56 © 2019 Adama Science & Technology University. All rights reserved Ethiopian Journal of Science and Sustainable Development e-ISSN 2663-3205 Volume 6 (2), 2019 Journal Home Page: www.ejssd.astu.edu.et ASTU Short Communication Isolation and Morphological Identification of Some Indigenous Microalgae from Ethiopia for Phycoprospecting Abate Ayele1, Arumuganainar Suresh1,*, Solomon Benor1, 2 1Department of Biotechnology, College of Biological and Chemical Engineering, Addis Ababa Science and Technology University, Addis Ababa-16417, Ethiopia 2Research, Community Service, Technology Transfer and TUIL Directorate, Ministry of Science and Higher Education, Ethiopia *Corresponding author, e-mail: blueyellowsnu@gmail.com Abstract Bioprospecting of microalgae is one of the latest promising industries because of its high photosynthetic efficiency. Microalgae prospects are under limelight not only for its value added applications but also for no competition for food, water and arable land usage. However, the product is still costlier when compared to other organisms which obstruct large-scale phycoprospecting. Indigenous microalgae can improve phycoprospecting and it is on high demand. There is a need to isolate and identify the potential native microalgae for local application with ease. Besides, biodiversity in Ethiopia is intense and not investigated much on microalgae. Therefore, this research aims to isolate microalgae from eight different sites including Akaki pond, Akaki River, Kality pond, Kality Gidb pond, Tuludimtu ditch, Awash Lake, Koka Lake and Sumit Ditch. The samples were inoculated to Bold’s Basal medium and incubated under natural Sun light at 25C for 15 days. Then microalgae were purified using agar plate and identified morphologically using light microscope. Eighteen species of algae were obtained from 12 genera. Pediastrum sp., Chlorella sp., Chlamydomonas sp., Scenedesmus sp., Chlorogonium sp., Oscillatoria sp., Anabaena sp., Microcystis sp., Microspora sp., Closterium sp., Synechocystis sp., and Navicula species were among the identified genera. Of these, 8 genera belong to eukaryotic protist and the other 4 comes under prokaryotic cyanobacteria. These Ethiopian native species of microalgae can be used effectively for its value added application locally. Keywords: Microalgae, Ethiopia, Cyanobacteria, Protista, Microscope, Phycoprospecting. 1. Introduction Microalgae are ubiquitous and have been evolving on Earth for billions of years and responsible for evolution of aerobic organisms including humans by using CO2 from the primitive atmosphere and released O2. Studies suggested that tiny algae have been producing 70% of atmospheric O2 (Walker, 1980). Microalgae (2-200 µm) are organisms highly capable of utilizing solar energy and CO2 to create biomass and they are the primary producer for majority life on the planet (Wilkie et al., 2011). Algal species were estimated between 250,000 to millions of which 35,000 species are scientifically recorded. Currently about 5,000 algal species are available through culture collections and only 10 to 20 species are cultivated industrially (https://subitec.com/en/fascination-algae-facts- on-microalgae; Raja et al., 2008). In addition, the estimated number of unknown species of algae is projected to be two orders of magnitude more than currently known species (Anderson, 1992; Norton et al., 1996). It is a stunning vision that how much potential remains waiting in undiscovered species because the group of discovered species is ever growing for its various applications. Many researchers recommend that microalgae may hold the key to solve many problems including pollution, hunger, energy, global warming, and diseases in sustainable manner (Wilkie et al., 2011). In fact, Stuart & Hessami (2005) found that a 4000 m3 pond under natural Sun light could sequester up to http://www.ejssd.astu.edu/ https://subitec.com/en/fascination-algae-facts-on-microalgae https://subitec.com/en/fascination-algae-facts-on-microalgae Abate Ayele et al. Ethiop.J.Sci.Sustain.Dev., Vol. 6 (2), 2019 57 2.2 kiloton of CO2 per year with no competition with food crops, not restricted to arable land and portable water, can be grown in salt water and wastewater (Suresh et al., 2018), easily adapt conditions, low energy requirement (Oswald, 2003), carbon neutral, renewable, used as single cell protein, nutrient supplements, pigments production, biogas (Suresh et al., 2013) and antioxidants. In addition, Chisti (2007; 2008) found that microalgae can produce 23–55 m3 oil per acre as compared to oil palm (high oil producing plant) which produces only 1.4 m3/acre. With these promising advantages, microalgae industry is not popular in many part of the world including Ethiopia due to lack of study on characterization and exploitation. Despite intense biodiversity in Ethiopia, its utilization is negligible due to inadequate studies. The existing few studies focused on the community structure and primary production of microalgae (Damtew Etisa et al., 2018; Adane Fenta & Almaz Kidanemariam, 2016) and for biodiesel (Abebe Girma et al., 2016) lately. A very low attention has been given to the indigenous microalgae and its potentials in Ethiopia. To propel algal biotechnological applications in any country, one should investigate the native phycological flora and its potential value to industrial scale. Therefore, the objective of this study was to isolate microalgae from various sites in Ethiopia and identify the isolates based on morphological characteristics. The present study is part of ongoing efforts to screen efficient native microalgal strains for their phycoremediation of industrial pollutants in Ethiopia. Focusing on microalgae-based processes, the unexplored country of Ethiopia is awaiting the opportunity to play its role in phycoprospecting and to contribute to the economy of the country in general. 2. Materials and Methods 2.1. Microalgae Sample Collection and Growth The microalgae water samples were collected from eight sites in Ethiopia (Akaki Pond-AKP, Akaki River- AKR, Kality Pond-KAP, Kality Gidb Pond-KGP (85344.99 N, 384720.98 E), Tuludimtu Ditch- TUD (85100 N, 384859.98 E), Awash Lake-AWL (85859.99 N, 40100.01 E), Koka Lake-KOL (82120.86 N, 3903.74 E) and Sumit Ditch-SUD (9019.44 N, 384548.99 E) and 1 mL was inoculated into a 250 mL flask counting 100 mL sterile Bold’s Basal medium (BBM) and incubated in the lab under natural Sun light (day and night cycle) at 25C for 15 days. The culture flasks were manually shaken twice a day. 2.2. Microalgae Purification and Identification BBM grown microalgae were purified using spread and streak plate method and then isolated colonies were inoculated in 100mL BBM and incubated same as mentioned above. Purified algae were identified by its morphology using light microscope (Labomed, USA) by wet slide mount method at 40x and 100x (oil immersion). The photomicrographs were taken with an iPhone camera via ocular lens and followed the Janse van Vuuren et al., (2006) manual to identify the microalgae genera. 3. Results and Discussion In this preliminary study a total of 18 microlagal species were isolated using the standard plating techniques, and based on distinguishable morphological characters under light microscopic examination. These strains were preliminary ascribed to the 12 genera, namely, Pediastrum sp., KGP, Chlorella sp., AKR, TUD, Chlamydomonas sp., AWL, Scenedesmus sp., KAP, KGP, Chlorogonium sp., AKP, Oscillatoria sp., SUD, TUD, Anabaena sp., KGP, KAP, Microcystis sp., KOL, Microspora sp., KGP, KAP, Closterium sp., AKP, Synechocystis sp., KAP, AKR and Navicula sp., SUD. Among this, 4 genera belongs to cyanobacterial group (Oscillatoria, Anabaena, Microcystis and Synechocystis) which are prokaryotes and other 8 belongs to protist algae of eukaryotes. Moreover, 3 out of 18 species were identified as filamentous algae and they resemble the genera of Oscillatoria, Anabaena and Microspora. Most of the genera belong to division of Chlorophyta (Pediastrum sp., Chlorella sp., Chlamydomonas sp., Scenedesmus sp., Chlorogonium sp., Microspora sp.). Interestingly, a diatom (Navicula sp.) and charophyta (Closterium sp.) species also screened. In general all isolates have some potential application except Microcystis sp., which is a disease causing microalgae by producing neurotoxins. Figure 1 shows the photograph of isolated microalgal species. Abate Ayele et al. Ethiop.J.Sci.Sustain.Dev., Vol. 6 (2), 2019 58 Microalgae are available in all existing earth ecosystems, and representing a diverse polyphyletic group of species living in a wide range of environmental conditions and have potential to solve problems in the world. Yet they are one of the most poorly understood, characterized and exploited groups of microorganisms on earth (Wilkie et al., 2011; Raja et al., 2018). In addition, Wilkie et al., (2011) suggested that algae has great diversity, however indigenous species are potential candidates for bioprospecting because native species has advantage over type culture and genetically engineered organisms. Isolating native microalgae with desirable properties gives robust biological platform for phycoprospecting. Native strains come equipped with millions of years of adaptation to the local biotic and abiotic stress (naturally engineered species). Through optimization of native species may yield superior organisms for bioresource production. Consequently, isolation is a fundamental process to obtain pure cultures and is the first phase towards the screening and selection of microalgae strains with the potential for the value added applications. Although morphological analysis is frequently used to identify microalgae, it is inaccurate and very difficult for the identification at the species level, because the relationship between diagnostic morphology and biological species boundaries are largely unknown in many micro-eukaryotes (Moniz & Kaczmarska 2010). Besides, according to Yu et al., (2012) the microalgae morphology for the same strain varies in relation to age and culture conditions. Recently in Ethiopia, Etista et al., (2018) found that the Chlamydomonas sp., was dominant in Lake Abaya in all seasons, therefore in our study expected the same species in all samples but observed only in Awash Lake sample. Abebe Girma Demissie et al., (2016) isolated very different genera such as Oedogonium sp., and Cladophora sp., from Lake Abaya and Chamo in Arab Minch, those genus were absent in our sampling sites in Addis Ababa. In addition of those unique genera some common genera of Chlorella sp., and diatoms also identified (Abebe Girma Demissie et al., 2016). In general, our observation disclosed that different sites shown one or two different microalgae species, whereas Kality Gidb pond sample was contained 4 different genera of microalgae such as Pediastrum sp., Scenedesmus sp., Anabaena sp., and Microspora sp. In case of Kality pond also shown 4 different genera namely Scenedesmus sp., Anabaena sp., Microspora sp., and Synechocystis sp. Attractively, the same genera of microalgae were noticed in different site with a little altered morphology, for example Scenedesmus sp., and Anabaena sp., from KAP and KGP sample (Figure 1). The cells of Scenedesmus sp., from Kality Pond (KAP) observed 4-celled coenobia, while Kality Gidb Pond (KGP) sample showed 8 celled coenobia which is in agreement with Van den Hoek et al., (1995) description about the Scenedesmus sp. In case of Anabaena sp. isolated from KAP sample shown rectangular cells in the filament while elliptical to sphere shaped cells in KGP sample. Adane Fenta and Almaz kidanemariam, et al., (2016) reported variation of microalgae between sites is due to the difference in water quality, changes in physico-chemical characteristics lead to concomitant qualitative and quantitative changes in microalgae. Therefore, the result shows that the sampling sites was characterized by eutrophic mainly as a result of high nutrient loading from surface runoff from domestic, industrial, agriculture and construction near the sampling sites. In another study, Metzger and Largeau (2005) reported that, within in each chemical race and for the same strain, the morphology of the alga could vary in relation to age and culture conditions. The morphological heterogeneity of the alga makes the microscopic examination difficult and in support this point, Trainor (1998) observed Scenedesmus species shown diverse morphology under different environmental conditions in accordance with our identification. Identification of microalgae is supported by molecular tools would enhance the authentication of classification (Moniz & Kaczmarska, 2010), therefore, further analysis on its molecular identification under process. Abate Ayele et al. Ethiop.J.Sci.Sustain.Dev., Vol. 6 (2), 2019 59 Figure 1. Microscopic images of microalgae isolated from the different water samples In these isolates, some algae have lot of potential on value added products like single cell proteins (Chlorella sp), feed (Chlorella sp., Navicula sp.), oil producers (Chlorella sp., Scenedesmus sp., and diatom), wastewater treatment (Chlorogonium sp., Scenedesmus sp., Oscillatoria sp.,) nitrogen fixer as biofertilizer (Anabaena sp), pollutants removal, bioindicator (Closterium sp.,) and model organisms for study (Synechocystis sp., and Chlamydomonas sp.). These Ethiopian native genera of microalgae can be used effectively for its value added application locally and it’s under investigation. 5. Conclusion In this study, eighteen microalgal species were isolated from eight different sites from Ethiopia and identified by morphological features and noticed those belong to 12 different genera. Among these, 8 genera belong to eukaryotic protist microalgae and other 4 comes under prokaryotic cyanobacteria it includes 3 filamentous algae. Some isolates are industrially important such as Chlorella, Anabaena, diatom, Scenedesmus. Given the great diversity of microalgae, it is suggested that many native species are potential candidates for local application. Further analysis on its molecular identification is needed and its potential phycoprospecting are under process. Acknowledgements The support of the Directorate of Research and Technology Transfer, Addis Ababa Science and Technology University, in funding Internal Research Grant (Code No. ICA 04/2011) to Dr. A. Suresh is gratefully acknowledged. Chlamydomonas sp., (AWL)Chlorella sp.,(AKR) Anabaena sp.,(KAP) Synecocystis sp., (AKR) Chlorogonium sp., (AKP)Pediastrum sp., (KGP) Scenedesmus sp., (KAP) Oscillatoria sp., (TUD) Scenedesmus sp., (KGP) Closterium sp., (AKP) Microcystis sp., (KOL) Microspora sp., (KAP) Navicula sp., (SUD) Synecocystis sp., (KAP) Microspora sp., (KGP) Anabaena sp., (KGP) Oscillatoria sp., (SUD) Chlorella sp., (TUD) Abate Ayele et al. Ethiop.J.Sci.Sustain.Dev., Vol. 6 (2), 2019 60 Reference Andersen, R.A. (1992). Diversity of eukaryotic algae. Biodiversity & Conservation, 1(4): 267-292. doi.org/10.1007/BF00693765 Chisti, Y. (2007). Biodiesel from microalgae. Biotechnology advances, 25(3): 294-306. doi.org/10.1016/j.biotechadv.2007.02.001 Chisti, Y. (2008). Biodiesel from microalgae beats bioethanol. Trends in biotechnology, 26(3): 126-131. doi.org/10.1016/j.tibtech.2007.12.002 Abebe Girma Demissie., Chinthapalli, B., Shumet Tenaw., Chitra, D.V. (2016). 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