中国科学院海洋研究所机构知识库

盐生杜氏藻（Dunaliella salina）是一种广盐性的单细胞、双鞭毛真核绿藻。在强光、高盐等胁迫条件下，能大量累积β-胡萝卜素，是目前天然β-胡萝卜素最佳来源。同时，盐生杜氏藻还含有丰富的蛋白质、甘油、多不饱和脂肪酸、维生素和矿物质等物质，具有广阔的应用前景。目前规模化培养盐藻生产β-胡萝卜素，主要采用二步串联法在自然浅塘（澳大利亚）或人工跑道池（以色列、中国、伊朗、印度等）中实现，即首先在适宜绿色盐藻细胞生长条件下，大量生产生物质或藻细胞，然后转入高盐度海水中，在自然强光、高温下大量积累β-胡萝卜素。理论上，高盐、强光是细胞大量积累β-胡萝卜素的必要条件；但从第一阶段向第二阶段转移时，已经适应温和条件的盐藻绿色细胞极易面对自然强光、介质盐度剧升等胁迫条件，发生光抑制甚至光氧化，导致藻细胞损伤甚至大量死亡，成为实现大规模培养盐生杜氏藻生产β-胡萝卜素的限制因素。高盐是否会加剧强光胁迫，是否有方法既能缓解可能存在的双重胁迫，又能维持较高的β-胡萝卜素产出尚不清楚。

本论文以盐生杜氏藻（IOCAS 879ss）为实验藻株，围绕其生长阶段和盐藻由生长阶段向β-胡萝卜素积累阶段的过渡阶段，开展了如下研究：首先，筛选出适宜盐藻生长的盐度、光照强度和温度范围，通过正交试验方法研究盐度、光照强度和温度对盐藻生长的交互作用影响，对比研究了盐藻比生长速率、色素含量、呼吸耗氧速率、光合放氧速率和叶绿素荧光参数的变化，并通过活体叶绿素荧光等方法挖掘各单因素或两因素交互作用的光合生理机制；其次，针对盐藻从生长阶段向β-胡萝卜素积累阶段转移时的盐度变化，利用快速光曲线分析不同盐度变化下的盐藻对光照的需求，对比研究了不同盐度梯度变化对盐藻生长、藻细胞光合能力、光强适应性等方面的差异，为明确不同程度盐度变化时，人工调节设置光照强度提供数据支撑；同时，通过添加有机碳源甘油和醋酸钠，探究盐藻在高光高盐条件下的细胞密度、色素含量、呼吸耗氧速率、光合放氧速率以及叶绿素荧光等参数的变化，对比研究了甘油和醋酸钠在高光高盐条件下分别对盐藻生长和色素积累的影响，并结合光合生理参数初步探究了最优碳源对盐藻生长的影响机制，验证有机物添加的作用效果。

主要结果如下：

1. 在生长阶段，藻细胞最大比生长速率是0.36 d^-1；盐度、光照强度和温度三因素对盐藻比生长速率的影响次序为温度＞盐度＞光照；最适合藻细胞增殖的条件为：盐度110、光照强度120 μmol·m^-2·s^-1、温度30℃。通过盐度、光照和温度交互作用的二元图分析发现，当盐度为160时，温度从20℃上升到30℃时，藻细胞的比生长速率增幅为0.12 d^-1，而盐度为110时，增幅为0.17 d^-1。同样的，光强为50 μmol·m^-2·s^-1时，藻细胞的比生长速率增幅为0.14 d^-1，光强为120 μmol·m^-2·s^-1时，增幅为0.17 d^-1，说明盐度与温度、光照与温度对藻细胞增殖存在交互作用。

2. 培养基盐度由110增加至160，盐藻叶绿素含量和类胡萝卜素含量分别增加了12.4%和12.6%，比生长速率增长了56.5%。不同盐度梯度变化实验结果显示：盐藻从NaCl终含量为80 g/L的培养基转移到NaCl终含量分别为160、240和320 g/L的培养基中，短期内藻细胞生长均受抑制。从80 g/L分别转移到240 g/L和320 g/L NaCl浓度的培养基中，藻细胞停止生长。随着培养时间的延长，NaCl终含量为160 g/L的类胡萝卜素总产量最高。这表明相对低盐度有利于藻细胞的快速增殖，相对高盐度有利于类胡萝卜素的积累，因此，若以快速获取藻细胞密度为目的，应选择盐度110，若以获取生物量为培养目的，盐度提升至160为宜。

3. 快速光曲线拟合结果显示，盐度梯度变化越大，藻细胞对光照的需求量越低。盐藻可以通过热耗散的形式来耗散掉多余的能量从而有效地应对盐度从80到160 g/L的突然上升。而当培养基盐度从80 g/L骤升到240，320 g/L时，由于Q_A^-的过度积累，PSII受体侧受到破坏，导致光合电子传递受到抑制。藻细胞通过β-胡萝卜素积累、热耗散等机制来应对盐度从80骤升到240 g/L。但由于盐藻受到非常强的光抑制以及其PSII受体侧的损伤，盐藻不能应对由80骤升到320 g/L的盐度变化从而导致藻细胞大批量死亡。因此，在瞬时盐度变化过程中，需要给藻细胞进行适当的遮光处理。当盐度从80 g/L NaCl骤升至160 g/L NaCl和240 g/L NaCl时，短期遮光至800 μmol·m^-2·s^-1和500 μmol·m^-2·s^-1比较合适。

4. 在培养过程中添加外源醋酸钠，可以有效缓解强光对藻细胞产生的光抑制，促进藻细胞的增殖和类胡萝卜素的积累。其中2 g/L醋酸钠对藻细胞增殖的效果最好，添加1 g/L醋酸钠可以有效促进藻细胞类胡萝卜素的积累。添加醋酸钠抑制了藻细胞的光合活性和总光合放氧速率，但明显促进了藻细胞的呼吸耗氧速率，推测醋酸钠可能是通过生成乙酰辅酶A增加了糖代谢途径中代谢物的通量从而促进了β-胡萝卜素的合成。在光照强度为240 μmol·m^-2·s^-1的条件下，添加1 g/L的外源醋酸钠，对照组藻细胞F_v/F_m值下降了26%，而添加醋酸钠组的降幅仅为2.4%，说明添加外源醋酸钠后有效的缓解了强光对藻细胞产生的光抑制。同时醋酸钠组的非光化学淬灭（NPQ）显著升高，说明醋酸钠是通过NPQ的构建将多余的光能以热耗散的形式耗散掉，从而保护了藻细胞的光合机构。

综上所述，盐度、光照和温度三因素对盐藻的生长存在交互作用，低盐度（110）有利于盐藻的增殖，高盐度（160）有利于盐藻类胡萝卜素的积累。且在培养过程中添加醋酸钠可以有效的促进盐藻细胞的生长和类胡萝卜素的积累，醋酸钠主要通过提高呼吸作用促进β-胡萝卜素的积累，且醋酸钠可以通过构建NPQ有效缓解强光对藻细胞产生的光抑制。

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 β-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 β-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 β-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 β-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 β-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 β-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 β-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 temperature 30°C, salinity 110 psu, and light intensity 120 μmol · 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 to 0.36 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-temperacture 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 NaCl was transferred to fresh medium with 160, 240 and 320 g/L NaCl in a short time. NaCl increased from 80 g/L to 240 g/L and 80 g/L to 320 g/L NaCl, 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 NaCl from 80 to 160 g/L by heat dissipation to dissipate the excess energy. Salinity with 240-320 g/L NaCl 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 NaCl from 80 to 240 g/L by accumulating amount of β-carotene per cell and heat dissipation. However, D. salina cells cannot cope efficiently with the sudden increase in NaCl 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 NaCl in production. It is suitable to shade D. salina cells to 800 μmol·m^-2·s^-1 and 500 μmol·m^-2·s^-1 when cells exposed to severe a NaCl shift from 80 to 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 β-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 β-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 β-carotene accumulation. Sodium acetate can effectively alleviate the photoinhibition of strong light on Dunaliella salina through the construction of NPQ.