The nutritional value of diatoms from giao thuy mangrove water of the red river delta biosphere reserve

JOURNAL OF SCIENCE OF HNUE Natural Sci., 2010, Vol. 55, No. 6, pp. 134-140 THE NUTRITIONAL VALUE OF DIATOMS FROM GIAO THUY MANGROVE WATER OF THE RED RIVER DELTA BIOSPHERE RESERVE Le Thi Phuong Hoa(∗), Dang Ngoc Quang and Nguyen Hoang Tri Hanoi National University of Education Nguyen Thi Hoai Ha and Pham Thi Bich Dao Vietnam National University, Hanoi (∗)E-mail: lephhoa@yahoo.com Abstract. Diatoms are predominant in marine water and considered to be the most important primary producers in sustaining marine food chains. Three diatom strains were successfully isolated from Giao Thuy mangrove water of the Red River Delta Biosphere Reserve. The morphology and the 18S rDNA sequence analysis revealed their identification as Navicula radiosa, Navicula tuscula and Melosira nummuloides. Among three media f/2, ASW and ESM, the best medium for the growth of N. radiosa and M. nummuloides is ASW whereas for N. tuscula is ESM. Fatty acid profiles indicated taxonomic similarity between three strains and other diatoms with high concentration of 16:0, 16:1n-7 and 14:0 (approximately 60 - 80% of total fatty acids). M. nummuloides contained higher content of unsaturated fatty acids (45.23%) than the other two diatoms. This strain also had remarkably high levels of polyunsaturated fatty acids, PUFAs (13.61%) and the highest proportion of eicosapentaenoic acid (EPA), 9.21%. These fatty acids play an essential role in cell membrane physiology and hormone metabolism. The result suggests that nutrition value as food of M. nummuloides is higher than that of the other Navicula strains. Keywords: Diatom, mangrove microalgae, fatty acid 1. Introduction Algae are important constituents of various ecosystems ranging from marine to freshwater environments, from hot water springs to snow and ice. They account for more than half the primary production at the base of the food chain [2]. In mariculture, microalgae have an important role as food for all stages of bivalves, for larval stages of crustaceans and fish, and as food for zooplankton which are fed to late larval and juvenile fish and crustaceans [2, 11, 12]. This is based on 134 The nutrition value of diatoms from Giao Thuy mangrove water... the fact that some species of marine microalgae contain large amounts of high- quality polyunsaturated fatty acids (PUFA), which are supposed to be essential in many marine animals due to their limited ability to synthesize from precursor fatty acids such as α-linolenic acid [7, 11]. The nutrition value including fatty acid composition of microalgae has been the subject of many intensive researches [1, 5, 7, 9, 11, 12]. The results provided insight into their role in food chain, aquaculture as well as other commercial applications and biotechnology. There is a lack of investigations of mangrove microalgae although they are thought to be an important energy sources in the mangrove ecosystem food chain [3]. In our study, diatom strains from Giao Thuy mangrove forest were isolated, identified and characterized the fatty acid components. This is the first report on fatty acid constituents of mangrove diatoms. 2. Content 2.1. Material and Methods ∗ Microalgae selection, isolation and identification: Samples were collected from different sites of Giao Thuy mangrove forest. Mi- croalgae were grown at room temperature and illuminated with neon lights (Philips daylight tubes) on 10:14h light: dark cycles. Diatom strains were selected and iso- lated based on their morphological properties [10]. Each strain was photographed under 1000-fold OLYMPUS CX41 microscopy. Total DNA was extracted and 18S rDNA-coding region were amplified and sequenced according to Fawley and Fawley [4]. The 18S rDNA sequences were analyzed using BLAST tool to get the identifi- cation of each algal strain. ∗ Culture medium selection and biomass culture of microalgal strains: In order to select the best suitable culture medium for the growth of algae, each strain was cultured in media f/2, ASW (artificial sea water) and ESM (modified Erd- Schreiber’s and Schreiber’s medium) [6]. Cells were cultured in 150 mL, unaerated media and harvested every two days. Cell density was determined with a Neubauer haemocytometer in three replicates. Biomass for fatty acid analysis was obtained from 1 litre to 4 litres of aerated culture suspensions in plastic containers. Cells were harvested at the early stationery phase. ∗ Determination of fatty acid composition: For determination of fatty acid composition, the algae samples were concen- trated by centrifugation, transferred into a plastic tube and weighted. The ex- traction products were obtained using methanol/chloroform (1:1, v/v). After the samples were evaporated, fatty acids were methylesterified as described by Krienitz et al. [7]. Subsequently, the methylated fatty ester were analyzed by gas chro- matography (Finnigan Trace GC) using an ultra-column BPX70. The identification 135 Le Thi Phuong Hoa, Dang Ngoc Quang, Nguyen Hoang Tri, Nguyen Thi Hoai Ha and Pham Thi Bich Dao of fatty acids was carried out by comparing retention times with retention times of a calibration standard solution. 2.2. Results and discussion 2.2.1. Selection, isolation, and classification of diatom species Based on morphological properties, three diatom strains were selected and isolated from Giao Thuy mangrove water. Two of them are solitary and belong to the genus Navicula, which is the largest and the most diverse genus of algae and the other belongs to the genus Melosira [8, 10]. They were signified as Navi1, Navi2 and M., respectively. - Phylum: Bacillariophyta - Phylum: Bacillariophyta - Class: Bacillariophyceae - Class: Coscinodiscophyceae - Order: Naviculales - Order: Melosirales - Family: Naviculaceae - Family: Melosiraceae - Genus: Navicula - Genus: Melosira SSU rDNA sequence comparison has proved to be a powerful alternative with morphology for inferring phylogenetic relationships at all taxonomic levels. The amplification and sequencing resulted in an 18S rDNA sequence of 1745 bp for Navi1 strain, 1359 bp for Navi2 and 1780 bp for M. strain. Sequence alignment using BLAST tool with NCBI database indicated 100% similarity of Navi1 strain with Navicula radiosa strain AM502034 and Navi2 strain with Navicula tuscula and M. strain with Melosira nummuloides (Figures 1, 2 and 3, respectively). Figure 1. Microscopic morphology of Navicula radiosa This strain is unicelullar with solitary frustules and nar- row valves. Lanceolate is grad- ually tapering from the middle to the acute ends. Axial area is indistinct and the central area is somewhat rounded. Cells are approximately 40 - 44 µm in length and 8 - 10 µm in breadth. Figure 2. Microscopic morphology of Navicula tuscula Cells can be solitary or two cells standing together. Axial area is broader. There are two laminate chromatophores sometimes splitting up into nu- merous small rounded granules. Cells are usually 25 - 25 µm in length and 9 - 16 µm in width. 136 The nutrition value of diatoms from Giao Thuy mangrove water... Figure 3. Microscopic morphology of Melosira nummuloides Cells are cylindrical to subspherical, with high man- tles and well developed gir- dles. They usually form long chains. The auxospore wall is composed of two distinct parts, an organic layer and a layer of siliceous scales. Cell diameter is about 10 - 40 µm. 2.2.2. Selection of Culture Medium Optimization of culture conditions for the selected strains is essential for biomass culture of algae. Furthermore, growth characteristics have been shown to have a significant impact on the lipid and fatty acid profiles [5, 7]. In this study, medium f/2, ASW and ESM were used for algae culture. The cells were collected and counted every two days. Table 1. Cell density of N. radiosa, N. tuscula, M. nummuloides in different culture medium Culture N. radiosa N. tuscula M. nummuloides day (cells x 106 mL−1) (cells x 106 mL−1) (chains x 103 mL−1) f/2 ASW ESM f/2 ASW ESM f/2 ASW ESM 1 3.2 3.2 3.2 2.5 2.5 2.5 0.007 0.027 0.003 3 4.8 8.0 8.0 4.8 3.5 5.2 0.167 0.667 0.167 5 32.8 35.2 35.2 13.5 9.2 15.6 0.2 0.767 0.235 7 60.8 49.6 44.8 31 20.2 43 0.23 8.1 0.33 9 88 137.6 128 78 50 129 3 9.3 2.33 11 43.2 107.2 68.8 48 32 98 0.67 5.33 0.43 The cell density of N. radiosa, N. tuscula, M. nummuloides was variable under different culture conditions (Table 1). The growth of three strains reached the high- est peak after 9 days culture. N. radiosa grew better in ASW medium than in the other two media. The number of cells in these two medium even decreased remark- ably after reaching the growth peak. Meanwhile, it is shown that ESM medium is the most suitable for the growth of N. tuscula, which had the cell density approxi- mately twice as high as the rest. M. nummuloides showed clearly high cell density when being grown in ASW medium, three or four times higher compared with f/2 and ESM medium, respectively. 137 Le Thi Phuong Hoa, Dang Ngoc Quang, Nguyen Hoang Tri, Nguyen Thi Hoai Ha and Pham Thi Bich Dao 2.2.3. Fatty acid composition Among organisms in aquatic food webs, algae is supposed to have the highest ability to synthesize long-chain PUFA. In contrast, most animals are not able to synthesize essential fatty acids [7]. Table 2. Percentage composition of fatty acids in three diatom species Total fatty acids (%) No. Fatty acid Chemical name Common name M. num- mu- loides N. tuscula N. radiosa 1 C 4:0 Butyric acid 1.17 0.12 2 C 10:0 Decannoic acid Capric 1.44 0.32 0.11 3 C 12:0 Dodecanoic acid Lauric 0.61 0.64 0.20 4 C 14:0 Tetradecanoic acid Myristic 7.76 9.69 3.12 5 C 14:1n-5 Tetradecenoic acid Myristoleic 0.80 0.22 6 C 15:0 Pentadecanoic acid Convolvulinolic 0.42 7 C 15:1n-5 Pentadecenoic acid Hormelic 0.70 0.35 8 C 16:0 Hexadecanoic acid Palmitic 37.90 52.56 58.30 9 C 16:1n-7 Axit 9-hexadecenoic Palmitoleic 24.40 13.69 25.29 10 C 17:0 Heptadecanoic acid Margric 0.99 1.20 0.18 11 C 17:1n-7 Heptadecenoic acid 2.49 1.49 0.23 12 C 18:0 Octadecanoic acid Stearic 4.52 3.77 1.26 13 C 18:1n-7 11-Octadecenoic acid Asclepic 8.62 1.93 14 C 18:1n-9 9-Octadecenoic acid Oleic 4.73 1.19 15 C 18:2n-6-t 9,12-Octadecadienoic acid Linoleic 1.30 1.27 16 C 18: 3n-3 9,12,15-Octadecatrienoic acid α-Linolenic acid (ALA) 0.48 17 C 18: 3n-6 6,9,12-Octadecatrienoic acid γ-Linolenic acid (GLA) 0.35 0.11 18 C 18: 5n-3 Octadecapentaenoic acid 1.56 19 C 20:0 Icosanoic acid Arachidic 1.15 20 C 20:4n-3 8,11,14,17- Eicosatetraenoic acid Eicosatetraenoic acid (ETA) 2.05 4.03 21 C 20:4n-6 5,8,11,14- Eicosatetraenoic acid Arachidonic acid (AA) 0.76 22 C 20:5n-3 5,8,11,14,17- Eicosapentaenoic acid Eicosapentaenoic acid (EPA) 9.21 23 C 22: 0 Docosanoic acid Behenic acid 1.04 0.80 24 C 22:4n-6 7,10,13,16- Docosatetraenoic acid Adrenic acid 0.34 25 C 22:6n-3 4,7,10,13,16,19- Docosahexaenoic acid Docosahexaenoic acid (DHA) 1.05 138 The nutrition value of diatoms from Giao Thuy mangrove water... The three diatom strains have a great variety of fatty acids ranging from satu- rated fatty acids (10:0 - 22:0) to polyunsaturated (18:2 - 22:6) fatty acids (Table 2). Among them, the saturated fatty acids palmitic (16:0) and the monounsaturated palmitoleic (16:1n-7) were dominant. They accounted for 60 - 80% of the total fatty acid. Myristic acid (14:0) was at high concentration. These fatty acids are pre- dominant in many diatoms in previous reports [5, 9, 12]. M. nummuloides have the highest content of unsaturated fatty acids (45.23%) in comparison with the two other diatoms, N. radiosa (29.58%) and N. tuscula (33.83%). Its concentration of PUFAs was also remarkably high (13.61% of total fatty acids), which can be an index of high nutritional value for many animals. Most animals and human are not able to syn- thesize essential fatty acids. Fatty acids such as EPA and DHA cannot be produced in sufficient quantities for metabolic functioning and mostly obtained from marine fish oil [5, 7]. M. nummuloides also contained significant amounts of eicosapen- taenoic acid (EPA, 20:5n-3), 9.21%, which plays an important role with arachidonic acid (AA) in the synthesis of eicosanoid compounds, paracrine hormones, including prostaglandins, thromboxanes, and leukotrienes [1]. The highest proportion of EPA as compared with other PUFAs was reported for another species of Melosira genus [9]. N. saprophila possessed high content of EPA under photoautotrophic condition. However, it was found in trace amount in two Navicula strains in this study [5]. Another PUFA, eicosatetraenoic acid (ETA) was detected in M. nummuloides and N. radiosa. Docosahexaenoic acid (DHA, 22:6n-3) plays an important role in the membrane lipids. In this study, it is found only in M. nummuloides at low level. The low level of DHA is reported for most diatoms [7, 12]. 3. Conclusion Three diatom strains were successfully isolated from Giao Thuy mangrove water and identified as Navicula radiosa, Navicula tuscula and Melosira nummu- loides. In order to optimize culture conditions for biomass production, the suitable medium was selected for each strain. Three diatoms showed a huge range of fatty acids among which palmitic (16:0) and palmitoleic acid (16:1, n-7) were the most dominant, as described for other diatoms. M. nummuloides contained remarkable amount of PUFAs and considerable level of EPA, suggesting high value as food. Diatoms are abundant in most aquatic habitats and are considered to be the most important primary producers in sustaining marine food chains. Our results add more data to the increasing search for high-quality food for marine animals as well as for humans. 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