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Available online at www.notulaebiologicae.ro Print ISSN 2067-3205; Electronic 2067-3264 Notulae Scientia Biologicae Not Sci Biol, 2013, 5(3):309-315 Comparative Salt Stress Study on Intracellular Ion Concentration in Marine and Salt-adapted Freshwater Strains of Microalgae Ahmad Farhad TALEBI1,2, Meisam TABATABAEI2*, Seyed Kaveh MOHTASHAMI2, Masoud TOHIDFAR2, Foad MORADI3 1 Ferdowsi University of Mashhad, College of Agriculture, Biotechnology and Plant Breeding Dept., Mashhad, Iran 2 Agricultural Biotechnology Research Institute of Iran (ABRII), Biofuel Research Team (BRTeam), Energy Crops Genetic Engineering Group, Microbial Biotechnology and Biosafety Dept., Karaj, Iran; [email protected] (*corresponding author) 3 Agricultural Biotechnology Research Institute of Iran (ABRII), Molecular Physiology Dept., Karaj, Iran Abstract Salinity imposes significant stresses in various living organisms including microalgae. High extracellular concentration of Na+ directly influences ionic balance inside the cell and subsequently the cellular activities. In the present study, the effect of such stress on growth and intracellular ions concentration (IIC) of Dunaliella salina and Chlorella Spp. was investigated. IIC was analyzed using Ion chromatography technique. D. salina showed the highest degree of resistance to increase in salinity as little changes occurred both in IIC and in growth parameters. D. salina could maintain the balance of K+ inside the cell and eject the excess Na+ even at NaCl concentrations above 1M. Moreover, D. salina accumulated β-carotene in order to protect its photosynthetic apparatus. Among Chlorella species, C. vulgaris showed signs of adaptation to high content of salinity, though it is a fresh water species by nature. Moreover, the response shown by C. vulgaris to rise in salinity was even stronger than that of C. salina, which is presumably a salt-water resistant species. In fact, C. vulgaris could maintain intracellular K+ better than C. salina in response to increasing salinity, and as a result, it could survive at NaCl concentrations as high as 0.75 M. Marine strains such as D. salina well cope with the fluctuations in salinity through the existing adaptation mechanisms i.e. maintaining the K+/N+ balance inside the cell, K+ accumulation and Na+ ejection, accumulation of photosynthetic pigments like β-carotene. Keywords: Chlorella, Dunaliella, ion chromatography, microalgae, salinity Abbreviations: Chl a - Chlorophyll a, Chl b - Chlorophyll b; IIC - Intracellular ion concentration Introduction large amounts of intracellular glycerol and efficient elimi- nation of Na+ by plasma membrane transporters. Rapid al- Exceptionally salt tolerant (halotolerant) organisms terations in cell volume donated by lacking a rigid cell wall could enrich our knowledge in knowing basic physiologi- in this genus makes it possible to respond to changes in salt cal mechanisms that may lead to enhance salinity tolerance concentration by intracellular ions and glycerol concentra- in crops. Algae are inhabitants of biotopes characterized by tion adjustments (Kacka and Donmez, 2008). varying salinities, and as a result they have attracted con- On the other hand, Chlorella is a genus of single-cell siderable attention in salt tolerance studies domain. They green microalgae, belonging to the same phylum as Du- have served as model organisms for better understanding naliella sp., Chlorophyta, but is of fresh water habitat. of salt acclimation in more complex physiological pro- Through photosynthesis, it multiplies rapidly, while re- cesses of higher plants (Alkayal et al., 2011). Among algal quiring only carbon dioxide, water, sunlight, and a small species, the unicellular green algae; Dunaliella salina, due amount of minerals. Algae species can be quit helpful for to its remarkable ability to adapt to highly saline condi- understanding the mechanisms involved in such resistance tions, could act as a valuable model for the identification since they show adaptation to fluctuations in salinity of of such mechanisms (Chen and Jiang, 2009). This organ- their aquatic biotope. ism can practically adapt to the entire range of salinities, As a matter of fact, increases in external concentrations well above the maximal salinity range for growth of most of inorganic ions impairs the osmotic balance between the plant species. The adaptation to extreme salinity involves cells and their surrounding medium and forces water ef- short-term and long-term responses in Dunaliella sp.; the flux (exosmosis) from the cells, leading to the loss of turgor former include osmotic adjustment by accumulation of pressure (Fricke and Peters, 2002); in this respect, plants, Received 28 June 2013; accepted 26 July 2013 Talebi A. F. et al. / Not Sci Biol, 2013, 5(3):309-315 310 including species of Chlorophyta, response to high con- Materials and methods centrations of salt by assimilation metabolites like those of fructose, sucrose and trehalose, which possess an osmolyte Strain collection and cultivation media function, or those of charged molecules, such as proline Four algae species were purchased from the Culture and glycine betaine in order to readjust osmotic equilib- Collection of Algae and Protozoa (CCAP) (Sams Re- rium by preventing water loss (Banu et al., 2009). search, Scotland). Those species included three fresh water Series of studies have been conducted to determine the species of Trebouxiophyceae: C. vulgaris 211/11B, C. em- osmotic responses of such Dunaliella sp. to the changes of ersonii 211/11A, C. salina 211/25, as well as one member salinity (Chen and Jiang, 2009). The results suggest that of the Chlorophyceae class, D. salina 19/18 which is a salt Dunaliella cells possess efficient mechanisms to elimi- water inhabitant. All the Chlorella species were cultivated nate Na+, accumulate K+ and to remain intracellular Ca+2 in Moh202, a medium developed in our previous stud- non-exchangeably with the extracellular pool (Pick et al., ies (Talebi et al., 2013) and was used for all fresh water + 1986b). Among these ions, K is the most contributing strains. Moh202 consisted of MgSO4 0.1 g/L, CaCl2 0.03 +2 to the osmotic balance while Ca play an important role g/L, KNO3 0.8 g/L, K2HPO4 0.15 g/L, KH2PO4 0.2 g/L, in cell permeability. Ca+2 contributes to osmotic balance Fe-citrate 0.02 g/L. NaCl concentration for control cul- maintenance in limited range since it is confined to specif- ture was set up at 0.5 mM in addition to the trace elements ic cell compartments like chloroplast matrix (Kirst, 1977). (standard Hunter’s trace-elements = 1ml/L). The culture The most known resistant species of this genus isD. salina medium for D. salina also included the same nutrient ele- which has developed special adaptation mechanisms to ments but NaCl concentration was set at 1, 1.5 and 2 M. overcome hyper-saline environment; lack of rigid cell wall, In this study, sodium bicarbonate was used instead of CO2 accumulation of glycerol in varied concentrations (Kacka injection due to its ease of use. and Donmez, 2008), fluctuations in photosynthetic pigments, and structural modifications in chloroplast Growth condition and growth survey (Stonynova and Toncheva, 2003). Rapid alterations in cell All strains were cultured under continuous illumina- volume donated by lacking a rigid cell wall makes D. salina tion (80 µmol photon m−2 s−1) at 22°C in a shaking growth possible to respond to changes in salinity, by intracellular chamber. During the cultivation period, growth kinetic ions and glycerol concentration adjustments (Kacka and parameters were measured in triplicates for all the strains. Donmez, 2008). Data comparison was then carried out using the ANOVA In an interesting investigation, aerobic decomposition test. The parameters analyzed included: of the unicellular green alga Chlorella salina CU-1 was 1) The cell density determined by measuring the absor- studied in freshwater and saline cultures. From the physio- bent of 1 ml of cell suspension by a spectrophotometer at logical point of view, Ca+2 in certain ratios to Na+ reversed 600 nm wavelength. most of NaCl stress symptoms in C. salina (Chan, 1985). 2) A step by step course of increasing salinity of cul- Ca+2, as second messenger play a significant role in induc- ture medium from 0.5mM (associated with fresh water tion of phosphorylation cascades leading to activation of cultivation) to 0.25, 0.5, and 0.75 M NaCl, was conducted genes responsible for adaptation to high salt resistance and and the adaptive changes of the populations of the above- reactive oxygen species (ROS). Application of gypsum in- mentioned microalgae strains were measured phenotypi- creased the chlorophyll and protein contents of C. vulgaris cally i.e. the changes in chlorophyll content and growth in all the concentrations of NaCl studied. This treatment rate and intracellular ion accumulation. could alleviate the adverse effects of saline stress (Mathad 3) Moreover, only for D. salina, total chlorophyll and and Hiremath, 2009). β-carotene content were measured by spectrophotometery Having considered the salient role of Dunaliella sp. in at 412, 431, 460 and 480 nm wavelengths (Eijckelhoff and studying the physiology of halophilous plants, this study Dekker, 1997). was set to better understand salt responsiveness of the in- Cultures were allowed to grow for 30 d in order to tracellular ion accumulation (IIC) by measuring common reach the stationary phase when cells were harvested for cations such as sodium, potassium and calcium using con- further analysis. ductivity detectors in an ion chromatography (IC). More- over, the present study was designed to look for putative Ion chromatography sample preparation evolutionary adaptive changes (high salt stress) by looking To determine the intracellular cation content of the into the populations of originally fresh water green algae; cells, 800 ml of algal suspension was centrifuged 30 d after Chlorella genus (i.e. C. emersonii, and C. vulgaris) and to the cultivation. The pellet was then washed with deionized determine their IIC while comparing with those of their water and the procedure was repeated.