Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.     

Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture

Production of astaxanthin by sequential heterotrophic-photoautotrophiccultivation of a green alga, Haematococcus pluvialis was investigated.This involved cultivating the cells heterotrophically to high cellconcentration, followed by illumination of the culture for astaxanthinaccumulation. The optimum pH and temperature for heterotrophic biomassproduction were 8 and 25 ^°C, respectively. There was no significantdifference in the specific growth rate of the cells when acetateconcentration was varied between 10 mM and 30 mM. However, cellgrowth was inhibited at higher acetate concentrations. A pH stat methodwas then used for fed-batch heterotrophic culture, using acetate as theorganic carbon source. A cell concentration of 7 g L^-1 wasobtained. Higher cell concentration could not be obtained because the cellschanged from vegetative to cyst forms during the heterotrophic cultivation.However, by using repeated fed-batch processes, the cells could bemaintained in the vegetative form, leading to more than two times increasein cell number output rate. When the vegetative cells were transferred tophotoautotrophic phase, there was a sharp decrease in the cell number andonly very few cells encysted and accumulated astaxanthin. On the otherhand, when the shift from heterotrophic to photoautotrophic condition wasdone when most of the cells had encysted, there was still a decrease in cellnumber but astaxanthin accumulation was very high. The astaxanthinconcentration (114 mg L^-1) and productivity (4.4 mg L^-1d^-1) obtained by this sequential heterotrophic-photoautotrophiccultivation method are very high compared to the data in the literature.     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

Protective role of astaxanthin against u.v.-B irradiation in the green alga Haematococcus pluvialis

Cyst cells of the green alga Haematococcus pluvialis accumulate astaxanthin with maturation of the resting stage. To study the protective role of astaxanthin against u.v. damage, both immature (astaxanthin-poor) and mature (astaxanthin-rich) cyst cells were exposed to u.v.-A or u.v.-B irradiation, and the residual cell viability and astaxanthin levels were determined. u.v.-B decreased both cell viability and astaxanthin level of cyst cells to a greater extent than u.v.-A. Tolerance of mature cyst cells to u.v.-B was 6-fold higher than that of immature cyst cells. These results indicated that astaxanthin in cyst cells functions as a protective agent against u.v.-B irradiation.     

Determination of an ethylene biosynthesis pathway in the unicellular green alga,Haematococcus pluvialis. Relationship between growth and ethylene production

In the freshwater ChlorophyceaeHaematococcus pluvialis, precursors of ethylene biosynthesis cycle are the same as those of higher plants: L-methionine S-adenosylmethionine 1-aminocyclopropane-1-carboxylic acid ethylene. However, the enzymatic complex of the last step of ethylene synthesis-ACCoxidase-differs from that of higher plants. It is stimulated by Co²⁺ (at least 10^-5 M), Mn²⁺ (at least 10^-6 M) and Ag²⁺ (at least 10^-4 M), inhibited by Cu²⁺ (at least 10^-5 M) and not affected by Zn²⁺, Fe²⁺ or Mg²⁺. ACCoxidase is also inhibited by salicylhydroxamic acid and by dark. Ethylene production is more important in young, mobile, green cells in active growth phase than in old, encysted and red cells in stationary growth phase. No peaks in ethylene production or respiration were observed during batch culture, as opposed to the situation with climacteric fruits.     

Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N-starvation and high light conditions

Haematococcus pluvialis gave the highest astaxanthin accumulation rate (2.7 mg l^–1 day^–1) and total astaxanthin content ( 22.7 mg g^–1 biomass). Astaxanthin accumulation in Neochloris wimmeri, Protosiphon botryoides, Scotiellopsis oocystiformis, Chorella zofingiensis and Scenedesmus vacuolatus was, respectively, 19.2, 14.3, 10.9, 6.8 and 2.7 mg astaxanthin g^–1 biomass, respectively.     

Characterization of astaxanthin esters in Haematococcus pluvialis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

After first being analyzed by HPLC, 4 free carotenoids, 15 astaxanthin monoesters, 12 astaxanthin diesters, and 3 astacin monoesters in Haematococcus pluvialis were identified by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-(APCI)MS). Identification of each compound was based on the characteristic fragment ions of the positive ion mode, negative ion mode, and MS(2). Astaxanthin esters were identified based on the loss of one or two fatty acids. In a positive ion mode, astaxanthin monoesters had characteristic fragment ions at m/z 597 [M+H-fatty acid](+) and m/z 579 and 561 that resulted from a continuous loss of water. The relative intensity of m/z 579 in MS(2) amounted to more than 80% of that of the molecular ion. In astaxanthin diesters, the intensity of m/z 561 occasionally was equal to that of m/z 579, but in general the former, amounting to 50 to 60% or more of the molecular ion, was stronger than the latter, which decreased to 20 to 30% of the molecular ion. In addition, a set of compounds with maximum absorbance at 400 nm, detected by high-performance liquid chromatography-diode array detector (HPLC-DAD), had strong characteristic fragment ions at m/z 871 and 593 in the positive ion mode MS(2). They were presumed to be linolenic acid or an isomer of omega-6-gamma-linolenic acid esters of astacin.     

Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth

The microalga Haematococcus pluvialis was cultured with NaNO₃ from 0 to 1 g l^–1 and optimal growth was obtained at 0.15 g l^–1. Sodium acetate and malonate (from 0 to 2% w/v) enhanced the accumulation of astaxanthin three and five times higher, respectively, than in autotrophic control cultures. However, high concentration of those compounds strongly inhibited growth. The ratio chlorophyll a/total carotenoids was a good indicator of the extent of nitrogen deficiency in the cells.     

The synthesis of astaxanthin esters, independent of the formation of cysts, highly correlates with the synthesis of fatty acids in Haematococcus pluvialis

The compositions and contents of astaxanthin esters and fatty acids in four types of Haematococcus pluvialis cells were studied by HPLC and GC-MS. Results showed that the synthesis and accumulation of astaxanthin was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of fatty acids, though it is an well known phenomenon that the accumulation of astaxanthin is usually accompanied by the formation of cyst. The red cysts contain more than 30% of fatty acids, with 81% of the unsaturated fatty acids. Taken together, besides a resource of astaxanthin, H. pluvialis would be a good resource of valuable fatty acids. Supported by the National Natural Science Foundation of China (Grant No. CNSF30570183), Chinese Academy of Science (KSCX2-YW-G-027) and Yunnan Provincial Sciences and Technology Department, China (2007AD009)     

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。     

Determination of the time transferring cells for astaxanthin production considering two-stage process of Haematococcus pluvialis cultivation

The two-stage culture system consisting of green vegetative growth and reddish inductive production stages has been widely accepted for the production of astaxanthin using Haematococcuspluvialis. However, little has been known about the appropriate cellular phase of H.pluvialis for transferring into the astaxanthin inductive conditions. In this study, we determined the optimal growth phase of H.pluvialis for transferring into the second production stage. Astaxanthin productivities were correlated with growth phases, as senescent green phases could increase more than 10-fold greater than juvenile green phases. Our results clearly demonstrated the appropriateness of the senescent vegetable cells for transferring into the production stage, due to the increased capacity to accumulate astaxanthin.     

Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of areporter gene, LacZ, in the unicellular green alga Haematococcuspluvialis. By employing the micro-particle bombardment method,motilecells in the exponential phase showed transient expression oflacZ. This was detected in bombarded motile cells undertherupture-disc pressures of 3103 KPa and 4137 KPa.Transient expression of LacZ gene could not be observed in non-motile cells ofthis alga under the same transformation condition. No LacZ background was foundin either the motile cells or the non-motile cells. The study suggests apromising potential of the SV40 promoter and the lacZreporter gene in genetic engineering of unicellular green algae.     

Optimization of culture conditions for growth of the green alga Haematococcus pluvialis

The effects of light intensity, inoculum volume, sodium nitrate and carbon dioxide concentrations on the growth of Haematococcus pluvialis were investigated using response surface methodology (RSM). All the four variables exhibited significant effects on growth and can be related (r 0.926, P 0.01) by a second-order polynomial consisting of linear, quadratic and interaction terms. The total quadratic effect (P 0.01) dominates over the total linear effect (P 0.01) but the role of interaction terms (P 0.10) is marginal. The optimum values of these variables were: carbon dioxide 1.54%, sodium nitrate 1.06 g/l, inoculum volume 24.97% and light intensity 2.42 klux; the predicted maximum value for the yield of biomass was 0.51 g/l (dry weight).     

Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray

The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3′-dihydroxy-β, β-carotene-4, 4′-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis 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 results 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. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably 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%). Supported by the Frontier Research Grant of the SCSIO, the Hundred Talents program of Chinese Academy of Sciences, and National Natural Sciences of China projects (Grant No. 40776087)     

Influence of sodium orthovanadate on the production of astaxanthin from green algae Haematococcus lacustris

Astaxanthin production is commonly induced under stress conditions such as nutrient deficiency (N or P), high light stress, and variations of temperature, high NaCl concentrations, and other factors. The objective of the present study is the analysis of the effect of oxidative stress by sodium orthovanadate (SOV), a nonspecific inhibitor of protein tyrosine phosphatases, on the cells growth and astaxanthin production of H. lacustris. In the presence of SOV (lower than 5.0 mM), maximum growth of H. lacustris obtained was 2.4 × 10⁵ cells/mL in MBBM medium at 24°C under continuous illumination (40 μE/m²/s) of white fluorescent light, with continuous aeration of CO₂ (0.2 vvm). Total carotenoids accumulated per cell biomass unit treated with 2.5 mM SOV has approximately shown 2.5 folds higher than the control after short period of SOV induction time as 2 days, despite that cells were grown under normal light. Meanwhile, maximal astaxanthin production from H. lacustris was 10.7 mg/g biomass in MBBM with 5 days of continuous illumination at 40 μE/m²/s, which has been established as optimal light intensity for the control culture of H. lacustris. Treating algae H. lacustris with sodium orthovanadate showed promoting the accumulation of astaxanthin by advancing either the inhibition of dephosphorylation or synthesis of ATP. Its potential role of PTPases in microalgae H. lacustris is discussed. The first two authors are equally contributed to this work.     

Effect of cultivation parameters on growth and pigment biosynthesis in flagellated cells of Haematococcus pluvialis

The volvocalean microalga Haematococcus pluvialis is used as a sourceof the ketocarotenoid astaxanthin for applications in aquaculture and thepharmaceutical and cosmetic industries. This green alga accumulatesastaxanthin, mostly esterified, canthaxanthin and echinenone in lipid vesiclesoutside the chloroplast. This accumulation process normally but notexclusively accompanies formation of the resting state in the developmentalcycle. With regard to increased bioavailability of the accumulated secondarycarotenoids, the fragility of the extracellular matrix makes the flagellatedstate of H. pluvialis an interesting alternative to the thick-walledaplanospore state. A two-step batch cultivation scheme was developed thatleads to accumulation of secondary carotenoids in flagellated cells of H. pluvialis (strain 192.80, Göttingen, Germany). Germination ofgreen aplanospores during the first step of cultivation proceeded optimallyunder 30 mol photon m^-2 s^-1 of whitefluorescent light at 20 °C. For optimal induction and enhancementof carotenoid biosynthesis, the flagellated cells formed were then exposedto a decreased level of nitrate (0.4 mM KNO₃) and to enhancedirradiance (150 mol photon m^-2 s^-1). Under theseconditions, which still permitted cell division and chlorophyll synthesisduring the first two days of exposure, carotenoid accumulation in theflagellated cells reached 2° of dry mass at the fourth day of exposure. Asa mixotrophic carbon source, addition of acetate at a concentration nothigher than 10 mM increased carotenoid synthesis only slightly whereaspartial or complete phosphate deficiency or salt stress (40 mM NaCl) didnot.     

Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors

Recent developments in photobioreactor technology havemade the production of astaxanthin from Haematococcus pluvialis commercially viable. The coreof our astaxanthin production chain is the AquasearchGrowth Module (AGM), a 25,000 L enclosed andcomputerized outdoor photobioreactor.At Aquasearch's newly expanded facility (dedicatedJanuary 1999), three AGMs (total volume 75,000 L) areused to produce large amounts of clean, fast growing,H. pluvialis. The H. pluvialis biomassproduced in the AGMs is transferred daily to a pondculture system, where carotenogenesis and astaxanthinaccumulation are induced. Following a 5-dayinduction period, the reddened H. pluvialiscells are harvested by gravitational settling. Theharvested biomass, which averages > 2.5 astaxanthinas percent of the dry weight, is transferred to aprocessing building where a high pressure homogenizeris used to rupture the cells' walls. Once the biomasshas been homogenized, it is dried to less than 5%moisture utilizing proprietary drying technology. Thedried product is then ready to be packaged accordingto customer needs.The photobioreactor research program has almostdoubled the performance of the AGMs in the first ninemonths of operations: standing biomass concentrationincreased from 50 to 90 g m^-2 and productionincreased from 9 to 13 g m^-2 d^-1 during thisperiod. Here, we discuss the significance of thesechanges in production parameters to the viability ofcommercial production of astaxanthin and other highvalue products from microalgae.     

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.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999