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.     

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.     

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。     

2,4-二氯苯氧乙酸(2,4-D)对雨生红球藻中虾青素积累的影响

本文初步研究了一定浓度范围内的2,4-D对雨生红球藻积累虾青素的影响.在对数生长 期的藻液中分别加入一系列不同浓度的2,4-D溶液,然后进行胁迫培养(25℃、24h、5000Lx连续光照 营养盐饥饿),诱导细胞内虾青素的合成积累.在诱导过程中,显微观察不同浓度2,4-D处理后细胞形态和虾青素积累的动态变化,并定期取样测定虾青素含量.结果表明,20.0mg/L的2,4-D能够明显促进雨生红球藻中积累虾青素.它不仅可以加快虾青素积累进程(比对照提前15 d),而且比对照能提高13.4%的虾青素产量.     

Interactions between irradiance and nutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis

Fully synchronised germination of Haematococcus pluvialis astaxanthin-replete aplanospores was induced by transfer to nitrogen-sufficient conditions under either high or low light intensities, and growth, pigment content and nitrogen consumption were monitored during the cell cycle. No germination of the aplanospores was achieved in the absence of nitrate, even when cells were transferred at low light intensities. On the other hand, cell density and chlorophyll concentration increased dramatically and astaxanthin concentration decreased in N-sufficient cultures due to the germination of 100% of the aplanospores, as demonstrated by flow cytometry. No significant effect of light intensity was observed on the degradation of astaxanthin during germination. In germinated cultures, nitrogen was depleted more rapidly under high light conditions, which resulted in earlier entry into the aplanospore stage and accumulation of astaxanthin. Germination of aplanospores accompanied by astaxanthin degradation could also be obtained in the dark in nutrient-sufficient conditions although at a much lower efficiency. The results demonstrate that nutrient availability is the main factor controlling the transition between red and green stages of H. pluvialis, with astaxanthin being accumulated only when cell division has ceased. High light levels accelerate the process by increasing the rate of nutrient depletion and providing more energy for astaxanthin synthesis.     

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。       

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.     

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。     

Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors

The feasibility of a one-step method for the continuous production of astaxanthin by the microalga Haematococcus pluvialis has been verified outdoors. To this end, influence of dilution rate, nitrate concentration in the feed medium, and irradiance on the performance of continuous cultures of H. pluvialis was firstly analyzed indoors in bubble column reactors under daylight cycles, and then outdoors, using a tubular photobioreactor. At the laboratory scale, the behavior of the cultures agreed with that previously recorded in continuous illumination experiences, and attested that the major factors determining biomass and astaxanthin productivity were average irradiance and specific nitrate supply. The rate of astaxanthin accumulation was proportional to the average irradiance inside the culture, provided that a nitrate limiting situation had been established. The accumulation of astaxanthin under daylight cycles was maximal for a specific nitrate input of 0.5 mmol/g day. The recorded performance has been modeled on the basis of previously developed equations, and the validity of the model checked under outdoor conditions. Productivity values for biomass and astaxanthin of 0.7 g/L day and 8.0 mg/L day respectively, were obtained in a pilot scale tubular photobioreactor operating under continuous conditions outdoors. The magnitude of the experimental values, which matched those simulated from the obtained model, demonstrate that astaxanthin can be efficiently produced outdoors in continuous mode through a precise dosage of the specific nitrate input, taking also into consideration the average irradiance inside the culture.     

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.     

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.     

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%).     

利用雨生血球藻(Haematococcus pluvialis)生产天然虾菁素的研究进展

本文综述了当前国内、外利用雨生血球藻生产天然虾菁素的优化条件、雨生血球藻的大规模培养方法以及虾菁素的最佳提取工艺。     

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。     

Stress-related differential expression of multiple beta-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis is used as a biological production system for astaxanthin. It accumulates large amounts of this commercially interesting ketocarotenoid under a variety of environmental stresses. Here we report the identification and expression of three different beta-carotene ketolase genes (bkt) that are involved in the biosynthesis of astaxanthin in a single strain of the alga. Bkt1 and bkt2 proved to be the crtO and bkt previously isolated from two different strains of H. pluvialis. Bkt3 is a novel third gene, which shared 95% identical nucleotide sequence with bkt2. Nitrogen deficiency alone could not induce the alga cells to produce astaxanthin in 3 days even though it enhances the expression of the bkt genes to three times of that in normal growing cells within 16 h. High light irradiation (125 micromol m(-2)s(-1)) or 45 mM sodium acetate greatly increased the expression of bkt genes to 18 or 52 times of that in normal growing cells, resulting in an accumulation of substantial astaxanthin (about 6 mg g(-1) dry biomass) in 3 days. It is suggested that the existence of the multiple bkt genes and their strong up-regulation by different stress conditions is one of the reasons that H. pluvialis accumulates large amounts of astaxanthin in an instant response to stress environments.     

Fed-batch culture of astaxanthin-rich Haematococcus pluvialis by exponential nutrient feeding and stepwise light supplementation

A fed-batch culture process followed by subsequent photoautotrophic induction was established for the high density culture of astaxanthin-rich Haematococcus pluvialis using a CO₂-fed flat type photobioreactor under unsynchronized illumination. Fed-batch culture was performed with an exponential feeding strategy of the growth-limiting nutrients, nitrate and phosphate, concurrently with the stepwise supplementation of light depending on the cell concentration. During the growth phase, a biomass of 1.47 g/L was obtained at a biomass productivity of 0.33 g/L/day. Photoautotrophic induction of the well-grown vegetative cells was performed consecutively by increasing the light intensity to 400 μmol photon/m²/s, while keeping the other conditions in the CO₂-fed flat type photobioreactor fixed, yielding an astaxanthin production of 190 mg/L at an astaxanthin productivity of 14 mg/L/day. The proposed sequential photoautotrophic process has high potential as simple and productive process for the production of valuable Haematococcus astaxanthin.     

Astaxanthin biosynthesis from simultaneous N and P uptake by the green alga Haematococcus pluvialis in primary-treated wastewater

An alternative microalgal system for biological wastewater treatment is proposed for both the removal of nitrogen and phosphorus from wastewater and the production of a valuable carotenoid, astaxanthin. The system consists of sequential photoautotrophic cultivation and induction processes using the green alga Haematococcus pluvialis. The Haematococcus process was applied to primary-treated sewage (PTS) and primary-treated piggery wastewater (PTP) with serial dilution. H. pluvialis grew well on PTS and PTP diluted four-fold, resulting in the successful removal of nitrogen and phosphorus from both wastewaters. At that time, cell growth rates were comparable to those in the algal-defined NIES-C medium. Following the cultivation stage, N-deprived vegetative cells were transformed under photoautotrophic induction by continuous feeding of both CO₂-mixed gas and intense light to red aplanospores with substantial astaxanthin contents. The resulting astaxanthin contents accounted for about 5.1 and 5.9% of the total biomass of the PTS and PTP cultures, respectively. Our results indicate the potential of the proposed Haematococcus process as a subsidiary wastewater treatment technology with the capability of biosynthesizing the high-value antioxidant astaxanthin.     

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。     

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.