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Isolation and Identification of Microalgae As Omega-3 Sources from Mangrove Area in Aceh Province

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Isolation and Identification of Microalgae As Omega-3 Sources from Mangrove Area in Aceh Province Proceedings of The 3rd Annual International Conference Syiah Kuala University (AIC Unsyiah) 20 3 In conjunction with The 2nd International Conference on Multidisciplinary Research (ICMR) 20 3 October 2-4, 20 3, Banda Aceh, Indonesia Isolation and identification of microalgae as omega-3 sources from mangrove area in Aceh Province Sri Haryani Anwar, M. Ikhsan Sulaiman, Syarifah ohaya, Novi Safriani Department of Agricultural Product Technology, Syiah Kuala University Banda Aceh 23 , Indonesia Corresponding Author: sri.haryani%unsyiah.ac.id ABSTRACT. Omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (D,A) are essential fatty acids with numerous health .enefits. The main sources of these fatty acids are fish and fish oils. ,owever, fish supply is limited and the availa.ility is uncertain. There are two main pro.lems with regard to fish supply: overfishing and mercury contamination. In addition, food enrichment with fish oil is apparently challenging due to sensitivity of fish oil to oxidation, and fishy smell which ma/e them less attractive to consumers. Based on a.ove pro.lems, alternative sources of EPA and D,A must .e found. An alternative for omega-3 fatty acids are microalgae. This research aims to isolate, identify, and find microalgae .iodiversity that are a.le to produce omega-3 fatty acids in their cells. 0icroalgae were isolated from fallen, senescent leaves of mangrove tree (Kandelia candel) in two locations in Aceh. The colonies were identified morphologically using light and stereo microscopes with 111x magnifications. The results were compared with those in literatures. This research has isolated and identified some genus of microalgae that were predicted .elong to order 2a.yrinthulida and family Thraustochytriidae. 0orphological o.servations have also confirm identification of one fungi, genus Mortierella. All identified microalgae and fungi are omega-3 fatty acids producers, mainly EPA and D,A. Key words: microalgae, omega-3 fatty acids, EPA, D,A, 2a.yrinthulid, Thraustochytrium. Introduction 3esearch in the field of polyunstaurated fatty acids (PU4A) produces enormous outcomes in regards to the .eneficial effects of PU4A to human health. The most important claims are: PU4A is effective in lowering cholesterol, decreasing the ris/ of arrhythmia, lowering the .lood pressure, preventing dia.etes in pregnancy, and relief of 5oints in arthritis (0c0urray, 2117). Both omega-3 and omega-7 PU4A are precursors of hormone-li/e compounds, which are involved in many important .iological processes in human .ody (Trautwein, 211 ). In early 811s, there was a term called 9:itamin 49 for omega-3 fatty acids which attracted research attention (Cannon, 2118). Incorporation of PU4A into food products is dominated .y omega-3 fatty acids (α-linolenic acid (A2A) C 8:3n-3, eicosapentaenoic acid (EPA) C21:5n-3, docosahexaenoic acid (D,A) C22:7n-3) (Augustin and Sanguansri, 2113). The main sources of omega-3 fatty acids are fish and seafood, particularly cold-water fish. ,owever fish sustaina.ility as the main source of essential fatty acids is now uncertain .ecause of mercury contamination issue and overfishing (Cannon, 2118). A.ove-mentioned pro.lems have encouraged researchers to find alternative sources for omega-3 fatty acids. It is well /nown that fish does not produce fatty acids instead they are coming from the microalgae as the fish feed. 0icroalgae are microorganisms that produce fatty acids in their cells. Singh et al., (21 ) reported that Botryococcus braunii, Crypthecodinium cohnii, Schi.ochytrium sp. (thraustochytrids) are a.le to produce 25-75%, 21%, and 51-77% oil (dry weight), respectively. Exploration of microalgae as omega-3 fatty acids producers is a relatively new field and research on this topic has increased considera.ly in the last few years. Isolation and identification of microalgae were carried out worldwide and the results sometimes specific according to the region and climate where they were isolated. In Indonesia, a study of 159 Proceedings of The 3rd Annual International Conference Syiah Kuala University (AIC Unsyiah) 20 3 In conjunction with The 2nd International Conference on Multidisciplinary Research (ICMR) 20 3 October 2-4, 20 3, Banda Aceh, Indonesia PU4As production from microalgae using glycerol had .een conducted in 21 1. This research found seven strains of microalgae that produce D,A. The amount of .iomass resulted was in the range of 2.48-8. 4 g/2 (Basu/i et al., 21 1). In Aceh Province, isolation and identification of microalgae from mangrove area have never .een done. Base on the fact that research on microalgae is growing significantly elsewhere, in Indonesia it seems that this field is not fully explore yet. A num.er of studies on .iodiesel from microorganisms are quite many however researches on microalgae as sources of omega-3 fatty acids are scarce. 0icroalgae contain .ioactive components that have significant .enefit to human health such as carotenoid, polysaccharides, antioxidants, and essential fatty acids. Some microorganisms reported to produce edi.le oils that contains omega-3 fatty acids are from genus Schi.ochytrium, Thraustochytrium and Ul/enia. The species of microalgae that are a.le to produce EPA including 0it.schia spp., 0annochloropsis spp., 0avicula spp., Phaeodactylum spp., Porphyridium spp., and Isochrysis galbana. Other species found with high amount of D,A in heterotrophic conditions are Crypthecodinium cohnii dan Schi.ochytrium spp. (Aard and Singh, 2115). Material and Methods Materials and e1uipment 3esearch materials used were natural sea water, distilled water, yeast extract peptone (BEP) media (consist of glucose (Difco), yeast extract (0erc/), mycological peptone (Sigma), .acto agar (Difco), anti.iotics (penicillin and streptomycin), lugol staining, and 2CB (2actophenol Cotton Blue). The equipment and apparatus to support isolation and identification process were light microscope (Olympus 0odel BC4 T4) equipped with camera (Olympus DP 2), light microscope (Ni/on Eclipse 51i), inverted or stereo microscope (Ni/on), autoclave, incu.ator, master refractometer (Atago-Eapan), p, meter, analytical scale, micropipette (Socorex- Swiss), paraffin, .ea/er glass, petri dish and other microbiological glassware. Isolation and identification process Samples were collected from two locations of mangrove area in Aceh Besar District: ,utan Kota BNI and in Neuheun-U5ung Batee, Aceh Besar. Three types of leaves .rought to the la.oratoryF dar/ .rown, .rown, and yellow-.rown colour leaves. Upon arrival in the la., they were cut into small pieces and grown into media which consisted of yeast extract peptone as descri.e .y 4an et al., (2112). Isolation process and microbiological experiments were carried out at 0icro.iology 2a.oratory, Dept. of Agricultural Product Technology, Syiah Kuala University. Identification process using microscopes were done at 2a.oratory of Pests and Plant Diseases, 4aculty of Agriculture and Pathology 2a.oratory-4aculty of :eterinary, Syiah Kuala University in Banda Aceh-Indonesia. The petri dishes were incu.ated for 2-3 days at 25°C and the colonies growth were o.served daily using light microscope. At particular, the coloniesG morphology (shape and colour) were monitored .y stereo microscope and each different colony found must .e su.- cultured immediately into the new media. The process was repeated until axenic cultures were obtained. If the colonies were not axenic after su.-culturing, then they must .e followed .y a serial dilution process. An axenic culture was characteriHed .y cells that appears in the same shape, colour, and there was no contamination at any part. These morphological characteristics were closely monitored using light microscope (Olympus 0odel BC4 T4) equipped with camera (Olympus DP 2) with 111C magnification. The results were compared to literature according to the morphological point of view. 160 Proceedings of The 3rd Annual International Conference Syiah Kuala University (AIC Unsyiah) 20 3 In conjunction with The 2nd International Conference on Multidisciplinary Research (ICMR) 20 3 October 2-4, 20 3, Banda Aceh, Indonesia esults and Discusion Morphological identification 0icroalgae /nown as omega-3 fatty acids producers are classified into phylum ,eterokontophyta (Stramenopiles), order 2a.yrinthulida, and family Thraustochytriidae. There are 2 genus in family Thraustochytriidae .ut genus which significantly produce EPA and D,A are Aplanochytrium, 2a.yrinthuloides, SchiHochytrium, Thraustochytrium, dan Ul/enia (Aard and Singh, 2115). The first group that firstly identified in this research using stereo microscope was colonies with core or pellet surrounding .y white filament as can .e seen in 4ig . 4igure shows identification steps of 0ortierella: (a) white colonies growth on solid Beast Extract Peptone media, (.) o.servation using stereo microscope, and (c) the cellsG morphology under light microscope with 111x magnification. Using stereo microscope, the colonies were seen in dar/ green colour particularly the cores ( .). If one single touch of the colony is transferred into a glass ob5ect followed .y lugol staining, the cells are as in 4ig. c. 4igure . 0orphological o.servation of 0ortierella According to the literatures, this group is /nown as filamentous microorganisms which can .e categoriHed into fungi (genus 0ortierella). The fungi is morphologically characteriHed .y Ihairy pelletG or IfilamentousG mycelia (Par/ et al., 211 ). 4igure 2 shows the mycelia of Mortierella alpina. 0ortierella
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