مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Effect of nitrogen concentrations and Salinity stress on β-carotene accumulation in Dunaliella salina
Effect of nitrogen concentrations and Salinity stress on β-carotene accumulation in Dunaliella salina
Fereshteh Fouladi,1,*Fatemeh Ehsani Besheli,2
1. Department of Chemical Engineering, Amirkabir University of Technology 2. Department of Chemical Engineering, Amirkabir University of Technology
Introduction: Natural carotenoids in the global application due to the widespread use of the market for them in nutrition, medicine, food color and cosmetics, as well as the natural and health priorities of today's consumers (M.R. Fazeli et al., 2006). B-carotene as a natural carotenoid is produced by plants and micro-algae. In addition to the best documented and proven activity of provitamin A (Olson and Hayashi, 1965) and an important role in photosynthesis (Telfer, 2002), this compound also has significant physiological effects, for example, as a concomitant Peroxyl radicals (Burton and Ingold, 1984), an immune response stimulus (Williams et al., 2000) and enhancement of the connection of the split bond (Stahl and Sies, 1997). Micro-algae seems to be a promising alternative to the third-generation biofuels that are very attractive and respected of the environment and easily replace fossil fuels (Xu and Boeing, 2014; Prieto. Pedro et al., 2011).Osmotic stress due to salinity is responsible for oxidative stress caused by reactive oxygen species (ROS) that results in the destruction of cellular macromolecules (Chockshi et al., 2015. Ines BenMoussa-Dahmen et al., 2016). Some of the species Dunaliella salina is able to produce high levels of b-carotene (up to 10-14% of dry weight) (Yang et al., 2013).
The aim of this study was to optimize culturing conditions for the lipid and B-carotene accumulation of algae species Dunaliella salina and studying the role of salinity stress in physiological and biochemical changes under conditions on the nitrogen concentration.
Methods: 2.1. Microorganism and culture conditions
Microalga Dunaliella salina separated from the Iranian Fisheries Organization to obtain their high lipid content. Before to the TAG accumulation experiment, a culture was preserved as an expression (Guido Breueret al., 2012). The original average growth (OAG) of microalgae biomass on the basis of chemical components in the following culture composition (Ben-Amotzet al., 1983): (mg/lit): 36 mgKNO3, 20 mg KH2PO4, 56 mg Na2HPO4, 10 mg MgSO4.7H2O, 0.1 mg ZnSO4, 1.5 mg MnSO4.H2O, 0.08 mg CuSO4.5H2O, 0.3 mg H3BO3, 0.3 mg (NH4)6Mo7O24.4H2O, 17 mg FeCl3.6H2O, 0.2 mg Co(NO3)2.H2O, 7.5 mg EDTA and 35 gr NaCl distilled water. Medium pH was adjusted to pH 7.5with NaOH and the medium was filter sterilized prior to use. Before the main trial, microalgae were cultivated in the OAG to the late-exponential growth phase and then centrifuged at 4500 rpm for 30 min, washed in distilled H2O and suspended in culture medium with a defined nutrient composition. The starting algal density was similar in all experiments of the central composite design: 5 * 107 cells mL_1, and two experiments with different densities. Five days before the start of the experiment, flasks were inoculated and placed in a shaking incubator, with an irradiance of 200 µmol m_2 s_1 and a headspace enriched with 4% CO2, operating at 300C and 4500 rpm. To ensure a sufficiently high cell density, the irradiance was increased to 200 µmol m_2 s_1 two days before the start of the experiment (Ben-Amotzet al., 1983).
2.2. β-carotene extraction and measurement
Β-carotene was extracted using the n-hexane by the method of Rodriguez (Rodriguez, 2001). After exposing the cell to the stress conditions, 50 ml of the micro-algal suspension was taken by centrifugation at 4500rpm for 15 minutes, then with ethanol /n-Hexane (2/1) and distilled water, degraded, and analyzed by the n-Hexane method. The concentration of β-carotene was measured by the colorimetric test. Beta-carotene content was determined at 450 nm using a UV / Visible spectrophotometer (PG instrument Ltd.).
The amount of β-carotene extracted in n-hexane was obtained by spectrophotometry (Eijckelhoff and Dekker, 1997) using the following equation:
(Eq. 1) β-carotene (mg/mL) = 25.2 × A450
Results: 3.1. Nitrogen starvation and salt concentration on cell growth
Nitrogen restriction is known to prevent cell division, so cells the cells divide slowly decompose by decreasing chlorophyll content. Also, cells produce more b-carotene to protect cells from potential radiation damage. Similar reactions were observed under conditions limiting sulfate and phosphate (Meng Chen et al., 2011. Kanchan Phadwal and P.K. Singh, .2003). The nitrogen starvation clearly influenced the growth rate, lipid, and β-carotene in D.salina.
Fig.1.a D. salina obtained the maximum β-carotene at 0.01 mg/L (89pg cell-1) and the lowest concentration at 0.36 mg/L (52pg cell-1). The number of fixed cells and changes in the cell occur, which include lipid accumulation and beta-carotene accumulation.
Fig.1.a. β-carotene cures of D. salina under conditions of nitrogen concentration with 0.36mg/L, 0.18 mg/L and 0.01 mg/L.
As Joseph Msanne asserts, nitrogen depletion studies could be made to “These results suggest
that turnover of nitrogen-rich compounds such as proteins may provide carbon/energy for TAG biosynthesis in the nutrient-deprived cells. In Chlamydomonas, several genes coding for diacylglycerol: acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regulated. Understanding the interdependence of these anabolic and catabolic processes and their regulation may allow the engineering of algal strains with improved capacity to convert their biomass into useful biofuel precursors.” (Joseph Msanne et al., 2012). Besides, decreasing Cn have two opposite effects on the lipid production: (1) leads to an increase in microalgae lipid (positive effect), and (2) decreased cell growth (negative effect). These metabolic pathways lead to more oil production but it decreases cell growth of microalgae (Fatty Acid Oxidation to produce energy or the synthesis of lipids which is called Lipogenesis). As Joseph Msanne asserts, studies could be extended to “N depletion also affects the expression of lipid biosynthesis genes” (Joseph Msanne et al., 2012).
Conclusion: The achieving of large-scale photobioreactors for natural oil large production from microalgae needs a complete understanding of the phenomena of the key operating variables involved in biomass growth and lipid formation. This problem was investigated using D. salina specie as a study model. The β-carotene of microalgae was strongly related to nitrogen and salt concentration. Various levels of conditions cultivation were used to investigate its effects on β-carotene. The maximum β-carotene obtained was 89pg cell-1 when salt concentration was (NaCl) of 90 g/L. various feed timing and concentrations of nitrate were carried out to investigate the cell growth rate, β-carotene accumulation of D. salina.