RESEARCH ARTICLE Isolation and Characterization of Dunaliella Species from Sambhar Lake (India) and its Phylogenetic Position in the Genus Dunaliella Using 18S rDNA Pooja Sharma • Varsha Agarwal • M. Krishna Mohan • Sumita Kachhwaha • S. L. Kothari Received: 22 December 2011 / Accepted: 19 May 2012 Ó The National Academy of Sciences, India 2012 Abstract High b-carotene producing microalga Dunali- ella, isolated from Sambhar Lake, India, was identified, characterized and analyzed for its phylogenetic position in the genus Dunaliella. It was characterized on the basis of morphology and 1.7 Kb PCR product produced using the primers MA1 and MA2 which is specific to Dunaliella. Four stress parameters were used for characterization i.e. NaCl concentration, temperature, light and nitrogen con- centration. On these parameters total cell number, specific growth rate and total carotenoids/chlorophyll a ratio was analyzed. 1.7 M NaCl, 26 °C temperature, diurnal light and 4.9 mM N-concentration were identified as optimum conditions for growth whereas 3.5 M NaCl, 37 °C tem- perature, continuous light and without nitrogen source were identified as optimum conditions for carotenoid production. Highest cell number (19.6 9 10 5 cells/ml) and specific growth rate (0.505 division per day) were observed at 1.7 M salinity whereas highest total carotenoids/chloro- phyll a ratio (1.57) was observed at 3.5 M salinity. 17 sequences of 18S rDNA of Dunaliella, taken from NCBI nucleotide database, were used for phylogenetic analysis. A bootstrap consensus tree was constructed using neigh- bour-joining method. Keywords 18S rDNA  Carotenoids  Dunaliella  NCBI  Stress condition Introduction Dunaliella, is a chlorophycean alga which is unicellular, biflagellate and devoid of cell wall. It is ovoid, spherical or ellipsoid with size varying from 16 to 24 lm in length and from 9.5 to 13.3 lm in width [1]. It has remarkable capacity to grow and adapt to media ranging in salinity from 50 mM to 5 M NaCl. The major means of osmo- regulation is through production of intracellular glycerol [2] at a concentration that is proportionate to the external NaCl concentration [3]. It is an important organism that can accumulate high amounts of b-carotene up to 10 % of its dry biomass [4] resulting in orange-colored growth [5]. b-Carotene is marketed as a food coloring agent, pro- vitamin A in food and animal feed, an additive to cos- metics, for multivitamin preparation and, in the past dec- ade, for its purported anti-oxidant properties [6]. Several industrial production plants of Dunaliella salina are oper- ating in Australia, Israel, USA, China and Japan [7], where annual production is up to 1,200 tonnes dry weight [8]. Various species of Dunaliella are known, which have different requirement for growth, salinity tolerance and carotenoid production. Identification of Dunaliella is ambiguous when only morphological characteristics or physiological characteristics are considered, since it has diverse morphological and physiological characteristics depending on the conditions of growth. Molecular identi- fication is a useful tool to distinguish between inter- and intra-specific species with similar morphology [9]. It is well established that the type and degree of nutrient limitation result in dramatic changes in the cellular chemical P. Sharma  V. Agarwal  S. Kachhwaha  S. L. Kothari (&) Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur 302 004, India e-mail: slkothari28@gmail.com M. Krishna Mohan Birla Institute of Scientific Research, Statue Circle, Jaipur 302 001, India S. Kachhwaha  S. L. Kothari Department of Botany, University of Rajasthan, Jaipur 302 004, India 123 Natl. Acad. Sci. Lett. DOI 10.1007/s40009-012-0038-6