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Morphological, Molecular, and Biochemical Characterization of Astaxanthin-Producing Green Microalga Haematococcus Sp

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Morphological, Molecular, and Biochemical Characterization of Astaxanthin-Producing Green Microalga Haematococcus Sp J. Microbiol. Biotechnol. (2015), 25(2), 238–246 http://dx.doi.org/10.4014/jmb.1410.10032 Research Article Review jmb Morphological, Molecular, and Biochemical Characterization of Astaxanthin-Producing Green Microalga Haematococcus sp. KORDI03 (Haematococcaceae, Chlorophyta) Isolated from Korea Ji Hyung Kim1†, Md. Abu Affan1,2†, Jiyi Jang1, Mee-Hye Kang3, Ah-Ra Ko4, Seon-Mi Jeon1, Chulhong Oh1, Soo-Jin Heo1, Youn-Ho Lee3, Se-Jong Ju4, and Do-Hyung Kang1* 1Global Bioresources Research Center, Korea Institute of Ocean Science & Technology, Seoul 426-744, Republic of Korea 2Department of Marine Biology, Faculty of Marine Science, King AbdulAziz University, Jeddah 21589, Saudi Arabia 3Marine Ecosystem Research Division, Korea Institute of Ocean Science & Technology, Seoul 426-744, Republic of Korea 4Deep-sea and Seabed Resources Research Division, Korea Institute of Ocean Science & Technology, Seoul 426-744, Republic of Korea Received: October 15, 2014 Revised: November 4, 2014 A unicellular red microalga was isolated from environmental freshwater in Korea, and its Accepted: November 4, 2014 morphological, molecular, and biochemical properties were characterized. Morphological analysis revealed that the isolate was a unicellular biflagellated green microalga that formed a non-motile, thick-walled palmelloid or red aplanospore. To determine the taxonomical First published online position of the isolate, its 18S rRNA and rbcL genes were sequenced and phylogenetic analysis November 10, 2014 was performed. We found that the isolate was clustered together with other related *Corresponding author Haematococcus strains showing differences in the rbcL gene. Therefore, the isolated microalga Phone: +82-31-400-7733; was classified into the genus Haematococcus, and finally designated Haematococcus sp. Fax: +82-31-400-7857; KORDI03. The microalga could be cultivated in various culture media under a broad range of E-mail: [email protected] pH and temperature conditions. Compositions of the microalgal cellular components were † These authors contributed analyzed, and its protein, carbohydrate, and lipid compositions were estimated to be equally to this work. 21.1 ± 0.2%, 48.8 ± 1.8%, and 22.2 ± 0.9%, respectively. In addition, D-glucose and D-mannose were the dominant monosaccharides in the isolate, and its amino acids were composed mainly of aspartic acid, glutamic acid, alanine, and leucine. Moreover, several polyunsaturated fatty acids accounted for about 80% of the total fatty acids in Haematococcus sp. KORDI03, and the astaxanthin content in the red aplanospores was estimated to be 1.8% of the dry cell weight. To the best of our knowledge, this is the first report of an Haematococcus sp. isolated from Korea, which may be used for bioresource production in the microalgal industry. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2015 by Keywords: Haematococcus sp. KORDI03, astaxanthin, bioresource production, phylogenetic The Korean Society for Microbiology analysis and Biotechnology Introduction species have been taxonomically classified in the genus Haematococcus at AlgaeBase (http://www.algaebase.org/), The genus Haematococcus described by Flotow (1844) is a and H. pluvialis Flotow has been thoroughly investigated freshwater unicellular microalga belonging to the class among these species. This microalga tends to accumulate Chlorophyceae [10, 36]. It is widely distributed worldwide, the valuable carotenoid pigment, which is known as and its natural habitats are characterized by their unstable astaxanthin (3,3’-dihydroxy-β,β-carotene-4,4’dione), that temporary conditions, such as small rock pools, water can protect against high light intensity and oxygen radicals holes, and other small natural (or artificial) water bodies, [28, 38]. However, to the best of our knowledge, there have including birdbaths [10, 25]. To date, a total of seven been no reports on the isolation of Haematococcus in Korea. Feburary 2015 ⎪ Vol. 25⎪ No. 2 239 Kim et al. Astaxanthin is one of the most valuable ketocarotenoids and BG-11 medium under the same culture conditions described owing to its biological function as a vitamin A precursor above. The growth characteristics of the microalgal isolate was [16] and as a more efficient antioxidant than β-carotene and determined at a range of salinity, pH, and temperature conditions, vitamin E [24]. Although the majority of this carotenoid is with salinity varying from 0 to 50 psu (adjusted using NaCl), pH synthetically derived, it is generally used as a food coloring ranging from 3 to 11 (adjusted using NaOH or HCl), and temperature ranging from 10°C to 35°C under the same conditions agent or natural feed additive in aquaculture and poultry, as mentioned above, excluding the variants. and for medicinal and cosmetic applications owing to its efficient antioxidant capacity [28]. Astaxanthin is produced Microscopy by several microalgae and other microorganisms, such as The isolated microalga was examined under a light microscope Haematococcus [38], Chlorella zofungiensis [4], the red yeast (LM, Eclipse 80i; Nikon Co.). Images were obtained using a Phaffia rhodozyma [6, 32], and the marine bacterium camera (DXM 1200C; Nikon Co.), and the cellular size was Agrobacterium aurantiacum [15, 37]. The content of astaxanthin calculated with an image analyzer (NIS-Elements BR 3.0; Nikon reported in these microorganisms was between 0.04% and Co.). For scanning electron microscopy (SEM) analysis, the 2.7% of the dry cell weight [6, 11, 17, 21, 32], and the green microalgal cells were fixed in 2.5% glutaraldehyde, post-fixed in microalga Haematococcus is known as the richest source of 2% osmium tetroxide, dehydrated in an ethanol series (30.0%, natural astaxanthin production among these microalgae 50.0%, 70.0%, 80.0%, 90.0%, 95.0%, and 100%), dried with 100% [28]. hexamethyldisilazane, and then gold-coated for 90 sec in a Sputter Coater (SCD050; BAL-TEC). Coated samples were examined by Since 2008, we have screened several microalgae strains field emission SEM (AURIGA; Carl Zeiss). For ultrastructural with advantageous properties for the production of analysis, the cells were fixed, post-fixed, stained with 0.5% uranyl biodiesel and high-value products. In this study, we acetate, dehydrated as described above, transited with 100% identified and characterized a new strain of Haematococcus propylene oxide, and embedded in Spurr’s resin [34]. The samples (herein designated Haematococcus sp. KORDI03), which were sectioned using an ultra-microtome (EM UC7; Leica was isolated at the Korea Institute of Ocean Science & Microsystems), stained with 2% uranyl acetate and lead citrate, Technology (KIOST, Ansan, Korea). The morphological and examined using transmission electron microscopy (TEM; and biological properties of the isolated microalga were JEM1010; JEOL Ltd.). investigated, and its taxonomical position was analyzed based on the molecular characteristics. Furthermore, the Molecular Identification and Phylogenetic Analysis composition of the cellular components (including Genomic DNA was extracted using a DNeasy Blood & Tissue kit astaxanthin) in the isolate was determined, showing that (Qiagen) according to the manufacturer’s instructions. Amplification of the 18S rRNA gene was performed with primers SR1 [20] and this isolate is a strong candidate for the production of high- 18SC2M (5’-GCA GGT TCA CCT ACG GAR ACC-3’), which were value products. slightly modified based on the 18SC2 primer [3]. PCR conditions were as follows: initial denaturation at 94°C for 5 min, 30 cycles of Materials and Methods 94°C for 40 sec, 50°C for 40 sec, and 72°C for 2 min, and a final extension at 72°C for 10 min. To amplify the ribulose-1,5- Microalgal Isolation and Culture Conditions bisphosphate carboxylase/oxygenase large subunit (rbcL) gene In April 2009, pale-reddish standing water was observed by the from the isolate, primers ChlorRbclF (5’-GCN GGT GTW AAA naked eye on the rooftop surface of a building (longitude GAY TAY GG-3’) and ChlorRbclR (5’-TAC CAC CWG AAG CWA 126°83’E, latitude 37°29’N) at the KIOST, and it gradually turned CHG GCA-3’) were designed based on the conserved regions of dark red. Sparsely aggregated red microalgal populations were the known sequences from Chlorophyta, and PCR was performed observed in the standing water by light microscopy; therefore, the with the following steps: pre-denaturation at 95°C for 3 min, water (average temperature 23.8°C, pH 7.5) was sampled in May followed by amplification for 30 cycles at 95°C for 30 sec, 52°C for 2009. From the collected water sample, red microalgal cells were 60 sec, and 72°C for 90 sec, and a final extension at 72°C for 7 min. isolated using micropipettes, and serially diluted and incubated in The amplified approximately 1 kb PCR product was cloned into 96-well plates at 25°C under 12 h light/12 h dark cycles at the pCR2.1-TOPO vector using the TOPO TA Cloning kit 100 µmol photons/m2/s in BG-11 medium [1]. The uniagal wells (Invitrogen) and sequenced at Macrogen Inc. (Korea). Sequencing were then streaked on BG-11 agar plates and cultured under the of the middle regions in the amplified 18S rRNA fragments was same conditions mentioned above, and single colonies were performed using the 18SJMID primer (5’-CAA TAG CGT ATA repeatedly subcultured until a pure isolate was obtained. The TTT AAG TTG-3’). growth of the isolated microalga was examined on Tris-acetate The obtained 18S rRNA and rbcL sequences were compared phosphate (TAP) medium [22], Bold’s basal medium (BBM) [30], with the sequences of related taxa obtained from the GenBank J. Microbiol. Biotechnol. Haematococcus sp. KORDI03 Isolated from Korea 240 database at the National Center for Biotechnology Information initial oven temperature of 50°C, an injector temperature of 250°C, using a BLASTN search (http://www.ncbi.nlm.nih.gov/BLAST). and a detector temperature of 320°C. The oven temperature was The sequences were aligned with other sequences using MEGA then ramped at 10°C/min to 120°C and 4°C/min to a final 4.0, and nucleotide differences and genetic distances among the temperature of 300°C.
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