UV-B辐射对雨生红球藻光合特性和虾青素含量的影响及其响应

实验研究了不同强度的UV-B(280-320 nm)辐射对雨生红球藻(Haematococcus pluvialis)的光合活性、生物量、色素含量、活性氧(ROS)含量和抗氧化酶活性等的影响, 以探讨利用UV-B辐射诱导虾青素生物合成增强的可能性。结果发现, 经UV-B辐射处理后,雨生红球藻的光合活性降低、生物量增长被抑制。UV-B辐射对叶绿素影响不大, 但会改变细胞的类胡萝卜素和虾青素含量:0.1和0.3 W/m2强度的UV-B辐射使细胞中的这两种色素含量升高, 0.5 W/m2组的色素含量短暂升高后恢复到对照水平。中低强度的UV-B可以促进雨生红球藻单细胞虾青素含量的增加, 但由于其对细胞生长的抑制作用, 并不能使虾青素大量积累。随辐射时间延长, 细胞内ROS含量未明显增加,但抗氧化酶(过氧化氢酶和超氧化物歧化酶)活性下降, 雨生红球藻可能主要依靠虾青素来淬灭ROS。以上结果表明, UV-B辐射对雨生红球藻的主要生理生化过程有抑制作用, UV-B辐射既可以诱导虾青素的合成又会消耗一部分虾青素, 对虾青素含量的影响与其强度有关, 而利用虾青素来清除细胞内的ROS可能是雨生红球藻抵御这种不利环境条件的最重要的途径。       

雨生红球藻β－胡萝卜素酮化酶（bkt）启动子功能分析

单细胞绿藻———雨生红球藻在逆境条件下积累大量的虾青素。β-胡萝卜素酮化酶(bkt)催化在β-胡萝卜素和玉米黄素的β-紫罗酮环C-4位引入酮基的反应,是虾青素合成过程中的关键酶。我们利用凝胶阻滞的方法研究雨生红球藻中bkt基因309bp(-617/-309)启动子区域的转录因子结合位点并发现在-396/-338的59bp探针存在特异的核蛋白结合位点。通过序列分析,发现此59bp区域并不包含TATA或者CAAT-box,而是存在对光、缺氧、p-香豆酸及激素反应的G-box。     

Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 microE/m2.s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L.d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L.d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells.     

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。     

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.     

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 col-umn was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus plu-vialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The re-sults showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingien-sis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a re-markably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).     

雨生红球藻的光周期效应

雨生红球藻(Haematococcus pluvialis)是一种单细胞淡水绿藻, 是自然界已知的中虾青素含量最高的生物物种。通过分析3种光照强度(70、120和300 μmol·m^–2·s^–1)下雨生红球藻细胞形态、生长速率和虾青素含量的差异, 对其光周期效应进行了研究。结果表明, 不同光强下适宜雨生红球藻生长的光周期均为16小时光照/8小时黑暗, 光强为120 μmol·m^–2·s^–1时其细胞生长速率最大, 为0.43 d^–1; 细胞内虾青素含量随着光强和光照时间的增加而增加, 在300 μmol·m^–2·s^–1光强下连续光照15天后, 藻细胞呈亮红色, 平均直径为21.02 μm, 最大虾青素值达39.40 pg·cell^–1。     

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UVB(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...     

缺氮胁迫下雨生红球藻虾青素积累过程中的基因组MSAP分析

虾青素具有多种生物学活性,雨生红球藻为天然虾青素的最佳来源,缺氮胁迫会导致雨生红球藻积累虾青素。为了解缺氮条件下雨生红球藻虾青素积累的分子机制,该研究通过对雨生红球藻进行缺氮胁迫,结合MSAP法,研究了雨生红球藻在缺氮胁迫下虾青素积累过程中基因组甲基化水平的变化,结果表明:缺氮胁迫0~72 h期间,雨生红球藻生长速度减慢,而虾青素积累主要发生在缺氮处理12~24 h期间,随后积累速度减慢。同时,对缺氮胁迫0、24、72 h的雨生红球藻基因组DNA进行甲基化敏感扩增多态性分析,共得到了291个甲基化多态性位点,其中发生甲基化变化的位点在0~24 h和24~72 h分别占总位点的29.90%和53.95%。在缺氮胁迫24 h处DNA半甲基化率最大(为12.71%),全甲基化率最低(为26.80%);缺氮胁迫72 h处DNA全甲基化率最高(为28.52%),半甲基化率最低(为1.72%)。这表明DNA甲基化调节方式的改变是虾青素积累过程中的一种重要调控模式。     

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。     

Carotenogenesis in the green alga Haematococcus pluvialis: Cellular physiology and stress response

The unicellular green alga Haematococcus pluvialis Flotow has recently aroused considerable interest due to its capacity to amass large amounts of the ketocarotenoid astaxanthin (3,3'-dihydroxy- β , β -carotene-4,4'-dione), widely used commercially to color flesh of salmon. Astaxanthin accumulation in Haematococcus is induced by a variety of environmental stresses which limit cell growth in the presence of light. This is accompanied by a remarkable morphological and biochemical 'transformation' from green motile cells into inert red cysts. In recent years we have studied this transformation process from several aspects: defining conditions governing pigment accumulation, working out the biosynthetic pathway of astaxanthin accumulation and questioning the possible function of this secondary ketocarotenoid in protecting Haematococcus cells against oxidative damage. Our results suggest that astaxanthin synthesis proceeds via cantaxanthin and that this exceptional stress response is mediated by reactive oxygen species (ROS) through a mechanism which is not yet understood. The results do not support in vivo chemical quenching of ROS by the pigment, although in vitro it was shown to quench radicals very efficiently. The finding that most of the pigment produced is esterified and deposited in lipid globules outside the chloroplast further supports this assumption. We have suggested that astaxanthin is the by-product of a defense mechanism rather than the defending substance itself, although at this stage one cannot rule out other protective mechanisms. Further work is required for complete understanding of this transformation process. It is suggested that Haematococcus may serve as a simple model system to study response to oxidative stress and mechanisms evolved to cope with this harmful situation.     

Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions

The microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in salmon feed. This study was made to discover optimal conditions for biomass and astaxanthin production in H. pluvialis from Steptoe, Nevada (USA), cultured in batch mode. Growth was carried out under autotrophic (with NaNO3, NH4Cl and urea) and mixotrophic conditions (with 4, 8, 12 mM sodium acetate) under two photon flux densities (PFD) (35 and 85 mumol m-2 s-1). The carotenogenesis was induced by 1) addition of NaCl (0.2 and 0.8%), 2) N-deprivation and 3) high PFD (150 mumol m-2 s-1). Total carotenoids were estimated by spectrophotometry and total astaxanthin by HPLC. Ammonium chloride was the best N-source for growth (k = 0.7 div day-1, 228-258 mg l-1 and 2.0 x 10(5)-2.5 x 10(5) cells ml-1 at both PFD, respectively). With increasing acetate concentration, a slight increment in growth occurred only at 85 mumol m-2 s-1. Light was the best inductive carotenogenic factor, and the highest carotenoid production (4.9 mg l-1, 25.0 pg cell-1) was obtained in cultures pre-grown in nitrate at low light. The NaCl caused an increase in carotenoid content per cell at increasing salt concentrations, but resulted in a high cell mortality and did not produce any increment in carotenoid content per volume compared to cultures grown at 150 mumol m-2 s-1. The highest carotenoid content per cell (22 pg) and astaxanthin content per dry weight (10.3 mg g-1) (1% w/w) were obtained at 85 mumol m-2 s-1 with 0.8% NaCl.     

A novel double-layered photobioreactor for simultaneous Haematococcus pluvialis cell growth and astaxanthin accumulation

This study proposes a novel double-region photobioreactor to simplify the commercial two-stage process of astaxanthin production by the cultivation of Haematococcus pluvialis. The feasibility of the double-region photobioreactor has been investigated and found to achieve high biomass yield in the inner core region and simultaneous astaxanthin accumulation in the outer jacket region. Among many environmental factors, light condition and nitrate level were manipulated for selective cell growth and astaxanthin production. In the outer jacket region, efficient astaxanthin production was accomplished by excessive irradiation (770+/-20 microE m(-2)s(-1)) and nitrate starvation, resulting in a dramatic increase of astaxanthin productivity (357 mg l(-1)). Meanwhile, attenuated light energy (40+/-3 microE m(-2)s(-1)) and sufficient nitrates were supplied to the vegetative cells in the inner core region, which continued to grow to a high cell concentration of 4.0 x 10(5) cells ml(-1). The sequential batch run was performed by utilizing the high-density vegetative cells as inoculum for the next batch run. The cultivation results exhibited similar trends as the previous run, reaching high cell density (4.3 x 10(5) cells ml(-1)) in the inner core region and high astaxanthin content (5.79% on a dry weight basis) in the outer jacket region. The present study indicates that the double-region photobioreactor and its method of operation possess a good potential for commercial production of astaxanthin by H. pluvialis.     

乙酸钠诱导雨生红球藻合成虾青素的机理

添加乙酸钠时,雨生红球藻细胞内硝酸还原酶(NR)的活性提高了1.6倍,培养液中硝酸盐浓度的迅速下降。另一方面,乙酸钠的存在和氮缺乏,又抑制了叶绿素的合成和1,5-二磷酸核酮糖羧化酶(Rubisco)的活性。在虾青素开始合成的第4d,硝酸盐的浓度,叶绿素含量和Rubisco活性分别下降了96.7%,71.9%和80%。相比之下,在未添加乙酸钠的对照培养液中,实验结束时硝酸盐的浓度,叶绿素含量和Rubisco活性仅下降了47.2%,27.3%和4.4%,培养过程中没有虾青素积累。     

On the relative efficiency of two- vs. one-stage production of astaxanthin by the green alga Haematococcus pluvialis

Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.     

Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.     

Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis

Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.