UV-B对雨生红球藻生长及虾青素积累的影响

以雨生红球藻(Haematococcus pluvialis)为材料, 研究不同强度的UV-B对雨生红球藻生长、光合作用及虾青素积累的影响和其作用机理。设置5种紫外线强度, 分别在正常光照培养条件下补充不同强度UV-B(100—500 lx), 标记为CK、U100、U200、U300、U400和U500六组。结果表明, 经UV-B辐射后雨生红球藻细胞密度、PSⅡ最大光化学效率(F_v/F_m)、非光化学淬灭系数(NPQ)和叶绿素(Chl.a和Chl.b)含量等均呈现下降趋势, 且与辐射强度相关。相反, 虾青素含量在100—400 lx强度下随UV-B辐射强度的增加而升高。与对照相比, 高强度UV-B辐射(U400)36h和72h后藻细胞虾青素含量分别提高了35.68%和56.23%, 达到5.82和7.06 mg/L。qRT-PCR检测发现雨生红球藻虾青素合成关键酶基因(IPI、PSY、BCH和BKT)的表达量随紫外辐射强度和辐射时间的增加均有不同程度升高。UV-B辐射亦调控紫外光受体UVR8及其信号转导通路核心元件(COP1、SPA1、HYH和HY5)的基因表达, 暗示上述基因参与了UV-B诱导雨生红球藻虾青素积累的过程。研究揭示紫外光受体UVR8介导的信号转导通路可能参与虾青素合成的转录调控, 为建立应用UV-B辅助光源促进雨生红球藻富集虾青素的工艺提供了基础, 同时为解析光诱导雨生红球藻虾青素积累的转录调控机制提供了新的视角。     

外源2,6-二叔丁基对甲酚对非生物胁迫条件下雨生红球藻虾青素积累的影响

【背景】雨生红球藻是天然虾青素的最佳来源,广泛应用于虾青素的工业化生产。【目的】探究外源添加不同浓度的2,6-二叔丁基对甲酚(Butylated hydroxytoluene,BHT)对雨生红球藻虾青素积累的影响,以期建立BHT提高雨生红球藻虾青素产量的技术体系。【方法】选用不含硝态氮的BBM培养基,辅以强光照,培养雨生红球藻(Haematococcus pluvialis)LUGU,测试不同浓度BHT对雨生红球藻生物量、虾青素含量、活性氧、抗氧化系统和虾青素合成相关酶基因的影响。【结果】在0-3 mg/L BHT范围内,2 mg/L BHT对雨生红球藻虾青素积累的促进效果最佳,达到31.66 mg/g。2 mg/L BHT有效降低了雨生红球藻内的活性氧水平,增加了细胞内NO水平,提高了藻细胞内过氧化氢酶(Catalase,CAT)、过氧化物酶(Peroxidase,POD)和超氧化物歧化酶(Superoxidedismutase,SOD)活性以及谷胱甘肽(Glutathione,GSH)的含量,诱导了虾青素合成关键酶基因chy和lcy的高效表达。【结论】非生物胁迫条件下,外源添加适量的BHT能促进雨生红球藻中虾青素的积累,且与藻细胞内的信号分子活性氧(Reactive oxygen species,ROS)、NO水平及虾青素合成相关基因的表达调控相关。     

UV-B辐射对雨生红球藻生长和类胡萝卜素含量的影响

以BG11培养基,对雨生红球藻进行了室内培养,研究了增强UV-B辐射对雨生红球藻生长速率和虾青素含量的影响.室内培养的条件是UV-B辐射强度为0.1J·m-2·S-1,0.2J·m-2·S-1, 0.3J·m-2·S-1, 光照强度为60 μmoL·m-2·S-1(昼夜比为12 h:12 h),温度为20～26℃.测定了培养液细胞数目、叶绿素a、类胡萝卜素的含鼍,并对雨生红球藻进行了显微结构观察.结果显示在室内培养雨生红球藻增加UV-B辐射,能够提高其细胞内虾青素的含量,其显微结构显示类胡萝卜素颗粒明显增加的现象.本研究目的是在室内培养雨生红球藻提高虾青素产量的方法.     

温度对雨生红球藻(Haematococcus pluvialis)生物量和虾青素产量的影响

在用环形培养池模拟系统培养雨生红球藻的过程中,研究了温度对雨生红球藻生物量及虾青素产量的影响。结果表明,在15～25℃的范围内,不同温度下雨生红球藻生物量和虾青素含量及产量都经历了一个上升—最高—下降的过程。25℃与22℃时红球藻的虾青素产量、虾青素含量(干重)均显著高于其他温度的(P<0.01),但两者间差异不显著(P>0.05)。15℃时,红球藻生物量、虾青素含量和虾青素产量均最低,分别为1.4g、0.54%和2.49mg/L;25℃时,红球藻生物量和虾青素产量最高,分别为2.68g和13.53mg/L;22℃时,虾青素含量最高,为1.52%。     

褪黑素对非生物胁迫下雨生红球藻中虾青素积累的影响

以雨生红球藻Haematococcus pluvialis LUGU株为研究对象, 研究在高光照和缺氮胁迫条件下, 添加不同浓度褪黑素(melatonin, MLT)对雨生红球藻生长、虾青素积累、活性氧(ROS)、信号分子及dxs基因表达量的影响。结果表明, 外源添加10 μmol/L MLT可有效提高藻细胞中虾青素的含量, 最高可达31.32 mg/g, 是对照组(13.27 mg/g)的2.36倍; 抑制了细胞内ROS水平, 上调了信号分子一氧化氮(NO)和水杨酸(SA)的含量; 此外, dxs基因表达水平比对照组明显提高, 最高达11.3倍。研究表明, 在非生物胁迫条件下, 雨生红球藻中虾青素的大量积累可能与外源MLT调控细胞内ROS、信号分子及基因表达有关。     

氮源对雨生红球藻绿色生长期细胞生长的影响

【背景】雨生红球藻(Haematococcus pluvialis)细胞能合成积累具有超强抗氧化活性的虾青素,是生产高值天然虾青素的优异藻种。【目的】解析不同氮源对雨生红球藻生长的效应,以期建立优化氮素营养提高雨生红球藻生物量和虾青素产量的技术体系。【方法】选用Na NO3和NH_4Cl为氮源、辅以pH缓冲液Hepes,培养雨生红球藻藻株797,测试2种不同氮源对雨生红球藻藻液pH、营养生长期(绿色生长期)藻细胞生长、叶绿素含量和生物量等的影响。【结果】以Na NO3为氮源培养的雨生红球藻细胞的比生长速率、生物量、叶绿素a和叶绿素b含量均高于以NH_4Cl为氮源培养的藻细胞。不同氮源对雨生红球藻培养液的pH值有显著影响,NH_4Cl氮源导致培养液pH值降低,然而Na NO3氮源则导致培养液pH值上升。添加pH缓冲液Hepes能有效稳定培养液的pH值,并促进雨生红球藻的生长,尤以NH_4Cl为氮源添加Hepes的效果更显著。不同氮源导致雨生红球藻营养生长阶段细胞的生长和生物量等差异主要源于不同氮源引起藻液pH的变化。【结论】添加pH缓冲液Hepes可有效控制藻液pH,进而显著促进以Na NO3和NH_4Cl为氮源的雨生红球藻营养生长阶段细胞的生长和生物量积累。     

雨生红球藻ZL-1 生长和虾青素积累条件优化

分离鉴定了一株雨生红球藻ZL-1, 比较了不同接种密度和吲哚乙酸浓度对其生长的影响; 在此基础上, 探究了不同浓度水杨酸和盐度对雨生红球藻虾青素积累的影响。结果表明: (1)接种密度为2.00×10⁴ cell·mL^–1 时, 雨生红球藻生长快速, 最终生物量达到最大值0.43 g·L^–1; 不动细胞比游动细胞更快的积累虾青素, 高光诱导不动细胞得到最高虾青素产量为8.44 mg·L^–1; IAA 终浓度为1.5 mg·L^–1 时, 雨生红球藻生长速度最快, 最终细胞密度和干重分别比对照组提高了24.28%和27.11%; (2)水杨酸具有缓解高光胁迫和促进虾青素积累的双重作用, 15 和25 mg·L^–1水杨酸诱导下, 雨生红球藻生物量较高, 虾青素产量分别比对照组提高了18.18%和18.94%; 使用4‰的盐度胁迫雨生红球藻, 虾青素产量较对照组提高了17.42%, 但盐度也会引起藻细胞的漂白、死亡, 导致生物量显著降低。     

温度对雨生红球藻（Haematococcus pluvialis）生物量和虾青素产量的影响

在用环形培养池模拟系统培养雨生红球藻的过程中,研究了温度对雨生红球藻生物量及虾青素产量的影响。结果表明,在15～25℃的范围内,不同温度下雨生红球藻生物量和虾青素含量及产量都经历了一个上升一最高一下降的过程。25℃与22℃时红球藻的虾青素产量、虾青素含量(干重)均显著高于其他温度的(P＜0．01),但两者间差异不显著(P＞0．05)。15℃时,红球藻生物量、虾青素含量和虾青素产量均最低,分别为1．4g、0．54％和2．49mg／L,25℃时,红球藻生物量和虾青素产量最高,分别为2．68g和13．53mg／L,22℃时,虾青素含量最高,为1．52％。     

雨生红球藻培养过程中色素动态变化与光合生理特性研究

以雨生红球藻Haematococcus pluvialis CG-06为实验材料,分析测定在正常培养周期内藻细胞主要色素的变化动态、光合生理特性,以及培养基中硝态氮的含量。结果表明,雨生红球藻在绿色细胞阶段的主要色素包括:叶绿素、叶黄素、β-胡萝卜素,培养至红色细胞阶段增加了角黄素、海胆酮、虾青素单酯及双酯等次生类胡萝卜素。硝态氮浓度在培养初期下降迅速,第3 d降至4.875 mg/L,下降了85.3%,至第7 d下降为0.169 mg/L。雨生红球藻培养至第7 d时,细胞中开始检测出虾青素,含量为0.159 mg/g,此时虾青素合成速度较快,至第11 d虾青素含量上升为1.68 mg/g,在虾青素合成初期β-胡萝卜素的含量下降。藻细胞的光合速率、呼吸速率和NPQ在培养前期比较稳定,第7 d细胞光合速率开始下降,而呼吸速率和NPQ则上升,在整个培养周期中,藻细胞的Fv/Fm变化不明显。     

活性氧对雨生红球藻生长及虾青素含量的影响

在雨生红球藻培养液中分别添加活性氧1O2、H2O2和·OH的诱生剂,通过测定细胞密度、叶绿素a含量、虾青素含量,研究了这三种活性氧诱生处理对雨生红球藻生长和虾青素含量的影响,初步探索了利用活性氧诱生剂提高雨生红球藻虾青素含量的可行性。实验结果表明,适当浓度的MB能够促进虾青素含量增加,当MB浓度为10-7mol·L-1时,虾青素含量达到5.27μg·mL-1,比对照显著提高。活性氧诱生剂对雨生红球藻生长有抑制作用,但MB的抑制作用小于H2O2和·OH诱生剂。     

氮胁迫对雨生红球藻色素积累与抗氧化系统的影响

选用雨生红球藻CG-06为试验藻株,以BBM为基础培养基,分别设置了0、13.7、27.5、41.2mg·L-1四个硝态氮浓度梯度,分析并探讨在不同硝态氮浓度条件下雨生红球藻生长、生理特性、细胞内主要色素含量的变化以及抗氧化酶活性。结果表明：细胞中色素的积累量和积累速率与初始硝态氮浓度成反比,与抗氧化酶活性呈负相关。缺氮时,培养到第3天的藻细胞中虾青素含量达到4.95μg·mg-1,而对照组在培养到第9天的细胞中才开始产生虾青素,而且在整个培养周期内细胞中的虾青素最大含量仅为4.17μg·mg-1。酶活测定结果显示,虾青素含量较高的红色细胞中,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性明显高于绿色细胞,且GSH-Px活性最高。研究表明,雨生红球藻可能有两种过氧化防御机制,绿色细胞阶段以抗氧化酶作用为主,在培养后期启动虾青素保护机制,两种机制具有协同作用。     

Ultraviolet-B-induced oxidative stress and responses of the ascorbate-glutathione cycle in a marine macroalga Ulva fasciata

The regulation of the antioxidant defence system by ultraviolet-B (UV-B) was determined in a marine macroalga Ulva fasciata Delile exposed to low (0.5, 1 W m(-2)), medium (2.5, 5 W m(-2)), and high (10, 20 W m(-2)) UV-B irradiance. UV-B > or =2.5 W m(-2) increased H2O2 contents that are positively correlated with lipid peroxidation and total peroxide contents. Inhibition of the UV-B-induced H2O2 increase by a specific O2.- scavenger, 1,2-dihydroxy-benzene-3,5-disulphonic acid, shows that O2.- is the primary source of H2O2. Superoxide dismutase activity was increased by UV-B with a peak at 2.5 W m(-2), which did not match the H2O2 pattern. Alleviation of UV-B-induced oxidative damage by a H2O2 scavenger, dimethylthiourea, and a free radical scavenger, sodium benzoate, which inhibited UV-B-induced H2O2 accumulation, suggests that oxidative damage caused by UV-B > or = 2.5 W m(-2) is ascribed to accumulated H2O2. However, a decrease in growth rate and TTC reduction ability only at high UV-B doses indicates that the defence and repairing systems operate at low and medium UV-B doses. H2O2 not only can be excreted but can also be detoxified via the ascorbate-glutathione cycle. Increases in catalase, peroxidase, ascorbate peroxidase, and glutathione reductase activities and ascorbate (AsA) and glutathione pools, as well as AsA regeneration ability, function to keep the balance of cellular H2O2 under low UV-B doses. Dehydroascorbate reductase and monodehydroascorbate reductase are responsible for AsA regeneration under low and medium UV-B radiation, respectively. The appearance of oxidative damage in medium and high UV-B flux is attributable to a lower induction of the ascorbate-glutathione cycle as an antioxidant defence system. Overall, the availability of antioxidants and the induction of antioxidant enzyme activities for detoxifying reactive oxygen species (ROS) are regulated in U. fasciata against UV-B-induced oxidative stress, and experiments using ROS scavengers demonstrate that the antioxidant defence system is modulated by O2.- or H2O2.     

Haematococcus astaxanthin: applications for human health and nutrition

The carotenoid pigment astaxanthin has important applications in the nutraceutical, cosmetics, food and feed industries. Haematococcus pluvialis is the richest source of natural astaxanthin and is now cultivated at industrial scale. Astaxanthin is a strong coloring agent and a potent antioxidant - its strong antioxidant activity points to its potential to target several health conditions. This article covers the antioxidant, UV-light protection, anti-inflammatory and other properties of astaxanthin and its possible role in many human health problems. The research reviewed supports the assumption that protecting body tissues from oxidative damage with daily ingestion of natural astaxanthin might be a practical and beneficial strategy in health management.     

城市生活污水用于培养雨生红球藻的研究

虾青素是自然界广泛存在的一种橘红色类胡萝卜素,广泛应用于食品、药品和化妆品行业。在虾青素的制备中,雨生红球藻是生产虾青素的最有效来源,目前提高虾青素产量的方式主要为提高生物量和产物合成率。目前已有大量研究针对生物量的优化,但依然存在改善空间。为此,尝试用城市生活污水作为培养基对雨生红球藻进行培养。结果表明,生活污水能促进雨生红球藻的生长,其产量是现有BG11培养基的2倍;虾青素的合成时期显著提前（P<0.05）,且体内重金属含量未明显富集,处在安全浓度范围。此外,养藻后的城市生活污水中氮、磷含量显著降低（P<0.05）,高氮、磷富余的情形得到有效改善。证实利用污水培养雨生红球藻的双重效应,一方面有利于积累藻类生物量,另一方面有助于净化水质,在经济效益和生态效益上具有极好的发展潜力。     

Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors

The present paper makes a comparative analysis of the outdoor culture of H. pluvialis in a tubular photobioreactor and a bubble column. Both reactors had the same volume and were operated in the same way, thus allowing the influence of the reactor design to be analyzed. Due to the large changes in cell morphology and biochemical composition of H. pluvialis during outdoor culture, a new, faster methodology has been developed for their evaluation. Characterisation of the cultures is carried out on a macroscopic scale using a colorimetric method that allows the simultaneous determination of biomass concentration, and the chlorophyll, carotenoid and astaxanthin content of the biomass. On the microscopic scale, a method was developed based on the computer analysis of digital microscopic images. This method allows the quantification of cell population, average cell size and population homogeneity. The accuracy of the methods was verified during the operation of outdoor photobioreactors on a pilot plant scale. Data from the reactors showed tubular reactors to be more suitable for the production of H. pluvialis biomass and/or astaxanthin, due to their higher light availability. In the tubular photobioreactor biomass concentrations of 7.0 g/L (d.wt.) were reached after 16 days, with an overall biomass productivity of 0.41 g/L day. In the bubble column photobioreactor, on the other hand, the maximum biomass concentration reached was 1.4 g/L, with an overall biomass productivity of 0.06 g/L day. The maximum daily biomass productivity, 0.55 g/L day, was reached in the tubular photobioreactor for an average irradiance inside the culture of 130 microE/m2s. In addition, the carotenoid content of biomass from tubular photobioreactor increased up to 2.0%d.wt., whereas that of the biomass from the bubble column remained roughly constant at values of 0.5%d.wt. It should be noted that in the tubular photobioreactor under conditions of nitrate saturation, there was an accumulation of carotenoids due to the high irradiance in this reactor, their content in the biomass increasing from 0.5 to 1.0%d.wt. However, carotenoid accumulation mainly took place when nitrate concentration in the medium was below 5.0mM, conditions which were only observed in the tubular photobioreactor. A similar behaviour was observed for astaxanthin, with maximum values of 1.1 and 0.2%d.wt. measured in the tubular and bubble column photobioreactors, respectively. From these data astaxanthin productivities of 4.4 and 0.12 mg/L day were calculated for the tubular and the bubble column photobioreactors. Accumulation of carotenoids was also accompanied by an increase in cell size from 20 to 35 microm, which was only observed in the tubular photobioreactors. Thus it may be concluded that the methodology developed in the present study allows the monitoring of H. pluvialis cultures characterized by fast variations of cell morphology and biochemical composition, especially in outdoor conditions, and that tubular photobioreactors are preferable to bubble columns for the production of biomass and/or astaxanthin.     

Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to zeta-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

Efficiency assessment of the one-step production of astaxanthin by the microalga Haematococcus pluvialis

Continuous cultivation of Haematococcus pluvialis under moderate nitrogen limitation represents a straightforward strategy, alternative to the classical two-stage approach, for astaxanthin production by this microalga. Performance of the one-step system has now been validated for more than 40 combinations of dilution rate, nitrate concentration in the feed medium, and incident irradiance, steady state conditions being achieved and maintained in all instances. Specific nitrate input and average irradiance were decisive parameters in determining astaxanthin content of the biomass, as well as productivity of the system. The growth rate of the continuous photoautotrophic cultures was a hyperbolic function of average irradiance. As long as specific nitrate input was above the threshold value of 2.7 mmol/g day, cells performed green and astaxanthin was present at basal levels only. Below the threshold value, under moderate nitrogen limitation conditions, astaxanthin accumulated to reach cellular levels of up to 1.1% of the dry biomass. Increasing irradiance resulted in enhancement of astaxanthin accumulation when nitrogen input was limiting, but never under nitrogen sufficiency. Mean daily productivity values of 20.8 +/- 2.8 mg astaxanthin/L day (1.9 +/- 0.3 g dry biomass/L day) were consistently achieved for a specific nitrate input of about 0.8 mmol/g day and an average irradiance range of 77-110 microE/m(2) s. Models relating growth rate and astaxanthin accumulation with both average irradiance and specific nitrate input fitted accurately experimental data. Simulations provided support to the contention of achieving efficient production of the carotenoid through convenient adjustment of the determining parameters, and yielded productivity estimates for the one-step system higher than 60 mg astaxanthin/L day. The demonstrated capabilities of this production system, as well as its product quality, made it a real alternative to the current two-stage system for the production of astaxanthin-rich biomass.     

雨生红球藻虾青素酰基转移酶的鉴定与功能研究

为研究雨生红球藻(Haematococcus pluvialis)的甘油二酯酰基转移酶(Diacylglycerol acyltransferase, DGAT)是否具有催化虾青素酰基化的功能, 首先通过雨生红球藻的cDNA库克隆得到了一个II型DGAT编码区全长序列(DGTT2)。在甘油三酯(Triacylglycerol, TAG)合成缺陷型酵母Saccharomyces cerevisiae H1246中过表达DGTT2基因发现HpDGTT2不能回补H1246的表型, 即不具有典型的DGAT功能。利用分离得到的雨生红球藻的内质网成功地建立了一个体外的虾青素酰基转移酶酶活测定体系, 添加含有重组HpDGTT2的酵母细胞的微粒体后虾青素酯的含量显著高于对照, 初步表明HpDGTT2具有催化雨生红球藻中虾青素酰基化功能。以上结果为进一步探索雨生红球藻中DGTT2的功能及深入理解虾青素合成在代谢水平的调控奠定了基础。     

Influence of environmental and nutritional factors in the production of astaxanthin from Haematococcus pluvialis

Astaxanthin extracted from green algae is desirable in the food and pharmaceutical industries due to its antioxidant properties. The green unicellular clear water microalga Haematococcus pluvialis has a high production rate of astaxanthin; indeed, it contains more than 80% astaxanthin content in its cells. This remarkable astaxanthin production is commonly obtained under stress conditions such as nutrient deficiency (N or P), high NaCl concentrations, variations of temperature, and other factors. In this vein, a great research effort has been oriented to determine optimal conditions for astaxanthin production by H. pluvialis.The objective of the present study was the analysis of environmental factors, such as light intensity, aeration and nutrients on the growth and astaxanthin production of H. pluvialis. Maximum growth of H. pluvialis obtained was 3.5x10(5) cells/ml in BBM medium at 28 degrees C under continuous illumination (177 micromol photon m(-2)s(-1)) of white fluorescent light, with continuous aeration (1.5 v.v.m.). Meanwhile, maximal astaxanthin production was 98 mg/g biomass in BAR medium with continuous illumination (345 micromol photon m(-2)s(-1)), with 1 g/l of sodium acetate and without aeration.