Selection and isolation of some microalgae strains from mangrove in Xuan Thuy national park as food for bivalve larvae

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2012, Vol. 57, No. 8, pp. 56-65 This paper is available online at SELECTION AND ISOLATION OF SOME MICROALGAE STRAINS FROM MANGROVE IN XUAN THUY NATIONAL PARK AS FOOD FOR BIVALVE LARVAE Le Thi Phuong Hoa1, Dang Ngoc Quang2 and Nguyen Thi Hoai Ha3 1Faculty of Biology, Hanoi National University of Education 2Faculty of Chemistry, Hanoi National University of Education 3Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi Abstract. Five microalgal strains, selected and successfully isolated to a unialgal state from mangroves trees in Xuan Thuy National Park, were identified as Navicula tuscula VACC-001, Chaetoceros muelleri VACC-005, Amphiprora alata VACC-007, Chlorella vulgaris VACC-012 and Nannochloropsis oculata VACC-017, based on morphological properties and 18S rDNA sequence analysis. ASW medium was found to be best suitable medium for their growth and biomass production. The fatty acid profile of isolated strains showed taxonomic characteristics. C. muelleri and N. oculata had the highest concentration of polyunsaturated fatty acids (PUFA), accounting for 36 - 50% of the total fatty acids with a ratio of ω-3/ω-6 ranging from 2.5 - 4 and a high level of eicosapentaenoic acid (EPA). A. alata had a significant proportion of arachidonic acid (AA) and docosahexaenoic (DHA) whereas C. vulgaris was found to have a high concentration of essential fatty acids and N. tuscula was at the high level of 14:0 + 16:0 acids, which was necessary for rapid development of larvae. Carbohydrate content of isolated strains was around 5 - 16% dry weight. The results suggest their high potential for use as mixed food in bivalve larvae aquaculture due to algal size, supplementary nutrition value and feasible biomass production. Keywords: Microalgae, mangrove, fatty acid, bivalve larvae, aquaculture. Received June 4, 2012. Accepted September 11, 2012. Biology Subject Classification: 10 605. Contact Le Thi Phuong Hoa, e-mail address: lephhoa@yahoo.com 56 Selection and isolation of some microalgae strains from mangrove... 1. Introduction Xuan Thuy National Park is a part of Red River Delta Biosphere Reserve and it is large mangrove forest in which microscopic algae (microalgae) are highly diverse and provide important food sources for many aquatic animals [11]. Microalgae are natural food for bivalves at all growth stages, for crustaceans and some fish species at the larval and early juvenile stages and for adult zooplanktons [1, 10]. Approximately thirty percent of world algal production is used for animal feed [10]. The importance of microalgae in this application is that they are high in proteins, carbohydrates, lipids, carotenoids, vitamins and especially polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), arachidonic acid (AA) and docosahexaenoic acid (DHA), which cannot be produced in sufficient quantities for metabolic functioning in most marine animals [1, 5]. Many of them are thought to be of nutritional benefit to the larvae and improve their growth and survival rate [7]. Moreover, algae can grow heterotrophically on cheap organic substrates under well-controlled cultivation conditions, which would be beneficial in aquatic culture. A large number of microalgal strains have been selected and studied for aquaculture applications but thus far only a few,Nannochloropsis, Chaetoceros, Chlorella, for example, are widely used in aquaculture [7]. In this study, we attempted to select and isolate some microalgae strains from mangrove areas in Xuan Thuy National Park, to identify the algae, to identify suitable growth conditions and determine their fatty acid composition and carbohydrate content to recognize potential aquaculture applications. 2. Content 2.1. Material and methods 2.1.1. Selection, isolation and identification of microalgal strains Samples were collected from different mangrove sites in Xuan Thuy National Park, Nam Dinh Province, and cultured in 10 ml jars of f/2 medium. Microalgal strains were isolated to a unialgal state using micropipettes and agar plates according to Shirai and et al. [9]. Each strain was photographed using 400-fold OLYMPUS CX41 microscopy. Total DNA was extracted and PCR amplification was performed using Fawley and Fawley’s method [2] with following primers: forward primer 2F: (2-21) 5’-ATCTGGTTGATCCTGCCAGT-3’ or 1315 F: 5’-CGATAAGGAACGAGACCTT-3’ and reverse primer 1794R: (1794 - 1775) 5’-GATCCTTCCGCAGGTTCACC-3’. PCR products were directly sequenced in an ABM Prism 3100-Avant Sequencer. The obtained sequences were analyzed using a BLASTn tool to get the relative identification of each algal species. 57 Le Thi Phuong Hoa, Dang Ngoc Quang and Nguyen Thi Hoai Ha 2.1.2. Culture conditions of microalgal strains Microalgal strains were grown in f/2, ASW and ESM media in 150 mL unaerated flasks [4]. Cells were harvested every two days and counted in a Neubauer haemocytometer in three replicates. Medium providing best growth of algal cells was chosen for biomass culture of each strain. The effect of salinity on the growth of C. muelleriwas also examined with different levels of NaCl concentration from 0%◦ to 40%◦ in f/2 medium. Biomass culture for later analysis was carried out in 500 ml conical flasks and then in 4 L flat-bottom round flasks at room temperature illuminated by neon light (Philips daylight tubes) of 3000 - 4000 lux on 10:14 h light:dark cycles. 2.1.3. Determination of fatty acid composition Microalgal biomass was collected at the early stationery phase using continuous centrifugation at 10000 rpm at 4◦C for 15 min, freeze-dried and extracted thereafter with 10 mL of methanol/chloroform (1:1, v/v). The extracts were concentrated under vacuum and then added to 4 mL of CH3OH-H2SO4 (95:5, v/v) for methylate reaction and stirred at 80◦C for 4 hrs. After that, 2.0 mL of H2Owas added and methyl-esters were extracted with n-hexane [5]. The n-hexane extracts were analyzed using gas chromatography (Finnigan Trace GC) in ultra-column BPX70. Fatty acids were identified by comparing retention times with those of a calibration standard solution. 2.1.4. Determination of carbohydrate content Freeze-dried microalgal biomass was hydrolyzed in 2.5N HCl for 2 hours. The hydrolysate was diluted 4 times and centrifuged at 4000 rpm for 15 minutes. Total carbohydrate content was determined using the phenol-sulfuric acid method [6] using 96-well microplates for spectrophotometric measurement at 490 nm with glucose as the standard. Total carbohydrate content in the samples was calculated based on the standard plot. 2.2. Results and discussion 2.2.1. Isolation and identification of isolated microalgal strains Five microalgal strains, including 3 diatoms, 1 chlorophyte and 1 eustigmatophyte, were selected and isolated to a unialgal state according to standard literature procedures based on morphological properties [8] (Figure 1). Sequence analysis and alignment with sequences on NCBI database gave a positive identity for all strains. Data are shown in Table 1 together with a short description of their morphology. 58 Selection and isolation of some microalgae strains from mangrove... Figure 1. Microscopic morphology of microalgal strains isolated from mangroves in Xuan Thuy National Park Table 1. Characteristics of microalgal strains isolated from mangroves in Xuan Thuy National Park Strain code Sequence length Matched species Morphology VACC 001 359bp Navicula tuscula Cells can be solitary or in pairs and are approximately 20 - 25 µm long and 9 - 16 µm wide. The valve is symmetrical, with ends narrowed, protruded and rounded. The axial area is broad. There are two laminate chromatophores which sometimes split into numerous small rounded granules. VACC 005 652bp Chaetoceros muelleri Cells are usually solitary with oval frustules, about 5 - 10 µm long. Cells are hexagonal in girdle view, with a high mantle. Setae do not touch each other, but arise close to the corners and diverge perpendicularly to the colony axis. Each cell has one large chromatophore. 59 Le Thi Phuong Hoa, Dang Ngoc Quang and Nguyen Thi Hoai Ha VACC 07 678bp Amphiprora alata Cells usually appear as solitary with a thin valve, shrink in the middle and with the two ends rounded. The cell valve surface is a lozenge/wing-shaped S curve. The girdle is figure 8 and two pigments are flattened. VACC 012 692bp Chlorella vulgaris This species usually lives as single cells with spherical shape, about 2 - 10 µm in diameter and without flagella. VACC 017 675bp Nannochloropsis oculata Cells are spherical or slightly ovoid without flagellate, 2 - 4 µm in diameter. Each cell has a plastid lacking pyrenoid. All the isolated strains are of appropriate size for the ingestion of bivalve larvae. They are not poisonous and some of them have been used as live feed for mariculture such as N. oculata, C. muelleri [1,7]. 2.2.2. Selection of culture medium for isolated microalgal strains Microalgae are fed to stock in many aquaculture operations and management of microalgal populations is thus considered to be an integral part of aquaculture. The growth of microalgae and their fatty acid formation is affected by medium composition and environmental conditions [1, 5, 7]. In this study it is shown that growth of all strains was successful in culture media, especially in ASW and f/2 media (Figure 2). Algal growth rate increased on day 4 of culture and reached a maximum on day 9 - 11 in cultures of diatoms and on day 13 in cultures of chlorophytes and eustimanophytes. After this time, growth decreased. Growth rate is an important perameter indicating the relative ecological success of a strain in adapting to its natural or experimental environment [3]. The cell density of each of the isolated strains increased 20 - 40 times when reaching the maximum as compared to the starting point. The cell density of N. tuscula was higher when using an ESM medium but it dropped markedly compared to ASW and f/2 medium in the following days. Therefore, the ASW medium is best for growing all strains. This medium can be made without natural sea water and with simple ingredients suggesting the potential for large scale culture of these strains in experimental situations or in open areas. The effect of salinity on the growth of isolated strains was also examined using different levels from 0%◦ to 40%◦ (data not shown). Salinity is an environmental factor that has a significant effect on the growth and biochemical composition of marine algae [3, 7]. This is an important factor when culturing microalgae at an experimental scale. The growth of all isolated strains was completely inhibited at 0%◦ salinity. However, they were 60 Selection and isolation of some microalgae strains from mangrove... able to grow at 10 - 40%◦ indicating tolerance to a wide range of salinity concentrations. The most suitable concentration of salinity for growth is 20 - 30%◦, similar to the range of salinity presents in the waters in many areas of Xuan Thuy National Park, as previously reported [11]. Those strains can be cultured and used as live mixture food in aquaculture. Figure 2. The growth of isolated strains in different culture media 2.2.3. Fatty acid composition Aquatic animals require dietary lipids to synthesize highly unsaturated fatty acids, hormones and utilize them as an energy source for embryo and larvae development [1, 3]. Marine microalgae appeared to be one of the most promising PUFA producers [5, 7]. Table 2. Fatty acids composition (weight percentage of total fatty acids) of microalgal strains isolated from Xuan Thuy National Park No. Fatty acid Common name N.tuscula C.muelleri A.alata C.vulgaris N.oculata 1 4:0 Butyric 1.17 - - - - 2 10:0 Capric 0.32 - - - - 3 12:0 Lauric 0.64 - 0.63 - 0.41 4 14:0 Myristic 9.69 1.91 13.26 1.53 3.82 5 14:1n-5 Myristoleic 0.80 18.09 - - - 6 15:0 Convolvulinolic - 0.74 1.10 - 0.27 7 15:1n-5 Hormelic 0.70 0.096 0.34 - - 8 16:0 Palmitic 52.56 5.53 14.31 27.43 19.23 61 Le Thi Phuong Hoa, Dang Ngoc Quang and Nguyen Thi Hoai Ha 9 16:1n-7 Palmitoleic 13.69 15.23 13.15 5.15 24.67 10 16:1n-9 Ambrettolic - 2.20 4.47 1.2 - 11 17:0 Margric 1.20 9.52 5.01 2.69 0.42 12 17:1n-7 Heptadecenoic 1.49 - 0.82 6.15 - 13 18:0 Stearic 3.77 1.46 4.15 2.91 0.63 14 18:1n-7 Asclepic 8.62 3.74 4.65 - - 15 18:1n-9 Oleic - - - 20.06 - 16 18:2n-6 Linoleic - - - 8.42 6.15 17 18:2n-6-t Linelaidic 1.27 2.70 - - - 18 18:3n-3 α-linolenic (ALA) - - - 17.46 1.76 19 18:3n-6 γ-linolenic (GLA) 0.35 1.12 - - 0.55 20 18:4n-3 Octadecatetraenoic - 0.22 0.59 - 0.55 21 18:5n-3 Octadecapentaenoic 1.56 - 0.58 - - 22 20:0 Arachidic - 1.05 2.21 4.98 - 23 20:0 Isoarachidic - - 0.92 - - 24 20:1n-7 Paullinic - 0.26 - - - 25 20:1n-9 Gondoic - 0.10 1.45 - - 26 20:3n-6 Eicosatrienoic - - - - 0.24 27 20:4n-3 Eicosatetraenoic - - - - 0.24 28 20:4n-6 Arachidonic (AA) 0.76 7.84 7.97 - 4.57 29 20:5n-3 EPA - 24.76 9.12 - 36.48 30 22:0 Behenic 1.04 - - - - 31 22:4n-6 Adrenic 0.34 - - - - 32 22:5n-6 Docosatetraenoic (DPA) - - 3.65 - - 33 22:6n-3 DHA - - 5.25 - - 34 24:0 Lignoceric - 0.12 2.83 - - Saturated fatty acids 70.39 20.33 44.43 39.55 24.15 Unsaturated fatty acids 29.58 76.36 52.21 59.24 75.22 ω-3 1.56 24.98 15.61 17.46 39.03 ω-6 1.45 10.54 11.62 8.42 11.52 PUFAs 4.28 36.64 27.16 25.88 50.55 (-): not determined 62 Selection and isolation of some microalgae strains from mangrove... The fatty acid composition of isolated strains (Table 2) has shown systematic differences according to taxonomic groups as previously reported [1]. Three diatom strains have fatty acids ranging from 10C to 24C fatty acid. The predominant fatty acids found in diatoms were 14:0, 16:0, 16:1n-7 and 20:5n-3, which in total accounted for 50 - 75% of the total fatty acids (Table 2). The content of 16:0 and 14:0 in C. muelleri VACC-005 was exceptionally low comparing to other diatoms and C. calcitrans in previous reports [1, 7] whereas the content of 14:1n-5 was high. This possibly is due to the occurrence of δ9 desaturation of 14:0. Meanwhile, the predominant in the chlorophytes were 16:0, 18:1 n-9, 18:2 n-6 and 18:3 n-3 (73% of total fatty acids) and those in the eustimanophytes were 16:0, 16:1n-7 and 20:5n-3 (80% of total fatty acids). C. muelleri VACC-005 and N. oculata VACC-017 had highest level of unsaturated fatty acids, more than half of which was PUFAs, 36.64% and 50.55% total fatty acids, respectively. The fatty acid composition and content, especially PUFAs such as EPA and DHA, are of major importance in determining the nutritional value of microalgae. Species of microalgae rich in these PUFAs are generally assumed to be of high nutritive value [5, 10]. Many marine bivalves show a low capacity for synthesis of PUFAs, which are considered essential for growth, development, and cellular function and thus have to acquire them through dietary sources [6]. The ω-3/ω-6 ratio in these two strains is about 2.5 - 4.0. The high ω-3/ω-6 ratio is thought to be of high nutritive value for many animals [7]. C. muelleri VACC-005 and N. oculata VACC-017 had significantly high level of EPA. C. muelleri VACC-005 also contained remarkable amount of AA (20:4n-6), higher than previously reported in Chaetoceros strains [7]. EPA and AA are important nutritional factors which play a vital role in the synthesis of eicosanoid compounds such as prostaglandins, which are precursors of compounds known as tissue hormones [5, 7]. Based on the high proportion of PUFAs, including EPA and AA, C. muelleri and N. oculata strains can be grouped with algae which are of good food quality in the marine ecosystem and can be used as widely as C. calcitrans in aquaculture for bivalve molluscs, crustacean larvae and others, especially in the early stage due to their small size. The concentration of PUFAs in A. alata VACC-007 was not as high as in C. muelleri and N. oculata strains but this strain possessed a considerable proportion of very long-chain PUFAs, DPA and DHA, 3.6% and 5.2%, repectively. DHA is an important membrane component and plays a role in tissue regeneration. C20 and C22 PUFAs were not detectable in C. vulgaris VACC-012 as many other chlorophytes [1]. However, the level of linoleic and α-linolenic in this strain was highest. These are essential fatty acids which most animal cannot synthesize and must aquire from their daily food to form longer chain PUFA. Unlike the above strains, N. tuscula VACC-001 has a high level of saturated fatty acids, most of which is 16:0 (52.56% of the total fatty acids). It was reported that there is relationship between 14:0 + 16:0 content of microalgae and the growth rate of 63 Le Thi Phuong Hoa, Dang Ngoc Quang and Nguyen Thi Hoai Ha Pacific oyster larvae, Crassostrea gigas. It was thought that these saturated fatty acids were beneficial for rapid growth of larvae as they provided energy more efficiently than unsaturated fatty acids [1]. Therefore, a mixture of isolated strains provided at different growth stages of larvae can provide efficient fatty acids for their metamorphosis and development. 2.2.4. Carbohydrate content Figure 3. Carbohydrate content of microalgal strains isolated from mangrove in Xuan Thuy National Park The carbohydrate content of five strains ranged from 5 - 16% dry weight, similar to the result of an analysis in 40 microalgal species by Brown and et al. [1]. C. vulgaris VACC-012 and N. oculata VACC-017 had the highest content of carbohydrates. Carbohydrates play an important role in balancing the utilization of protein and lipids for biosynthesis against catabolism for energy production. It was reported that with an adequate supply of protein and lipids, the use of C. muelleri, containing high levels of carbohydrates, enhanced the growth of oyster juveniles, Ostrea edulis [7]. 3. Conclusion Microalgae are a unique source of high-value compounds such as polysaccharides and, in particular, long-chain PUFAs which animals cannot sufficiently synthesize themselves. Therefore, cultured microalgae remain a critical resource for commercial rearing of marine animals. Five microalgal strains isolated from mangrove of the Xuan Thuy National Park are suggested to be good candidates to serve as mixed live feed for bivalve larvae. They are of different sizes but they are ingestible and digestible for larvae at the various stages of their development. With a diverse fatty acid composition especially rich in PUFAs, they are presumably able to supplement one another and provide an adequate mix of fatty acids and carbohydrates. The success of indoor biomasss production of those strains would increase the potential of large-scale production in open hatcheries. 64 Selection and isolation of some microalgae strains from mangrove... Acknowledgements. This work was supported by the Ministry of Education and Training, Vietnam, through the Hanoi National University of Education (Project number B2009-17-198). REFERENCES [1] M. R. Brown, S. W. Jeffrey, J. K. Volkman, G. A. Dunstan, 1997. Nutritional properties of microalgae for mariculture. 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