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.     

亚热带季雨林植物罗伞气体交换对光质的反应

亚热带季雨林林下阴生植物罗伞(Ardisia quinquegona)叶片的气体交换速率(PN.μmol.m~(-2),s~(-1))随光强(PFD,μmol,m~(-2),s~(-1))增高而增大。在光强低于80μmol,m~(-2),s~(-1),PN=29.21PFD×10~(-3)+0.36。在光强150μmol,m~(-2),s~(-1)对出现气体交换的光饱和现象。在低光强下,气孔传导率(G,m mol,m~(-2),s~(-1)与光强(m mol,m~(-2),s~(-1)的关系为G=265.6 PFD+4.6。在低光强下。开阔地的阳生灌木桃金娘(Rhodmyrtus tomentosa)的气体交换速率和气孔传导率与光强关系曲线的直线部分斜率皆较罗伞的低,在红光上,罗伞叶片气体交换速率(μmol,m~(-2),s~(-1)与光强(μmol,m~(-2),s~(-1)的关系为PN=32.4 PFD×10~(-3)-0.04。气孔传导率(m mol,m~(-2),s~(-1)与光强(m mol,m~(-2),s~(-1)的关系为G=339.08 PFD+7.37。同时气体交换速率的饱和红光光强亦较白光的高。在蓝光光强低时,气体交换速率(μmol,m~(-2),s~(-1))与光强(μmol,m~(-2),s~(-1))的关系为PN=13.54 PFD×10~(-3)—0.17,而气孔传导率(m mol,m~(-2),s~(-1))与光强(mμmol,m~(-2),s~(-1))的关系为G=80.5 PFD+4.35。在低的蓝光下,体交换速率和气孔传导率与光强关系曲线的直线部分斜率显著较在白光和红光下的低。罗伞叶片气体交换对红光的反应敏感。     

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。       

苋菜的光合特性

宽菜Amaranthus cruentus cv.生长在调控的温室条件。在光强0至800μmol.m~(-2)S~(-1),光合速率(PN,μmol.CO_2m~(-2)、s~(-1))随光强(PFD,μmol、m~(-2)、s~(-1))增高而增大,其关系为PN=56.82 PFD×10~(-3)—2.13。光补偿点为60μmol.m~(-2)、s~(-1)。叶片在1400 μmol.m~(-2)、s~(-1)达到光合光饱和点。在叶温35℃,叶片/空气水蒸汽压陡度20 m Pa、Pa~(-1)和外界CO_2浓度340μ1、1~(-1),光饱和光合速率为51.63±4.90μ mol.CO_2、m~(-2)、S~(-1)。在光强0至600μmol.m~(-2)、s~(-1),气孔传道率随光强增高而增大。光强高于600μmol.m~(-2)、s~(-1),气孔传道率变化较小。细胞间CO_2浓度为120μ1.1~(-1)由于细胞间CO_2浓度在光合速率——CO_2关系曲线的转折点,可能表明光合作用不受气孔限制。结果表明,苋菜适于高光强环境生长,在干旱条件下具有高的光合速率。     

Cosmetic benefits of astaxanthin on humans subjects

Two human clinical studies were performed. One was an open-label non-controlled study involving 30 healthy female subjects for 8 weeks. Significant improvements were observed by combining 6 mg per day oral supplementation and 2 ml (78.9 μM solution) per day topical application of astaxanthin. Astaxanthin derived from the microalgae, Haematococcus pluvialis showed improvements in skin wrinkle (crow's feet at week-8), age spot size (cheek at week-8), elasticity (crow's feet at week-8), skin texture (cheek at week-4), moisture content of corneocyte layer (cheek in 10 dry skin subjects at week-8) and corneocyte condition (cheek at week-8). It may suggest that astaxanthin derived from H. pluvialis can improve skin condition in all layers such as corneocyte layer, epidermis, basal layer and dermis by combining oral supplementation and topical treatment. Another was a randomized double-blind placebo controlled study involving 36 healthy male subjects for 6 weeks. Crow's feet wrinkle and elasticity; and transepidermal water loss (TEWL) were improved after 6 mg of astaxanthin (the same as former study) daily supplementation. Moisture content and sebum oil level at the cheek zone showed strong tendencies for improvement. These results suggest that astaxanthin derived from Haematococcus pluvialis may improve the skin condition in not only in women but also in men.     

Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial β-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (～38%), and low (～1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g(-1) dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g(-1) dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin.     

Physiological responses of the CAM epiphyte Tillandsia usneoides L. (Bromeliaceae) to variations in light and water supply

In an effort to understand the mechanisms that sustain rootless atmospheric plants, the modulation of Crassulacean acid metabolism (CAM) in response to variations in irradiance and water supply was investigated in the epiphyte Tillandsia usneoides. Plants were acclimated to three light regimes, i.e. high, intermediate and low, with integrated photon flux densities (PFD) of 14.40, 8.64 and 4.32 mol m-2 d-1 equivalent to an instantaneous PFD of 200, 100, and 50 mumol m-2 s-1, respectively. Daily watering was then withdrawn from half of the plants at each PFD for 7 d prior to sampling. In response to the three PFD treatments, chlorophyll content increased in plants acclimated to lower irradiances. Light response curves using non-invasive measurements of chlorophyll fluorescence demonstrated that photosystem II efficiency (phi PSII) was maintained in high PFD acclimated plants, as they exhibited a larger capacity for non-photochemical dissipation (NPQ) of excess light energy than low PFD acclimated plants. Net CO2 uptake increased in response to higher PFD, reflecting enhanced carboxylation capacity in terms of phosphoenolpyruvate carboxylase (PEPc) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities. After water was withdrawn, nocturnal net CO2 uptake and accumulated levels of acidity declined in all PFD treatments, concomitant with increased respiratory recycling of malate. Examining the strategies employed by epiphytes such as T. usneodies to tolerate extreme light and water regimes has demonstrated the importance of physiological mechanisms that allow flexible carboxylation capacity and continued carbon cycling to maintain photosynthetic integrity.     

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...     

Photosynthetic utilization of radiant energy by CAM Dendrobium flowers

14CO2 fixation was observed in orchid Dendrobium flowers; its rate decreased with the flower development. Chlorophyll (Chl) fluorescence in different developmental stages of flowers was compared to other green plant parts (leaf, inflorescence stalk, and fruit capsule). The photochemical efficiency of photosystem 2 (PS2) (Fv/Fm) of a leaf was 14-21 % higher than that of a mature flower perianth (sepal, petal, and labellum) which had a much lower total Chl content and Chl a/b ratio. A higher quantum yield of PS2 (ΦPS2) than in the mature flowers was observed in all green parts. Flower sepals had higher Chl content, Chl a/b ratio, and Fv/Fm values than the petal and labellum. During flower development the Chl content, Chl a/b ratio, Fv/Fm, and qN decreased while ΦPS2 and qP remained constant. An exposure of developing flowers to irradiances above 50 µmol m-2 s-1 resulted in a very drastic drop of ΦPS2 and qP, and a coherent increase of qN as compared to other green plant organs. A low saturation irradiance (PFD of 100 µmol m-2 s-1) and the increase in qN in the flower indicate that irradiation stress may occur since there is no further protection when the flower is exposed to irradiances above 100 µmol m-2 s-1. A low Chl/carotenoid ratio in mature flower perianth as a consequence of Chl content reduction in the course of flower development suggests a relief of irradiation stress via this mean.     

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.     

Evaluation of factors promoting astaxanthin production by a unicellular green alga,Haematococcus pluvialis,with fractional factorial design

Factors affecting the astaxanthin production by a unicellular green alga, Haematococcus pluvialis UTEX 16, were evaluated with sequential fractional factorial design. To simulate an actual production mode, a two-stage process was adapted for astaxanthin production: the alga was first cultivated under vegetative growth conditions, and then astaxanthin production was induced by applying various induction methods. A high dose of irradiation was most effective for the production of astaxanthin both in weight (mg/g) and in cellular (pg/cell) contents. A combination of nitrogen deficiency and acetate addition also significantly increased the astaxanthin content of cells on a dry weight basis. Meanwhile, the acetate addition alone increased only the cellular content of astaxanthin. Although the addition of ferrous ion alone had a negative effect on the weight content of astaxanthin, simultaneous addition of ferrous ion and acetate was effective for increasing the cellular content of astaxanthin.     

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%).     

Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.     

Regulation of Photosynthetic Induction State by the Magnitude and Duration of Low Light Exposure

This study was undertaken to examine the dependence of the regulatory enzymes of photosynthetic induction on photon flux density (PFD) exposure in soybean (Glycine max L.). The induction state varies as a function of both the magnitude and duration of the PFD levels experienced prior to an increase in PFD. The photosynthetic induction state results from the combined activity of separate processes that each in turn depend on prior PFD environment in different ways. Direct measurement of enzyme activities coupled with determination of in situ metabolite pool sizes indicated that the fast-induction component was associated with the activation state of stromal fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) and showed rapid deactivation in the dark and at low PFD. The fast-induction component was activated at low PFD levels, around 70 [mu]mol photons m-2 s-1. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 2.7.1.19) deactivated very slowly in the dark and required higher PFD for activation. Both enzymes saturated at lower PFD than did photosynthesis, around 400 [mu]mol photons m-2 s-1. Ribulose-5-phosphate kinase (EC 2.7.1.19) appeared never to be limiting to photosynthesis, and saturated at much lower PFD than either FBPase or Rubisco. Determination of photosynthetic metabolite pool sizes from leaves at different positions within a soybean canopy showed a limitation to carbon uptake at the stromal FBPase and possibly the sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) in shade leaves upon initial illumination at saturating PFD levels.     

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。