isolation of heterotrophic marine microalgae thraustochytrid producing carotenoid in ca mau province

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY ISOLATION OF HETEROTROPHIC MARINE MICROALGAE THRAUSTOCHYTRID PRODUCING CAROTENOID IN CA MAU PROVINCE SUPERVISOR STUDENT TRAN THI XUAN MAI DUONG TAN PHAT Student’s code: 4085175 Session: 34 (2008- 2013) Can Tho, 2013 APPROVAL SUPPERVISOR TRAN THI XUAN MAI STUDENT DUONG TAN PHAT Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract Microalgae are popular organisms employed for carotenoid production. Heterotrophic microalgae Thraustochytrid group is one of potential sources of carotenoid. Heterotrophic cultivation meets more profits than phototrophic cultivation such as cheap, simple for growing and easily to maintain on a large scale. In this study, nine isolates of heterotrophic marine microalgae were isolated from fallen submerged leaves in marine zones at Ca Mau province. Three isolates CM1, CM3 and CM6 appeared as pigmented colonies and revealed high carotenoid producing ability with total carotenoid as 6056, 4274 and 4769µg/kg dry weight, respectively. They were considered as suitable candidates for carotenoid production. Also, the isolates were identified by using a set of universal primers designed according to 18S rRNA gene sequences of several Thraustochytrium microalgae striatum, spiecies including Thraustochytrium aureum, Thraustochytrium aggregatum, multirudimentale, Thraustochytrium Thraustochytrium pachydermum, and Schizochytrium aggregatum. The identification showed that all the isolates might belong to the target species. Key words: carotenoid, Thraustochytrid, universal primers i heterotrophic microalgae, CONTENTS ABSTRACT………………………………………………...........i CONTENTS..………………………..……………...……....…...ii 1. INTRODUCTION ..................................................................... 1 2. MATERIALS AND METHODS .............................................. 3 2.1. Materials ............................................................................. 3 2.2. Methods .............................................................................. 3 2.2.1. Isolation of heterotrophic microalgae............................ 3 2.2.2. Creation of calibration curve of OD value and dry weight ............................................................................................ 4 2.2.3. Determination of carotenoid content ............................. 5 2.2.4. Designing universal primers.......................................... 6 2.2.5. Identification of the isolates of microalgae by universal primers........................................................................................... 6 2.2.6. Statistical analysis ......................................................... 7 3. RESULTS AND DISCUSSIONS ............................................ 8 3.1. Isolation of heterotrophic microalgae............................... 8 3.2. Creation of calibration curve of OD value and dry weight ............................................................................................ 9 3.3. Determination of carotenoid content .............................. 10 3.4. Designing universal primers ........................................... 12 3.5. Identification of the isolates of microalgae by universal primers......................................................................................... 13 4. CONCLUSIONS AND SUGGESTIONS ............................... 16 ii 4.1. Conclusions...................................................................... 16 4.2. Suggestions ...................................................................... 16 REFERENCES ............................................................................ 17 iii 1. INTRODUCTION Carotenoid is a form of organic pigment found in plants, algae, some fungi and bacteria. There are about 600 types of carotenoid currently and they are arranged in two main groups including xanthophyll (with oxygen in their hydrocarbon structures) and carotene (without oxygen in their hydrocarbon structures). Carotenoid plays important roles in activity of immune system, preventing carcinogens and maintaining visual system. However, mammals are unable to synthesize these pigments and have to absorb them from food. To date, carotenoid has been applied extensively in food industry, cosmetics and pharmaceuticals and used as a source of supplement food for livestocks, poultry and aquatic animals as well. Demands of consumers on natural products and pigments produced from biology sources have resulted in rise of microalgae culture. Phototrophic microalgae are popular organisms used in carotenoid production but there are limitations on their growth. Recently, heterotrophic marine microalgae in Thraustochytrid group have attracted much interest because of their capacity of production of polyunsatured fatty acids and carotenoid. Also, their easy cultivation and simple growth condition without requirements of light, CO2 are notable (Ratledge, 1993). Researches on Thraustochytrid have been considered only few years ago in Vietnam, yet there is no report about these microalgae in Mekong Delta and their applications in production of supplement food for human and animals. 1 Objectives To isolate and select several heterotrophic marine microalgae strains in Thraustochytrid group with high carotenoid content at Ca Mau province. 2 2. MATERIALS AND METHODS 2.1. Materials Submerged leaves of mangrove (Rhizophora apiculata blume) and crabapple mangrove (Sonneratia caseolaris) at various stages of decay in national beach and mangrove forest in Ca Mau province. Medium for isolating and growing: GYPS agar, GYPS broth (Glucose 0.3%, yeast extract 0.2%, peptone 0.1%, natural sea water : distilled water (1:1)) (Perveen et al., 2006), and added antibiotics (Streptomycin 300 mg/l and Penicillin 200 mg/l) before use. Chemicals and equipments in Genetic Plant engineering. Laboratory. 2.2. Methods 2.2.1. Isolation of heterotrophic microalgae This activity was carried out based on Bremer’s process with some modifications to adjust lab condition. a. Sample preparation Collected some intact pieces of leaf and removed decayed parts. Cleaned the leaf twice by natural sea water (NSW) solution including seawater and water (rate of 1:1). Removed stem, vein, and then cut the leaf in small pieces. Soaked leaf samples in sterilized NSW about 15 minutes. Poured off liquid, took those 3 samples into solution of sterilized NSW and distilled water (rate of 1:1) and cleaned the leaf samples by stirring for 20 minutes. Repeated this step 2 times. Continued to stir-to-clean the samples in solution of sterilized NSW and bi-distilled water (rate of 1:1) in sterile condition. Repeated this step 2 times. b. Isolation procedure Cultured the treated leaf samples in medium GYPS agar plates. Incubated these plates in dark, at room temperature, for 48 hours. Then, these plates were wrapped by parafilm and incubated in dark, at room temperature, for 48 hours. After that, transferred colonies around the leaf samples to a fresh GYPS agar plate. Observed and selected colonies with microalge-like morpholony. Continued to transfer the selected colonies to another fresh medium and repeated the subculture until axenic cultures were obtained. 2.2.2. Creation of calibration curve of OD value and dry weight This activity created a calibration curve which was applied with OD values to determine biomass dry weight rapidly. Biomass was obtained by growing the isolate in medium GYPS broth for days. First of all, suspended the isolates colonies in 5 ml of sterilized medium GYPS broth in tubes. The culture was incubated at room temperature and shaken at 200 rpm for 4 days. Then, inoculated all the microalgae liquid to 200 ml of sterilized medium GYPS broth in flasks. The culture was incubated at room temperature and shaken at 200 rpm for 4 days. Measured optical 4 density of the isolates liquid at 600nm of wavelenght. Centrifuged the microalgae liquid at 5000 rpm for 10 minutes, at 8oC to harvest biomass of the isolates. Microalgae biomass was dried in a drier at 65oC for 1-2 days. And quickly weighed the dried biomass after being removed from the drier. Used OD values and dry weights for creating the calibration curve by Excel program. 2.2.3. Determination of carotenoid content This activity examined the carotenoid producing capability of the isolates. Microalgae biomass was crushed with glass beads and extracted with 10ml acteone 90%. Put the crushed mixture at 3-4oC. Filtered this mixture on GF/C filterpaper and filled to volume of 10ml by acetone 90%. Proceeded to measure optical density of the obtained transparent liquid at 480nm of wavelenght and calculated carotenoid content according to the following formula (Strickland and Parson, 1972): Pigment content (µg/l) = C / V Where: V: volume of microalgae liquid employed for filtering C (total carotenoid) = 4.0 * E480 E480: OD value at 480nm of wavelenght. This activity was carried out in triplicates. 2.2.4. Designing universal primers A set of universal primers was designed based on similar regions in 18S rRNA gene sequences of several target microalgae species in Thraustochytrid group including Thraustochytrium 5 striatum, aggregatum, Thraustochytrium aureum, Thraustochytrium Thraustochytrium multirudimentale, Thraustochytrium pachydermum and Schizochytrium aggregatum. For designing universal primers, nucleotide sequences of 18S rRNA gene of target species were taken from GenBank of the website NCBI. Used DNAMAN 4.0 software to align the sequences and found out similar region. According to this region, designed a set of universal primers for all of the target species. Ordered for primers production and checked the size of product amplified from the primer sequences. 2.2.5. Identification of the isolates of microalgae by universal primers a. DNA extraction Microalgae were grown in 5 ml of GYPS broth for 4 days before DNA extraction. DNA of the isolates were extracted according to method of Hong et al. (2008) with some modifications to adjust lab condition. Centrifuged microalgae liquid and remove supernatant to harvest cultured cells. After that, the samples were immediately suspended in Extraction buffer (10 ml EB and 7µl beta mercapthoethanol) and incubated at 55oC for 15 minutes. Gently shook the solution at 4oC for 1 hour. Then, centrifuged the solution at 13000 rpm at 4oC for 5 minutes. Collected and added the supernatant 0.1V Sodium acetate 3M (pH 5.2) and 2V absolute ethanol to precipitate total DNA for 15 minutes of incubation at room temperature. Centrifuged it at 13000 rpm for 6 10 minutes to obtain DNA pellet. Washed DNA twice with ethanol 70% by centrifugation at 13000 rpm at 4oC for 5 minutes. Dried and resuspended the pellet in TE 0.1X and preserved it at -20oC. b. PCR technique and gel electrophoresis Polymerase chain reactions were done to amplify the target region of the isolates DNA with the set of universal primers. The PCR mixture (25 µl) contained 50-100ng of DNA, 2.5 µl of buffer 10X, 4µl of dNTPs, 3 µl of MgCl2 25mM, 0.4µM of each primer, 0.25 µl of Taq polymerase (5U/µl), and distilled water. Thermal program was set based on the designed universal primer sequences. Thermal cycler was basically set for amplification as follows: 95°C - 5 minutes; 95°C - 45 seconds (denaturation); 55°C - 45 seconds (annealing); 72°C - 1 minute (DNA synthesis, elongation); repeated for 35 cycles; 72°C - 5 min; and 10°C hold. PCR products were analyzed by agarose gel electrophoresis. Used gel 1.5% for analyzing DNA. The electrophoresis was run at stable voltage. Observed product under UV light and compared the product size with Ladder. Identification was successful when the size of amplified product was similar to theoretical size. 2.2.6. Statistical analysis Data were analyzed statistically by Statgraphics Centurion XV software. 7 3. RESULTS AND DISCUSSIONS 3.1. Isolation of heterotrophic microalgae All heterotrophic microalgae strains were isolated from submerged leaves at various stages of decay in the marine zone. As a result, there were nine microalgae strains isolated successfully and named as CM1, CM2, CM3, CM4, CM5, CM6, CM7, CM8 and CM9. According to morphological characteristics of heterotrophic microalgae, shapes of colonies and cells were described by observing through naked eyes and light microscope 100X. From crabapple mangrove leaves, there were five isolates including CM3, CM4, CM5, CM6 and CM7 with most of their milky white colonies. In particular, isolates CM3 and CM6 showed their pigmented colonies with orange and yellow in color, respectively. The colony of these five isolates appeared in several forms such as smooth-convex, rough-convex and rough-flat. The shapes of their cells mainly were oval or spherical. Four remaning isolates originated form mangrove leaves including CM1, CM2, CM8 and CM9. Most of their colonies were milky white and convex and smooth. In particular, isolate CM1 had light pink colonies. The margins of these four isolates were various, with entire, filiform or curled forms. The shapes of their cells mainly were oval or suboval. Morphology of the isolates colonies and cells were displayed briefly in Table 6’. 8 Table 6’. Morphological characteristics of the isolates Isolates Colony morphology (color, shape, margin form) Cell morphology CM1 Light pink, smooth-convex, entire Oval, small CM2 White, smooth-convex, curled Oval, small CM3 Orange, rough-convex, curled Spherical, big CM4 White, rough-flat, filiform Oval, small CM5 White, rough-convex, curled Spherical, big CM6 Yellow, smooth-convex, entire Spherical, big CM7 White, rough-flat, filiform Oval, small CM8 White, smooth-convex, entire Oval, series, small CM9 White, smooth-convex, filiform Suboval, small Generally, the isolates cell was oval or spherical, and several isolates (CM1, CM3 and CM6) owned pigmented colonies. These are also the characteristics of heterotrophic microalgae Thraustochytrid strains in previous studies (Honda et al., 1999; Aki et al., 2003; Chatdumrong et al., 2007; Hoàng Thị Lan Anh et al, 2010; Arafiles et al., 2011) 3.2. Creation of calibration curve of OD value and dry weight Dry weight of biomass was determinated according to description in section 2.2.2. Calibration curve of each isolate was created based on OD of microalgae liquid at 600nm and its dry weight. According to results of creation of calibration curve, the regression equations of the isolates were exhibited as following Table 7’. 9 Table 7’. Regression equation of calibration curve of isolates Isolates Regression equation R2 CM1 Dry weight = OD x 0.0935 – 0.0011 0.997 CM2 Dry weight = OD x 0.0816 – 0.0033 0.999 CM3 Dry weight = OD x 0.1886 + 0.0004 0.998 CM4 Dry weight = OD x 0.1062 – 0.0049 0.9978 CM5 Dry weight = OD x 0.176 + 0.0052 0.9976 CM6 Dry weight = OD x 0.0346 + 0.0027 0.9945 CM7 Dry weight = OD x 0.0537 – 0.0014 0.9973 CM8 Dry weight = OD x 0.0504 – 0.0031 0.9984 CM9 Dry weight = OD x 0.0265 + 0.0018 0.9953 Those calibration curves play important role in rapid determination of dry weight of biomass through measuring optical density of microalgae liquid of the isolates. 3.3. Determination of carotenoid content After 4 growing days in GYPS broth, microalgae biomass was employed for extracting carotenoid. Table 8’ showed total carotenoid of the isolates. As a result, total carotenoid contents extracted from the isolates were relatively different. The highest total carotenoid content (6056 µg/kg dry weight) was present in isolate CM1. The lowest content (528 µg/kg dry weight) belonged to isolate CM8. Especially, the isolates which contained high carotenoid content had pigment in their colonies and converted their growth medium liquid color to dark orange or yellow. This issue demonstrates the intracellular accumulation of carotenoid. Comparison of carotenoid content of isolate CM1 and 10 heterotrophic strain Thraustochytrium sp. TN22 isolated by Hoang Thi Lan Anh et al. (2010) indicated that isolate CM1 are able to produce a higher total carotenoid cotent. Table 8’. Carotenoid content in the isolates Isolates Total carotenoid (µg/kg dry weight) CM1 6056a ± 689 CM2 2984c ± 478 CM3 4274b ± 476 CM4 1486d ± 426 CM5 3017c ± 291 CM6 4769b ± 141 CM7 811de ± 189 CM8 528e ± 79 CM9 4067b ± 795 Note: Data are expressed as mean of triplicate flasks. Means followed by the same letter in the same column were not significantly different. Furthermore, comparing to some autotrophic microalgae which are commonly used as a source of carotenoid production such as Dunaliella salina and Haematococcus pluvialis, the isolate CM1 is much lower in total carotenoid content. However, procedure of growing those autotrophic microalgae is much more complicated and expensive than that of the isolates in this study. 11 Moreover, light density employed for autotrophic microalgae growth must be fit, otherwise quality and quantity of carotenoid pigments are going to be reduced (Fazeli et al., 2006). 3.4. Designing universal primers Universal primers were designed according to 18S rRNA gene sequence Thraustochytrium for target microalgae striatum, species including Thraustochytrium aureum, Thraustochytrium aggregatum, multirudimentale, Thraustochytrium Thraustochytrium pachydermum and Schizochytrium aggregatum with their Accession numbers on GenBank - NCBI as AB022112, AB022110, AB022109, AB022111, AB022113 and AB022106, respectively. Based on similar region on 18S rRNA gene sequence of the target species, a set of universal primers was designed at two different positions of consensus regions (Figure 11) and ordered. The sequences of universal primers were as follows: - Forward primer: Thrau-Schi-F 5’ AAA GAT TAA GCC ATG CAT G 3’ - Reverse primer: Thrau-Schi-R 5’ GCT GGC ACC AGA CTT GCC CTC 3’ This set of universal primers was able to amplify a target region within 530-540 bp of 18S rRNA gene sequence. 12 Figure 11. Positions of universal primers on 18S rRNA gene of the target species in DNAMAN 4.0 software 3.5. Identification of the isolates of microalgae by universal primers DNA of the isolates were extracted and employed for identification by PCR technique resorting to the universal primers. Thermal cycler was basically set for amplification with the set of universal primers as in section 2.2.5b. PCR products were analyzed by agarose gel electrophoreis with gel 1.5%. The result showed that thermal cycler program was not compatible due to presence of unexpected bands. 13 Therefore, an optimization of PCR thermal program was proceeded. Three thermal programs were set for amplification of target region of 18S rRNA gene sequence with the set of universal primers. The PCR programs were run as follows: (1) 94°C - 5 minutes, 94°C - 30 seconds, 50°C -30 seconds, 72°C - 1 minute, 72°C - 5 min, and 10°C hold; (2) 94°C - 5 minutes, 94°C - 30 seconds, 57°C -30 seconds, 72°C - 1 minute, 72°C - 5 min, and 10°C hold; (3) 94°C - 5 minutes, 94°C - 45 seconds, 58°C -45 seconds, 72°C - 1 minute, 72°C - 5 min, and 10°C hold. The amplification of each program was repeated for 35 cycles. As a result, the thermal program (3) solved the above problem. The PCR product expressed only one band at position 530-540 bp compared with Ladder in agarose gel under UV light (Figure 13’). While unexpected bands still appeared with the program (2), and there was no amplified product with the program (1). 14 500bp Figure 13’. Image of gel electrophoresis of the isolates DNA amplification Therefore, 18S rRNA gene sequences of all the isolates were amplified a region with 530-540 bp (theoretical size). Perhaps, all the isolates in this study belonged to the target heterotrophic microalgae species. 15 4. CONCLUSIONS AND SUGGESTIONS 4.1. Conclusions Nine marine heterotrophic microalgae were isolated which were composed of three isolates with high total carotenoid and pigmented colony. And the isolate CM1 could be a potential candidate for carotenoid production. All of the isolates might belong to Thraustochytrid group. 4.2. Suggestions - The optimization of growing conditions should be studied to increase biomass for the isolates. - Sequencing of 18S rRNA gene of the isolates CM1, CM3 and CM6 should be carried out using molecular technique. - More heterotrophic microalgae should be isolated from other sources and locations. 16 REFERENCES Vietnamese Hoàng Thị Lan Anh, Đinh Thị Ngọc Mai, Ngô Thị Hoài Thu và Đặng Diễm Hồng. 2010. Phân lập chủng vi tảo biển dị dưỡng mới thuộc chi Thraustochytrium giàu DHA và carotenoid từ đầm ngập mặn Thị Nại - Bình Định. Tạp chí Công nghệ Sinh học. 8(3A). tr:459465. English Aki, T., K. Hachida, M. Yoshinaga, Y. Katai, T. Yamasaki, S. Kawamoto, T. Kakizono, T. Maoka, S. Shigeta, O. Suzuki and K. Ono. 2003. Thraustochytrid as a potential source of carotenoids. J. Am. Oil Chem. Soc. 80, 789-794. Arafiles, K.H.V., J.C.O. Alcantara, J.A.L. Batoon, F.S. Galura, P.R.F. Cordero, E.M. Leaño and G.R. Dedeles. 2011. Cultural optimization of thraustochytrids for biomass and fatty acid production. Mycosphere 2(5), 521–531. Bremer, G. 2000. Isolation and culture of Thraustochytrids. In: Hyde K, Pointing S (eds) Marine mycology- a practical approach. Fungal Diversity Press, Hong Kong: 49-61. Chatdumrong, W., W. Yongmanitchai, S. Limtong and W. Worawattanamateekul. 2007. Optimization of docosahexaenoic acid (DHA) production and improvement of astaxanthin content in a Shizochytrium limacinum isolated from mangrove forest in Thailand. Kasetsart Journal (Nat. Sci.). 41: 324-334. Fazeli, M.R., H. Tofighi, N. Samadi, H. Jamalifar and A. Fazeli. 2006. Carotenoids accumulation by Dunaliella tertiolecta (lake urmia 17 isolate) and Dunaliella salina (ccap 19/18 & wt) under stress conditions. DARU. Volume 14, No. 3, pp. 146-150. Honda, D., T. Yokochi, T. Nakahara, S. Raghukumar, A. Nakagiri, K. Schaumann and T. Higashihara. 1999. Molecular phylogeny of Labyrinthulids and Thraustochytrids based on the sequencing of 18S ribosomal RNA gene. J. Eukaryot. Microbiol. 46(6), pp. 637647. Hong, D.D., H.T.M. Hien, N.H. Thu, H.L. Anh and L.Q. Hai. 2008. Phylogenetic analyses of Prorocentrum spp. and Alexandrium spp. isolated from Northern coast of Vietnam based on 18S rDNA sequence. Journal of Environmental Biology. 29(4), pp. 535-542. Perveen, Z., H. Ando, A. Ueno, Y. Ito, Y. Yamamoto, Y. Yamada, T. Takagi, T. Kaneko, K. Kogame and H. Okuyama. 2006. Isolation and characterization of a novel thraustochytrid like microganism that efficiently produces docosahexaenoic acid. Biotechnol Lett. 2006 Feb; 28(3). pp:197-202. Ratledge, C. 1993. Single cell oils—have they a biotechnological future? Trends Biotechnol; 11:278–284. Strickland, J.D.H. and T.R. Parsons. 1972. A practical handbook of seawater analysis, 2nd ed. Bull Fish Res Bd Can 167. pp:1-311. 18 [...]... 0.0018 0.9953 Those calibration curves play important role in rapid determination of dry weight of biomass through measuring optical density of microalgae liquid of the isolates 3.3 Determination of carotenoid content After 4 growing days in GYPS broth, microalgae biomass was employed for extracting carotenoid Table 8’ showed total carotenoid of the isolates As a result, total carotenoid contents extracted... Centurion XV software 7 3 RESULTS AND DISCUSSIONS 3.1 Isolation of heterotrophic microalgae All heterotrophic microalgae strains were isolated from submerged leaves at various stages of decay in the marine zone As a result, there were nine microalgae strains isolated successfully and named as CM1, CM2, CM3, CM4, CM5, CM6, CM7, CM8 and CM9 According to morphological characteristics of heterotrophic microalgae, ... of calibration curve of OD value and dry weight Dry weight of biomass was determinated according to description in section 2.2.2 Calibration curve of each isolate was created based on OD of microalgae liquid at 600nm and its dry weight According to results of creation of calibration curve, the regression equations of the isolates were exhibited as following Table 7’ 9 Table 7’ Regression equation of. .. expressed as mean of triplicate flasks Means followed by the same letter in the same column were not significantly different Furthermore, comparing to some autotrophic microalgae which are commonly used as a source of carotenoid production such as Dunaliella salina and Haematococcus pluvialis, the isolate CM1 is much lower in total carotenoid content However, procedure of growing those autotrophic microalgae. .. total carotenoid and pigmented colony And the isolate CM1 could be a potential candidate for carotenoid production All of the isolates might belong to Thraustochytrid group 4.2 Suggestions - The optimization of growing conditions should be studied to increase biomass for the isolates - Sequencing of 18S rRNA gene of the isolates CM1, CM3 and CM6 should be carried out using molecular technique - More heterotrophic. .. shapes of their cells mainly were oval or spherical Four remaning isolates originated form mangrove leaves including CM1, CM2, CM8 and CM9 Most of their colonies were milky white and convex and smooth In particular, isolate CM1 had light pink colonies The margins of these four isolates were various, with entire, filiform or curled forms The shapes of their cells mainly were oval or suboval Morphology of. .. Ono 2003 Thraustochytrid as a potential source of carotenoids J Am Oil Chem Soc 80, 789-794 Arafiles, K.H.V., J.C.O Alcantara, J.A.L Batoon, F.S Galura, P.R.F Cordero, E.M Leaño and G.R Dedeles 2011 Cultural optimization of thraustochytrids for biomass and fatty acid production Mycosphere 2(5), 521–531 Bremer, G 2000 Isolation and culture of Thraustochytrids In: Hyde K, Pointing S (eds) Marine mycology-... highest total carotenoid content (6056 µg/kg dry weight) was present in isolate CM1 The lowest content (528 µg/kg dry weight) belonged to isolate CM8 Especially, the isolates which contained high carotenoid content had pigment in their colonies and converted their growth medium liquid color to dark orange or yellow This issue demonstrates the intracellular accumulation of carotenoid Comparison of carotenoid. .. 13’ Image of gel electrophoresis of the isolates DNA amplification Therefore, 18S rRNA gene sequences of all the isolates were amplified a region with 530-540 bp (theoretical size) Perhaps, all the isolates in this study belonged to the target heterotrophic microalgae species 15 4 CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions Nine marine heterotrophic microalgae were isolated which were composed of three... universal primers on 18S rRNA gene of the target species in DNAMAN 4.0 software 3.5 Identification of the isolates of microalgae by universal primers DNA of the isolates were extracted and employed for identification by PCR technique resorting to the universal primers Thermal cycler was basically set for amplification with the set of universal primers as in section 2.2.5b PCR products were analyzed by agarose