实验海洋生物学重点实验室知识库

天然虾青素（Astaxanthin）是一种安全高效的着色剂和抗氧化剂，在水产养殖、化妆品、食品和医药等领域具有非常大的应用潜力。雨生红球藻（Haematococcus pluvialis，以下简称红球藻）是天然左旋虾青素的优质来源，是已知虾青素含量最高的生物，养殖该藻是目前获取天然虾青素的最佳途径。不断提高红球藻生物量和虾青素含量是提高虾青素生产效率的关键，这两方面的研究因兼具理论和应用价值，备受国内外关注。然而，目前与实际生产应用相结合的研究较少，且缺乏针对不动细胞阶段促进虾青素累积的代谢规律研究。

本论文以雨生红球藻H₆为实验藻株，重点关注不动细胞阶段藻细胞的虾青素累积情况，及其生理特性和代谢水平。首先基于产业规模化培养过程，利用光响应曲线（the photosynthesis-irradiance (P-I) curve），分析管道式光生物反应器中，不同培养时期的红球藻细胞对户外高光的响应机制。

其次，目前已确定添加醋酸钠能显著促进红球藻生物量积累和虾青素含量，但对醋酸钠如何影响藻细胞的生理特性及其代谢过程认知不足。本研究在不动细胞阶段添加醋酸钠，（1）通过检测呼吸速率和光合速率、最大光化学效率（Fv/Fm）及非化学光淬灭（NPQ）等，分析添加醋酸钠后红球藻虾青素积累过程中的生理变化；（2）结合代谢组学分析，并检测油脂含量及虾青素几何异构体组成，来探究外源醋酸钠对红球藻虾青素积累过程中细胞内代谢过程的影响。通过以上研究来进一步挖掘添加醋酸钠影响红球藻内物质的合成代谢机制，为在工业化培养中推广不动细胞阶段添加醋酸钠促进虾青素累积提供基础数据和理论支持。

此外，前期研究已证实抑制光呼吸会显著抑制红球藻虾青素积累，本研究结合代谢组学分析又进一步探究了光呼吸途径与虾青素合成的代谢相互关系，阐明光呼吸在虾青素积累过程中的重要作用。

本研究还基于实际生产应用中的问题，初步摸索最适接种比例。接种比例是指原藻液（旧液）体积与新培养基体积的比例，在大规模工业化的培养过程中，接种比例会影响红球藻虾青素生产效率。因此，研究不同接种比例在绿色游动细胞阶段对增殖生长的影响，及其后续在红色不动细胞阶段对虾青素积累的影响，以及不同接种比例的藻细胞在整个培养过程中的生理变化。该研究对于产业化培养红球藻生产虾青素是非常有必要的。

主要结果如下：

1、根据细胞形态和色素变化，雨生红球藻在管道式光生物反应器中的户外培养过程可以分为细胞转化阶段（由绿色游动细胞转变为绿色不动细胞）和虾青素积累阶段（逐步形成富含虾青素的红色不动细胞）。在前一阶段，干重和原生质体直径显著增加；在后一阶段，单个藻细胞的虾青素含量快速增加并形成多层细胞壁。此外，细胞转化阶段的净光合速率，最大净光合速率，光饱和点等光合参数都高于虾青素积累阶段，说明前一阶段具有更好的光合能力。由此推测在细胞转化阶段，光合作用的物质和能量主要用于生物量的积累和细胞的增大；在随后的虾青素积累阶段，虾青素积累及合成多层细胞壁所需的底物和能量主要来源于其他物质的转化。

2、在不动细胞阶段添加醋酸钠，虾青素含量增加，藻细胞的光合活性受到抑制，而其呼吸速率提高。可以推测，在添加醋酸钠后，呼吸作用的增强对促进虾青素积累起到了重要作用。此外，与凌晨相比，中午Fv/Fm的下降程度代表光抑制程度，添加醋酸钠Fv/Fm的下降程度在培养第4天和第6天均低于空白对照组，说明添加醋酸钠后红球藻所受的光抑制水平降低。同时，NPQ值在醋酸钠处理组藻细胞中显著增加，表明添加外源醋酸钠可诱导缓解藻细胞受光抑制的保护机制。综上所述，在不动细胞阶段添加外源醋酸钠，既增强了虾青素的积累，又提高了红球藻的光保护能力。

3、在不动细胞阶段添加醋酸钠（Ac），游离虾青素单体及虾青素酯的含量均显著增加，且对虾青素几何异构体的组成比例有影响。通过GC-MS共鉴定出78种代谢产物，其中氨基酸27种，有机酸16种，脂肪酸12种，多元醇7种，磷酸3种，糖3种，胺2种，其它化合物8种。根据藻液颜色收集红球藻藻细胞样品，并将其划分为绿色（I，第0天）、棕色（II，第6天）和红色（III，第12天）三个阶段。加入Ac后，在棕色和红色阶段丙酮酸大量积累，参与TCA循环的柠檬酸、延胡索酸、琥珀酸和苹果酸也大量积累，能与乙醛酸相互转化的草酸、甘油酸也大量积累；同时果糖和葡萄糖含量降低，蔗糖显著增高。此外，尽管Ac组的游离脂肪酸含量在红色阶段显著低于CK组，然而Ac组的总脂含量和虾青素酯含量在棕色和红色阶段均显著高于CK组，脂肪酸合成的前体物质丙二酸含量也显著高于CK组，说明Ac能促进脂肪酸的生物合成，而脂肪酸则可进一步用于合成油脂或虾青素酯。根据以上结果推测，外源添加的醋酸钠进入藻细胞后转化为乙酰辅酶A，参与TCA循环和乙醛酸循环，为虾青素的生物合成提供底物和能量（NAD(P)H、ATP）；同时，以乙酰辅酶A的形式可能还参与了脂肪酸的生物合成，并且合成的脂肪酸与已合成的游离虾青素单体酯合形成虾青素酯，进一步直接提高虾青素含量；此外，脂肪酸合成加快也提高了油脂含量，形成油滴保护虾青素从而间接促进虾青素累积。

4、在不动细胞阶段，光呼吸抑制剂CM对光呼吸的抑制作用并不影响藻细胞的生物量，对其叶绿素含量影响较小，而对虾青素的生物合成有显著的抑制作用。根据藻液颜色收集红球藻藻细胞样品，并将其划分为绿色（I，第0天）、棕色（II，第6天）和红色（III，第12天）三个阶段。通过多元统计分析（主成分分析、偏最小二乘判别分析）和层次聚类分析揭示了代谢物的聚类。加入CM后，甘氨酸和乙醇酸在棕色和红色阶段大量积累，说明CM确实抑制了红球藻的光呼吸途径，并且在虾青素积累过程中光呼吸始终被抑制。同时加入CM后果糖和葡萄糖含量降低，参与TCA循环的中间产物（柠檬酸、延胡索酸、琥珀酸和苹果酸）在棕色阶段也减少，推测抑制光呼吸也影响了TCA循环。然而，在光呼吸被抑制后，许多胞内的细胞保护代谢物，如氨基酸、多胺、多元醇和蔗糖，都显著增加。以上结果表明，在虾青素积累过程中，光呼吸途径可能影响TCA循环，从而影响虾青素合成所需的底物和能量，因此光呼吸在红球藻虾青素积累过程中发挥重要作用。

5、接种比例是指原藻液（旧液）体积与新培养基体积的比例，对绿色游动阶段的细胞生长和红色不动阶段的虾青素积累都有较大影响。接种比例较高（2:1，1:1）时生物量、细胞密度和虾青素含量都较高，但接种比例较低（1:7，1:3）时藻细胞的比生长速率较大，虾青素/叶绿素比率较高。在绿色阶段，接种比例对光合电子传递链（OJIP曲线）的影响随培养时间逐渐减小；而在红色阶段，接种比例对光合电子传递链有较大影响。叶绿素荧光参数（光系统II的实际光化学效率（F_PSII）、光化学淬灭（qP）、光系统II的最大光化学效率（Fv/Fm）及光能转换效率（Fv'/Fm'））表明，绿色阶段的光合能力要显著高于红色阶段，而接种比例为2:1时在红色阶段藻细胞的光合活性也优于其他低接种比例组。因此，在大规模商业化培养红球藻生产虾青素过程中，若需要尽快达到较高生物量和虾青素含量，可选择高接种比例2:1；若需要获得快速增殖的藻细胞和尽快收获虾青素产品，可选择低接种比例1:7。

以上研究有助于更深入地理解促进红球藻不动细胞累积虾青素的代谢规律，在生产中更有目的的通过人工调控避免光抑制发生、定向提高虾青素合成能力，创造更高的经济价值；也为通过基因工程等手段对红球藻进行遗传改良，提高其虾青素合成能力提供新思路和新靶点。

Astaxanthin is a safe and efficient colorant and antioxidant, which has great potential in aquaculture, cosmetics, nutraceutical and medicine. Haematococcus pluvialis, a unicellular Chlorophyceae, is widely known as the principal source of natural astaxanthin, is mass-cultivated in industrial-scale production. Continuous improvement of enhancing biomass and astaxanthin content of H. pluvialis is the key to develop astaxanthin production, and the researches on these two aspects have both theoretical and practical value. It has been confirmed that exogenous addition of sodium acetate significantly enhanced biomass accumulation and astaxanthin content of H. pluvialis, however, there is little information about physiological changes and the effects on the photoinhibition level and photoprotection capacity, by adding exogenous sodium acetate at the non-motile stage. Moreover, photorespiration has been proved to playing an important role in astaxanthin accumulation process, however, the metabolic responses allowing photorespiration to affect astaxanthin accumulation still need to be clarified. Furthermore, based on industrial-scale production, inoculation proportion also has an important impact on cell growth and subsequent astaxanthin accumulation.

The alga H. pluvialis (strain H₆) was used in this study, combined with two-stage culturing strategy. This study was focused on physiological characteristics and metabolic changes of the second stage, which is the astaxanthin accumulation stage. Firstly, the photosynthesis-irradiance (P-I) curve was used, to analyze the photoacclimation response of algal cells in the tubular photobioreactors, under the outdoor, changeable and high light conditions. Secondly, the effects of exogenous sodium acetate on the physiological changes during astaxanthin accumulation process, and on the photoprotection capacity at the non-motile stage were investigated. Finally, metabolomics analysis in absence or presence of sodium acetate during astaxanthin accumulation period were performed, using gas chromatography-mass spectrometry (GC-MS), and the composition of astaxanthin geometrical isomers and the total lipid content were determined, in order to further explore the mechanism of exogenous sodium acetate affecting the biosynthesis and metabolism in H. pluvialis. The above researches would provide basic data and theoretical support for the promotion of adding sodium acetate at the astaxanthin accumulation stage in industrial-scale production.

Moreover, to further investigate the interrelation between photorespiration and astaxanthin accumulation, metabolomics analysis of H. pluvialis during astaxanthin accumulation period were performed, in absence or presence of carboxymethoxylamine (CM), an inhibitor of the photorespiratory pathway. Furthermore, it is necessary to study the effects of different inoculation proportion on cell growth during the green motile stage, and astaxanthin accumulation during the subsequent red non-motile stage, combined with changes of biomass and physiological level.

The main results were as follows:

1. Based on morphological and pigment changes, the process can be divided into the cell transformation stage (from green, motile cells to green, astaxanthin-deficient, non-motile cells) and the astaxanthin accumulation stage (forming red, astaxanthin-enriched, non-motile cells). Dry weight and the diameter of the cellular protoplasm increased significantly in the former stage, while astaxanthin content of algal per cell significantly increased and the multilayered cell wall was formed in the latter stage. The net photosynthetic rates, maximum net photosynthetic rate, and saturation irradiance during the cell transformation stage were higher than those during the astaxanthin accumulation stage, indicating the former stage possessed better photosynthetic activities. We speculate that photosynthetic strategy of different stages were as follows: (i) during the cell transformation stage, the flux of photosynthetic substance and energy mainly flowed into biomass (especially the rounded and enlarged protoplast); (ii) during the subsequent astaxanthin accumulation stage, photosynthesis performance reduced, and the substrates and energy required for astaxanthin accumulation and cell wall thickening were mainly derived from the transformation of other substances, suggesting adding exogenous carbon source would enhance astaxanthin accumulation at this stage. This study would provide physiological background for optimizing astaxanthin production using H. pluvialis in industry.

2. The astaxanthin contents in H. pluvialis cells with 10 mM sodium acetate increased more than 1.26-fold as compared with that in cells without sodium acetate after 6 days of incubation, indicating that exogenous sodium acetate accelerated astaxanthin accumulation at the non-motile stage significantly. Addition of sodium acetate inhibited photosynthetic rates, indicating that exogenous sodium acetate suppressed photosynthetic activity at the non-motile stage. However, additional sodium acetate increased respiratory rates. It can be speculated that the enhanced respiration plays an important role in the acceleration of astaxanthin accumulation in the presence of sodium acetate. Moreover, the level of photoinhibition in H. pluvialis decreased after adding sodium acetate at non-motile stage, which is indicated by the fact that the decreased value of Fv/Fm determined at midday, compared with that determined at predawn, declined on day 4 and day 6. NPQ increased significantly with additional sodium acetate on day 4 and day 6, indicating that additional sodium acetate induced a mechanism to protect H. pluvialis cells against photoinhibition. Taken together, exogenous sodium acetate enhances astaxanthin accumulation and the photoprotection capacity of H. pluvialis at the non-motile stage.

3. Adding exogenous sodium acetate (Ac) at the astaxanthin accumulation stage significantly enhanced the astaxanthin content, including both free astaxanthin monomer and astaxanthin ester, and had a certain influence on its geometrical isomers. A total of 78 metabolites were identified by GC-MS, including 27 amino acids, 16 organic acids, 12 fatty acids, 7 polyols, 3 phosphoric acids, 3 sugars, 2 amines, and 8 other compounds. After adding Ac, pyruvic acid significantly accumulated, as well as citric acid, succinic acid, fumaric acid and malic acid which involved in the tricarboxylic acid cycle (TCA cycle) were increased, moreover, the contents of oxalic acid and glyceric acid which involved in the glyoxylae cycle were higher than those in CK. At the same time, fructose and glucose content decreased, sucrose increased significantly. In addition, the detected free fatty acids in Ac was significantly lower than those in CK at the red phase, while the total lipid content and astaxanthin esters content in Ac were significantly higher than those in CK at both brown and red phases, and the content of malonic acid, the precursor of fatty acid biosynthesis, was also significantly higher than that in CK. The above results indicated that exogenous Ac promoted the biosynthesis of fatty acids and further be used to synthesize lipid or astaxanthin ester. It is speculated that acetate can be utilized by TCA cycle and glyoxylae cycle to generate the carbon skeletons and NAD(P)H for astaxanthin synthesis directly; it can also directly participate in fatty acid biosynthesis in the form of acetyl coenzyme A to increase total lipid content, and the synthesized fatty acids combined with free astaxanthin monoesters to form astaxanthin ester, to further enhance astaxanthin content indirectly.

4. The inhibition of photorespiration by CM did not affect the biomass, had a slight effect on chlorophyll at the end of the incubation, but significantly suppressed astaxanthin accumulation. Algal samples were collected on days 0, 6 and 12, which were named as the green phase with green, non-motile cells, the brown phase with astaxanthin-synthesizing, non-motile cells, and the red phase with astaxanthin-enriched, red, non-motile cells, respectively. Multivariate statistical analyses (principal component analysis, partial least squares-discriminant analysis), and hierarchical cluster analysis revealed the clustering of the metabolites. Glycine and glycolic acid had accumulated substantially at both the brown and red phases, indicating that photorespiration was inhibited by CM. In presence of CM, the TCA cycle was restricted at the brown phase due to decreased intermediates, specifically, decreased levels of fructose and glucose. However, inhibiting photorespiration enhanced the levels of many intracellular cytoprotective metabolites, such as amino acids, polyamines, polyols and sucrose. A hypothetical metabolic regulation model of the photorespiratory pathway affecting astaxanthin accumulation of H. pluvialis is proposed. This study provides the first metabolomic evidence that photorespiration enhances astaxanthin accumulation.

5. The inoculation proportion had a great influence on both cell growth during the green motile stage and astaxanthin accumulation during the subsequent red non-motile stage. The biomass and cell density were higher when inoculation proportion was higher, as well as astaxanthin content. However, the specific growth rate and the of ratio of astaxanthin/chlorophyll were higher when inoculation proportion was lower, and the color of algal cultures in lower inoculation proportion was redder. At the green stage, the effect of inoculation proportion on photosynthetic electron transfer chain (OJIP curve) decreased over time, while at the red stage, initial inoculation proportion had significant effects on photosynthetic electron transfer chain (OJIP curve). The photosynthetic performance during the green stage was significantly higher than that during the red stage, however, when the inoculation proportion was 2:1, the algal cells still had high photosynthetic activity than other groups of lower inoculation proportion during the red stage. Therefore, in the large-scale commercial cultivation of H. pluvialis, it is necessary to select the appropriate inoculation proportion, in order to achieve better production efficiency of astaxanthin.