Study On The Physiological Response Of Dunaliella Salina To Strong Light And Hypersaline And The Methods To Alleviate The Stress

Dunaliella salina,a unicellular biflagellate eukaryotic green alga,could accumulates large amounts of carotenoids under specific conditions.Dunaliella salina is the best commercial source of natural?-carotene.Meanwhile,Dunaliella salina is rich in protein,glycerol,polyunsaturated fatty acids,vitamins and minerals,which has a wide range of applications in the pharmaceutical,food and cosmetics industries.At present,the large-scale cultivation of?-carotene by Dunaliella salina is mainly achieved through outdoor shallow ponds(Australia)or artificial raceway ponds(Israel,China,Iran,India,etc.)by two-phase cultivation mode.That is the alga were initially cultivated at low irradiances and low salinity for accelerating the growth of D.salina,and then transferred to high irradiances accompanied with hypersaline condition to enhance carotenogenesis in open ponds.Hypersaline and strong light are the necessary conditions for Dunaliella salina to accumulate?-carotene in theory.However,when transferring from the first stage to the second stage,Dunaliella salina which have adapted to the mild conditions will be very easy to produce photoinhibition or even photooxidation under high annual irradiance,resulting in cell damage and death.These are the limiting factors for large-scale cultivation of Dunaliella salina to produce?-carotene.It is unclear whether hypersaline would aggravate the strong light stress and whether there are some ways to alleviate the possible double stress and maintain high?-carotene production.The alga Dunaliella salina(IOCAS 879ss)was used in this study.We carry out the following research around its initially cultivation stage and the transition stage from initially cultivation stage to?-carotene accumulation stage.First of all,the suitable salinity,light intensity and temperature range for the growth of Dunaliella salina were selected.To confirm the effects of salinity(A),light intensity(B),temperature(C),and the interaction of the two factors A×B,A×C,and B×C on the growth of Dunaliella salina,a three-factor,two-level orthogonal experiment was conducted.The specific growth rate was obtained from eight groups of batch culture for 7 days at different factor combinations as further evaluated index.The photosynthetic oxygen evolution rate,respiratory oxygen consumption rate,chlorophyll fluorescence,and pigment content were also measured to clarify the physiological mechanism of the interactions.Secondly,in view of the changes of salinity when Dunaliella salina is transferred from the initially cultivation stage to the?-carotene accumulation stage,the rapid light curves are used to analyze the light intensity requirements of Dunaliella salina under hypersaline shock.And the effects of hypersaline shock on the growth,the photosynthetic capacity and the adaptability to light intensity of this alga were studied.To provide basic data and theoretical support for the selection of light intensity for the cultivation of Dunaliella salina with hypersaline shock;Finally,to explore the effects of glycerol and sodium acetate on the growth and pigment contents of Dunaliella salina under high light intensity,the cell density,pigment contents,respiration rate,photosynthesis rate and chlorophyll fluorescence parameters were measured to clarify the physiological mechanism of Dunaliella salina by additional organic carbon source-glycerol and sodium acetate.The main results were as follows:1.In the initially cultivation stage of Dunaliella salina,the orthogonal experiment results showed that the maximal specific growth rate of Dunaliella salina is 0.36 d^-1.Temperature is the most significant impact factor,followed by salinity,light intensity.That is,the optimal condition of the algal proliferation was settled as temperature30°C,salinity 110 psu,and light intensity 120?mol·m^-2·s^-1.At 160 psu,the specific growth rate increased from 0.11 d^-1 at 20?to 0.23 d^-1 at 30?.Meanwhile,the amplitude raised to 0.17 d^-1 at 110 psu with the same temperature(from 0.19 d^-1 to0.36 d^-1).Similarly,the promotion degree of growth by increasing light intensity was from 0.14 d^-1 at 20?to 0.17 d^-1 at 30?.The binary diagram showed that the interaction of salinity-temperature and the interaction of light intensity-temperature both independently affected the specific growth rate of Dunaliella salina.2.It was found that the salinity increased from 110 psu to 160 psu,and the content of chlorophyll and carotenoid increased by 12.4%and 12.6%,and the specific growth rate increased by 56.5%,respectively.The experimental results of hypersaline shock showed that the growth of Dunaliella salina was inhibited when the algae pre-adapted to 80 g/L Na Cl was transferred to fresh medium with 160,240 and 320 g/L Na Cl in a short time.Na Cl increased from 80 g/L to 240 g/L and 80 g/L to 320 g/L Na Cl,the growth of algae cells arrested.It indicated that high salinity inhibited the growth of Dunaliella salina.These phenomenons indicate that 110 psu is beneficial to the proliferation of this strain,and 160 psu may be more effective for biomass accumulation.Therefore,110 psu should be selected for the purpose of rapid acquisition of algal cell density,and 160 psu for the purpose of acquiring biomass.3.The results of the rapid light curves fitting showed that light requirement of D.salina was down-regulated under hypersaline shock conditions.D.salina cells can cope efficiently with the cells sudden increase in Na Cl from 80 to 160 g/L by heat dissipation to dissipate the excess energy.Salinity with 240-320 g/L Na Cl led to over production of Q_A^-and the destruction of the destruction of PSII acceptor side,causing inhibition of photosynthetic electron transport.D.salina cells can cope efficiently with the sudden increase in Na Cl from 80 to 240 g/L by accumulating amount of?-carotene per cell and heat dissipation.However,D.salina cells cannot cope efficiently with the sudden increase in Na Cl from 80 to 320 g/L due to the stronger inhibition and impaired of PSII acceptor side,resulting in a damage even a death of the cells.Thus,it is necessary to treat cells with proper shading when it exposed a sudden increase in Na Cl in production.It is suitable to shade D.salina cells to 800?mol·m^-2·s^-1 and 500?mol·m^-2·s^-1 when cells exposed to severe a Na Cl shift from 80to 160 g/L and 80 to 240 g/L.4.Exogenous sodium acetate can effectively alleviate the photoinhibition of high light on D.salina.It not only can promote the proliferation of D.salina,but also promote the accumulation of carotenoids during the culture process.The results show that 2 g/L sodium acetate is beneficial to the proliferation of this strain,and 1 g/L may be more effective for?-carotene accumulation.Exogenous sodium acetate inhibited the photosynthetic activity and total photosynthetic oxygen evolution rate of D.salina,while significantly promoted the respiratory oxygen consumption rate of D.salina.It is speculated that sodium acetate may increase the metabolic flux of glucose metabolism pathway through the formation of acetyl coenzyme A,thus promoting the synthesis of?-carotene.The F_v/F_m value of D.salina decreased only by 2.4%,while the CK is 26%when adding 1 g/L sodium acetate,indicating that the exogenous sodium acetate effectively alleviated the photoinhibition by strong light on this alga.And the non-photochemical quenching(NPQ)is significantly increased,which indicated that sodium acetate dissipated the excess light energy by heat dissipation through the construction of NPQ,so as to protect photosynthetic apparatus.In conclusion,Salinity,light intensity and temperature have interaction on the growth of Dunaliella salina.110 psu should be selected for the purpose of rapid acquisition of algal cell density,and 160 psu for the purpose of acquiring biomass.In addition,sodium acetate can effectively promote the growth and biomass accumulation of Dunaliella salina.It can be speculated that the exogenous sodium acetate enhanced respiration plays an important role in acceleration of?-carotene accumulation.Sodium acetate can effectively alleviate the photoinhibition of strong light on Dunaliella salina through the construction of NPQ.