Phenotypic and Genotypic characterization of Chlorella species isolated from Environmental samples.

Application and Standardization of Uniplex PCR targeting Chlorella species

  • PREMINA S. Madras Christian College, Chennai
  • NIREN ANDREW S. Madras Christian College, Chennai
  • SUNDARALINGAM R. Madras Christian College, Chennai
  • SHARANYA THERESA V. Loyola College, Chennai


Microalgae with beneficial compounds available in the environment are rich source for cost effective pharmaceutical and food industries. Isolation and identification of Chlorella species from the environment through phenotypic method requires technical expert with great knowledge on the Algal biology. In this 21st century, advancement of Molecular identification plays vital role in identification of any Microorganisms from genus to  species level with higher accuracy and confidence rate than the phenotypic identification, which may give us false report based on the expertise of individual taxonomist. In this context, this study was aimed to design primer targeting Chlorella and other closely related algal species targeting 18s ribosomal RNA, ITS1 region. We have standardized the in-house designed primer and optimized the PCR conditions to characterize Chlorella isolated from the environmental water samples. Further, we carried out Sanger sequencing for the identification of algae upto the genus and species level. Out of 2 algae samples identified phenotypically, one isolated showed to be Chlorella vulgaris and other one showed as chlorella sorokiniana. We have submitted the Chlorella vulgaris to NCBI and published the same. To conclude, this study provided a primers with PCR conditions to characterize algal samples through molecular identification with 100% accuracy than the phenotypic method.

Keywords: Chlorella species;, Chlorella species; Microalgae; PCR; Primer Designing; Sanger Sequencing; Genotyping; Environmental samples; PCR; PRIMER DESINGING;, SANGER SEQUENCING; GENOTYPING


Download data is not yet available.


1. El-Sheekh, M., Abu-Faddan, M., Abo-Shady, A., Nassar, M. Z. A., & Labib, W. (2020). Molecular identification, biomass, and biochemical composition of the marine chlorophyte Chlorella sp. MF1 isolated from Suez Bay. Journal of Genetic Engineering and Biotechnology, 18(1), 1-10.
2. "Chlorella vulgaris". NCBI taxonomy. Bethesda, MD: National Center for Biotechnology Information. Retrieved 5 December 2017. Other names: synonym: Chlorella vulgaris var. viridis Chodat includes: Chlorella vulgaris Beijerink IAM C-27 formerly Chlorella ellipsoidea Gerneck IAM C-27
3. Duval B., Margulis L. (1995). "The microbial community of Ophrydium versatile colonies: endosymbionts, residents, and tenants". Symbiosis. 18: 181–210. PMID 11539474.
4. Safi, C., Zebib, B., Merah, O., Pontalier, P. Y., & Vaca-Garcia, C. (2014). "Morphology, composition, production, processing and applications of Chlorella vulgaris: A review" Renewable and Sustainable Energy Reviews. 35: 265–278. doi:10.1016/j.rser.2014.04.007.
5. Kitada, K., Machmudah, S., Sasaki, M., Goto, M., Nakashima, Y., Kumamoto, S., & Hasegawa, T. (2009). "Supercritical CO2 extraction of pigment components with pharmaceutical importance from Chlorella vulgaris". Journal of Chemical Technology and Biotechnology. 84 (5): 657–661. doi:10.1002/jctb.2096.
6. Wang B, Li Y, Wu N, Lan CQ. CO2 bio-mitigation using microalgae. Appl Microbiol Biotechnol. 2008;79:707–718.
7. Satpati GG, Pal R. Microalgae-biomass to biodiesel: a review. J Algal Biomass Utln. 2018;9(4):11–37.
8. Müller J, Friedl T, Hepperle D, Lorenz M, Day JG. Distinction between multiple isolates of Chlorella vulgaris (Chlorophyta, Trebouxiophyceae) and testing for conspecificity using amplified fragment length polymorphism and ITS rDNA sequences. J Phycol. 2005;41(6):1236–1247.
9. Baytut Ö, Gürkanli CT, Gönülol A, Özkoç I. Molecular phylogeny of Chlorella-related chlorophytes (Chlorophyta) from Anatolian freshwaters of Turkey. Turk J Bot. 2014;38(3):600–607.
10. Huss V, Frank C, Hartmann E, Hirmer M, Kloboucek A, Seidel B, Wenzeler P, Kessler E. Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta) J Phycol. 1999;35:587–598.
11. Wan M, Rosenberg JN, Faruq J, Betenbaugh MJ, Xia J. An improved colony PCR procedure for genetic screening of Chlorella and related microalgae. Biotechnol Lett. 2011;33:1615–1619.
12. Tear C, Lim C, Wu J, Zhao H. Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris. Microb Cell Factories. 2013;12(1):106–112.
13. El-Sheekh MM, Gheda SF, El-Sayed A, Abo Shady A, El-Sheikh M, Schagerl M. Outdoor cultivation of the green microalga Chlorella vulgaris under stress conditions as a feedstock for biofuel. Environ Sci Pollut Res. 2019;26:18520–18532.
14. Machado RR, Lourenço SO. Propriedades nutricionais de microalgas usadas como alimento de moluscos bivalves: uma revisão. Museu Nacional, Série Livros. 2008;30:281–304.
15. Borges-Campos V, Barbarino E, Lourenço SO. Crescimento e composição química de dez espécies de microalgas marinhas em cultivos estanques. Cienc Rural. 2010;40:339–347.
16. Murugan, N., Malathi, J., Therese, K. L., & Madhavan, H. N. (2018). Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes. The Kaohsiung journal of medical sciences, 34(2), 79-88.
17. Bharathi, M. J., Murugan, N., Rameshkumar, G., Ramakrishnan, R., Reddy, Y. C. V., Shivkumar, C., & Ramesh, S. (2013). Comparative Evaluation of Uniplex, Nested, Semi-nested, Multiplex and Nested Multiplex PCR Methods in the Identification of Microbial Etiology of Clinically Suspected Infectious Endophthalmitis. Current Eye Research, 38(5), 550-562.
18. Muthukumar A, Elayaraja S, Ajithkumar TT, Kumaresan S, Balasubramanian T. Biodiesel production from marine microalgae Chlorella marina and Nannochloropsis salina. J Pet Technol Altern Fuel. 2012;3(5):58–62
19. Rosenberg JN, Kobayashi N, Barnes A, Noel EA, Betenbaugh MJ, Oyler GA. Comparative analyses of three Chlorella species in response to light and sugar reveal distinctive lipid accumulation patterns in the microalga C. sorokiniana. PLoS One. 2014;9(4):e92460
20. Montoya EYO, Casazza AA, Aliakbarian B, Perego P, Converti A, De Carvalho JCM. Production of Chlorella vulgaris as a source of essential fatty acids in a tubular photobioreactor continuously fed with air enriched with CO2 at different concentrations. Biotechnol Prog. 2014;30(4):916–922.
21. EL-Mohsnawy E, El-Sheekh MM, Mabrouk M, Zoheir W. Enhancing accumulation of omega 3 and 9 fatty acids in Chlorella vulgaris under mixotrophic nutrition. The Journal of Animal and Plant Sciences. 2020;30(2):485–492
22. Premina S, Sundaralingam R, Niren Andrew S. Biosynthesis, Characterization and Antimicrobial Potential studies of copper oxide nanoparticle produced from Chlorella vulgaris. Paripex-Indian Journal of Research. 2020 ; 9:3
36 Views | Downloads
How to Cite
S., P., N. ANDREW S., S. R., N. MURUGAN, and S. THERESA V. “Phenotypic and Genotypic Characterization of Chlorella Species Isolated from Environmental samples.: Application and Standardization of Uniplex PCR Targeting Chlorella Species”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 13, no. 7, May 2021, doi:10.22159/ijpps.2021v13i7.41701.
Original Article(s)