (SEM) (Crawford et al., 2001). Due to the presence of organic material on the frustule and cell contents, the characteristics of the valve are obscured thus it is necessary to clean diatom frustules from organic matter to provide a permanent slide and observe the frustule ornamentations under SEM. Recently molecular tools such as 18S rRNA gene sequence are used to confirm the morphological identifications (Dawson and Pace, 2002). The objective of this research was to identify the isolated cell and determine the effect of a prolonged culture period (21 days) on the fatty acid profile of Navicula salinicola, collected from the Caspian Sea, Iran. Materials and Methods Isolation and culture conditions: The samples were collected from the Gohar-Baran port, the southern Caspian Sea, Iran (36°49'51.1''N, 53°11'38.4''E). The samples were left for 24 hrs to settle the organisms in IBRC microalgae laboratory and then cultivated in filter sterilized seawater enriched with Guillard f/2 medium (NaNO3 8.82104 M; KH2PO4 3.62105 M; FeCl3.6H2O 1.17105 M; Na2EDTA.2H2O 1.17105 M; CuSO4.5H2O 3.93108 M; Na2MoO4.2H2O 2.60108 M; ZnSO4.7H2O 7.65108 M; CoCl2.6H2O 4.20108 M; MnCl2.4H2O 9.10 107 M; thiamine HCl 2.96107 M; biotin 2.05 109 M; cyanocobalamin 3.691010 M, 100mg.L-1 Imipenem - all chemicals purchased from Sigma Aldrich Inc., St Louis, MO, USA) (Guillard, 1975; Presieg and Andersen, 2005). The pH of the medium was set at 7 and the sample was grown at 18±1°C under a light/dark cycle (12h: 12h) in f/2 medium that prepared from seawater under a low intensity of 50 µmol m–2 s–1 (Preisig and Andersen, 2005). First, the axenic colonies of diatom on the plate were transferred to the triple 150 ml flask after 7 days, subsequently they were inoculated with 10% (v/v) into triple 500 ml fresh f/2 medium over another 7 days and kept to grow for 7 days at the same condition (prolonged culture). Then, one of the triple sample of normal and prolonged culture were randomly chosen and centrifuged at a speed of 3,000 rpm for 30 min, then the supernatant were removed. The pellets were washed twice with Distilled Water and dried at 60°C to obtain their constant weight. Then, they were weighted and stored at 4ºC for lyophilizing step. Finally, before starting a lipid extraction process, the pellets were lyophilized and weighted. Morphological identification: Morphological identif- ication was carried out through micromorphology under a light microscope (LM microscope, Olympus, CX31) and SEM (VEGA 3, TESCAN). For precise morphological identification, the permanent slide was used with H2O2 treatment. Following Cox and Mann’s method (Cox, 1990), the permanent slide was made with Naphrax and pictures were captured with a DIC and phase contrast microscope (Nikon eclipse 80i). Molecular identification: Total genomic DNA from the cultured sample was isolated based on Liu method (Liu et al., 2000) and stored at -20°C for further analysis. The primers SSU1 5'-AACCTGGTTGATC CTGCCAGT-3' (Kociolek, 2013) and ITS1DR 5'- CCTTGTTACGACTTCACCTTCC-3' (Kociolek, 2013; Edgar and Theriot, 2004) and amplification of the target gene (18S rRNA) were done according to Kociolek protocol (Kociolek, 2013) by PCR (PCR Bio-Rad My Cycler Personal Thermal Cycler, USA). The genomic DNA and PCR product were separated by agarose gel electrophoresis (Bio-Rad Power Pac Universal power supply Electrophoresis, USA). PCR product was sequenced based on the Sanger method and edited by ChromasPro and compared with registered sequences at NCBI by using Basic Local Alignment Search Tool (BLAST). Lipid analysis: Lipid of the normal and prolonged sample was qualitatively analyzed with modified 270 Etesami et al./ Effect of prolonged culture on fatty acid profile of Navicula salinicola Sudan Black B staining method (Jape, 2014; Thakur et al., 1989). The procedure initiated by fixing the air dried slides and flooding the slide in Sudan Black B stain for 20 min at room temperature (prepared about 0.3% Sudan Black B in 70% ethanol). Finally, the slide was stained by Safranin (0.5% aqueous solution) for 5 to 8 seconds (Thakur et al., 1989). The lipid of the lyophilized samples (normal and prolonged) which were in late stationary growth phase were extracted via CHCI3: MeOH (2:1 v/v) solvent mix method (Folch et al., 1957; Axelsson and Gentili, 2014). The samples were homogenized and the residues were removed by filtration after homogenization. 0.73% NaCl solution was added which led to the production of final solvent (2:1:0.8 chloroform: methanol: water (v/v/v)). Solvents were removed by evaporation and the dry extracts were weighted for determination of total lipid percentages in normal and prolonged condition. As a final process, the dry extract of prolonged condition sample dissolved in dichloromethane for further quantitative analysis by Gas Chromatography (GC). In the following, the extracted oil was trans- esterified by two-step procedure (2-TE) (Cavonius, 2014) and the fatty acid profile was determined by GC- young lin 6000 with an oven temperature 145°C isothermal, the injector temperature 280°C, detector temperature 300°C, capillary column-length: 50 m, internal diameter: 0.25 mm, Varian-CP-Sil 88, 60 m. The GC analysis was performed at the Department of Food Science, Iranian National Standards Organiza- tion (INSO) Karaj, Iran, based on the national standard 4090 and 4091. Results Morphological and molecular analysis: Identification of the isolated N. salinicola from the Gohar-Baran port in the Caspian Sea were confirmed according to the morphological and molecular characters explained in Stoermer et al. (1999) and Kociolek (2005). In N. salinicola, the valves were linear-lanceolate, rounded ends with the length-width 7-20, 2-4.5 μm, respectively. Filiform raphe and striae features were slightly paralleled to radiate, convergent at the ends (Fig. 1B, D, E, F). The amplified sequence of small subunit 18S rDNA which BLASTed in NCBI revealed 99% similarity to N. salinicola (FR865499) identified from, San Francisco, California, USA. As a result, this strain was identified as N. salinicola strain IBRC-M 5083 in IBRC (Iranian Biological Research Centre) microalgae collection. Lipid analysis: Determination of total lipids percentages of this strain were obtained 31.83% (DCW) in normal condition and 43.72±1.4% under prolonged culture. In addition, primary screening of lipid vacuoles of N. salinicola under prolonged condition by staining with Sudan Black B indicated blue–black color droplets (Fig. 1C). Finally, the fatty acid composition of prolonged cultivation of N. salinicola was demonstrated by GC analysis (Table 1). The result revealed the high level production of EPA (25.58% TFA) under prolonged culture in this strain. Discussion The diatoms could be an affordable and appropriate source for biotechnological applications, however, studies on diatoms are rare in Iran and need more attention. For this purpose, the isolated sample should be identified in the first step. Taxonomic of diatoms are based upon morphology characteristics such as frustules structure (Round et al., 1990) as well as ribosomal DNA sequencing (Kociolek et al., 2013). It needs to clarify that the ornamentations of frustule have enough information for diatom identification, however, the gene sequences of diatoms in databases Table 1. The Fatty acid profile of Navicula salinicola extracts under prolonged culture (21 d) by GC Strain Fatty acid composition (% of total fatty acids) Navicula salinicola C16:0 C16:1 C18:1 C18:2 C18:3 (n-6) C20:5 4.79 9.28 2.25 0.48 0.07 25.58 271 Int. J. Aquat. Biol. (2017) 5(4): 268-274 are not complete yet and in many cases, it is not feasible to compare the sequence of an isolate with the sequence information in NCBI. The results of this study revealed that the isolate algae is N. salinicola based on morphological and DNA data. Algae produce carbohydrates, lipids and protein in photosynthesis process that depend on environmental factors (Juneja et al., 2013). Environmental conditions like nutrient availability are closely effective on quantity and content of carbon fixation and they lead to change of fatty acids composition as well (Juneja et al., 2013; Sharma et al., 2012). A study about evalua- tion of the nutrients in culture medium on lipid metabolism has shown that limitation of macro- nutrients such as nitrogen, silicon, phosphate can effect on lipid metabolism and contents of neutral lipids (TAGs) (Hu et al., 2008). It should be noted that determining the algae products under variable Figure 1. (a) Navicula salinicola under LM, (B) frustule features in P ermanent slide, (C) appearance of lipid droplets via Sudan Black B staining, (D) striae slightly parallel, (E) a filiform raphe in N. Salinicola., and (F) linear-lanceolate and rounded ends valves. 272 Etesami et al./ Effect of prolonged culture on fatty acid profile of Navicula salinicola environmental factors could be important for understanding how influence these factors on algae. Under prolonged incubation time of microalgae like diatoms, environmental factors could be altered and these changes effect on fatty acid composition and enhance TAGs during stationary phase in many algal species (Hu et al., 2008); for instance, the total lipid content of Nacicula sp. under normal condition was determined as 41.10 (% DCW) which could be improved to 60.28±4.92 (% DCW) under nitrogen starvation (Thajuddin et al., 2015). According to previous study, lipid content of N. salinicola was commonly induced up to 58% under nitrogen deficiency and to 22-49% under silicon deficiency (Nagle and Lemke, 1990). Based on the results, the total lipid content of isolated N. salinicola (IBRC-M 5083) was 43.72±1.4 (% DCW) which was higher than the total lipid content of Nacicula sp. under normal condition but it was lower than under nitrogen limitation conditions. The content of lipids e.g. neutral lipids in the biomass like TAGs are important for biotechnological applications (d’Ippolito et al., 2015) such as aquafeed, Omega3 production and biofuel sources. For example in biofuel sources, linolenic acid and four double bonds in fatty acid methyl esters should not increase to 12% and 1%, respectively according to biodiesel standard EN14214 methods (Juneja et al., 2013). The GC analysis data in our work showed that the amount of linolenic acid in N. salinicola (IBRC-M 5083) is lower than 12%, which encounters the requirements of the European Standard EN 14214 for biodiesel. The above reports and absence of C18:3 demonstrated that N. salinicola (IBRC-M 5083) could be an appropriate source of biodiesel (Matsumoto et al., 2010; Thajuddin et al., 2015). In addition, Nacicula sp. has suitable level of the other fatty acids such as C16:0, C16:1, C17:01 and C18:0 (Matsumoto et al., 2010; Thajuddin et al., 2015). The pervious works showed C16:0 (21.4% TFA) and C16:1 (25.1% TFA) under 200 hrs incubation in Nacicula sp. (Matsumoto et al., 2010) and C17:01 (29.36% TFA) and C18:0 (26.48% TFA) in Naviculoid for 336 hrs (14 days) incubation period under normal condition (Thajuddin et al., 2015). The valuable polyunsaturated fatty acids, especially Omega 3 is one of the valuable products of microalgae. Studies revealed that Omega3 such as Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) have proper effect on human health (Simopoulos, 1991). The results of this research demonstrated that the prolonged culture of N. salinicola produces a high level of EPA (25.58% TFA) after 21 days which agrees with the previous studies (Kitano et al., 1997; Khatoon, 2006; Zheng et al., 2007). However, the level of Omega3 were lower than the results of this study (Kitano et al., 2007; Khatoon, 2006; Zheng et al., 2007). 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(2017) 5(4): 268-274 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2017 Iranian Society of Ichthyology چکیده فارسی تاثیر کشت طوالنی مدت بر محتوای از دریای خزر و بررسی Navicula salinicola (Hustedt 1939) جداسازی اسید چرب آن 5،1، سید ابوالحسن شاهزاده فاضلی4، غالمرضا بخشی خانیکی3،1محمد علی آموزگار ،2*مصطفی نوروزی ،1فرخنده صبا ،1اعتصامی ناحسا .ایران ،مرکز ملی ذخایر ژنتیکی و زیستی ایران، تهران ها،بانک میکروارگانیسم 1 .ایران ،تهران ،س الزهرا دانشگاه ،علوم زیستی دانشکده، بیوتکنولوژی گروه2 .دانشگاه تهران، ایرانعلوم، سیپرد ،موجودات زنده ییقطب تبارزاروبیولوژی، دانشکده زیست شناسی، گروه میک 3 .ایران ،تهران ،پیام نور تهران شرق دانشگاه ،علوم دانشکده، بیوتکنولوژی گروه4 .ایران ،تهران ،علم و فرهنگ دانشگاه علوم پایه و دانشکدهبیولوژی سلولی و ملکولی، گروه5 چکیده: در این تحقیق دیاتوم ناویکلوئید از بخش جنوبی . شوندمی محسوب طبیعی اشباع غیر چرب هایاسید تولیددارای پتانسیل منابع از یکی هادیاتوم و شناسیریخت مشاهده صفات کمک به شده جداسازی آرایهو توانایی تولید و تجمع لیپید در آن مورد ارزیابی قرار گرفت. دریای خزر جداسازی در IBRC-M 5083 دسترسی شماره باو گرفت قرار شناسایی مورد Navicula salinicola عنوانهب 18S rRNA ژنومی طقهمن مولکولی آنالیز در سویه این( روز 21) مدت طوالنی کشت و نرمال کشت تیمارهای اعمال و کشت از پس. نگهداری شد ذخایر ژنتیکی و زیستی ایرانمرکز ملی رنگ توسط لیپیدی ذرات تولید همچنین. آمد بدست سلول خشک وزن درصد 43.72 و 31.83 ترتیب به کل چربی تولید میزان ،f/2 کشت محیط گازی، کروماتوگرافی روش از استفاده با سویه این مدت طوالنی کشت چرب اسید محتوای سنجش. شد مشخص سویه این در Bبلک سودان آمیزی در 3 امگا مناسب محتوای و لیپید تجمع توانایی به توجه با. داد نشان را اسید ایکوزاپنتانوئیک( چرب های اسید کل درصد) 58/25 میزان N. salinicola، زیستی سوخت همچنین و دارویی آبزیان، خوراک صنایع در سویه این از استفاده جهت مناسبی بستر بیشتر تحقیقات با توانمی .نمود فراهم .اسید، دوکوزاهگزانوئیک اسید، ایکوزاپنتانوئیک 3جلبک، امگا :کلمات کلیدی