The Purification And Research Of Biologic Characteristic Of Dunaliella Salina

In this thesis, the biological characteristic of Dunaliella salina, which included the axenic purification, physiology characteristics and genetics characteristics, was studied systematically. Three different methods including the combination of antibiotics and centrifugal technique, the combination of centrifugal washing technique and dilution method, and high concentration of salt were designed to proceed for purification of D. salina. Five axenic strains of D. salina were obtained, and axenic status was confirmed by different methods.The influence of different nutrition factors, the salt concentration, vitamins, hormones, organic carbon sources, organic nitrogen sources and extracts of animal and plant, on the growth of strain A1 of D. salina were investigated, The results showed that D. salina can adapt to 0~ 292.5g·L-1 NaCl, and abrupt increase of NaCl would induce lag phase of the growth of D. salina, and the length of the period has been related with concentration of NaCl. The growth of D. salina was promoted in some way by VB1, VB6, VB12, VC or VH, and the best concentrations were 150μg·L-1, 160μg·L-1, 0.5μg·L-1, 320mg·L-1and 2.0μg·L-1, respectively. The promoting effect of glucose was better than potassium acetate, and the most suitable concentrations in liquid and solid medium were 10~15g·L-1and 10~30g·L-1respectively. The promoting effect of urea was better than glycine, and the most suitable concentration in liquid medium was 0.1g·L-1. Inositol, NAA, 6-BA, KT and GA could also promote the growth of D. salina, and the best concentrations were 55, 3, 4, 0.1 and 0.2mg·L-1 respectively. 20 ~ 40ml·L-1 fish soup, 100ml·L-1 bean sprout juice, 100ml·L-1potato juice and 4~6g·L-1peptone could promote the division of D. salina. The Johnsons medium with fish soup can be regarded as a complete medium of D. salina and might be used to select the auxotroph strain of D. salina. The orthonogal test was designed to search the optimum concentration of major affecting factors of NaCl, VC, urea, and glucose. The result showed the optimum medium for mixotrophic growth of strain A1 was NaCl 58.5g·L-1, VC 320mg·L-1, urea 0.2g·L-1 and glucose 15g·L-1.The sensitive degree of D. salina to antibiotics, herbicide and metabolite analog was different. The minimum inhibited concentrations (MIC) of Cm, Em and PPT in liquid ferment amounted to 120, 28 and 5mg·L-1, respectively, while the MICs of Cm, Em, PPT and Act in solid medium were only 60, 16, 3 and 0.4mg·L-1, respectively. The MICs of Sm, Km, Amp, Nm were over 1000mg·L-1 in solid medium. The MICs of isoniazid, L-canavanine, L-azetidine-2-carboxylic acid and mercaptopurine for D. salina in liquid medium were over 200mg·L-1, while the MIC of ρ-Fluorophenylalanine was only 180mg·L-1.D. salina has no complete fibrous cell wall, but glycoprotein coat on the external lamella of the plasmalemma. The protoplasts can be obtained by pronase B in buffer of 5mmol·L-1 HEPES pH7.5 with 0.5mol·L-1 sorbitol as osmotics, and the effect of pronase B was little better than those of proteinase K, papain, and pepsin. The best enzymolysis parameters were as following: 400mg·L-1pronase B, 1h of isolation time, 30℃.The resistant ability of D. salina to the ultraviolet ray was stronger than other microalgaes. The irradiation at a dose over 60J·m-2 could cause death of D. salina completely. The 90% lethal dose was 13.92J·m-2, and the dose was suitable for the isolation of mutant of D. salina. The preservation of axenic strain of D. salina was investigated with different ways. The results showed the algae could be preserved in liquid medium, plate, and slant.