Use of response surface methodology to optimise carotenogenesis in the microalga,Haematococcus pluvialis

The factors controlling biomass production and the synthesis of astaxanthin esters in the microalga Haematococcus pluvialis (CCAP 34/7) have been investigated using a statistical approach employing response surface methodology (RSM). The culture conditions required for optimal growth and carotenogenesis in this alga are very different. Of particular importance is the photon flux density: for growth the optimum is 50–60 μmol m⁻² s⁻¹ whereas the optimum for astaxanthin synthesis is much higher at ∼-1600 μmol m⁻² s⁻¹. The addition of low levels of NaCl to the medium also stimulates to a small extent synthesis of astaxanthin, but photon flux density remains the overriding factor. The optimal temperature for this strain is quite low at 14–15 °C. RSM has been shown to be a rapid and effective technique leading to the optimisation of algal culture conditions. This statistical approach can be applied readily to the majority of microalgae and their products.     

雨生红球藻虾青素合成研究进展

虾青素是一种重要的次级类胡萝卜素,具有高活性的抗氧化功能,广泛应用于食品保健、医药、水产养殖等领域。雨生红球藻是一种在胁迫条件下能够大量积累虾青素的微藻。文中回顾了雨生红球藻虾青素的生物合成研究的进展,包括虾青素生物合成的诱导与调控、虾青素合成与光合作用及脂类代谢的关系等研究现状。     

Studies on Haematococcus pluvialis for improved production of astaxanthin by mutagenesis

Cultures of Haematococcus pluvialis were exposed to mutagens like u.v. and EMS (ethyl methanesulphonate). The results showed that the survival rate decreased with the increase in u.v. exposure time and increase in EMS concentration. These mutants were further screened using inhibitors of the carotenoid biosynthetic pathway viz. diphenylamine (15–90 M), nicotine (160–320 M) and compactin (1.5–3.0 M). The mutants thus obtained showed early enhanced (2.2–3.2-fold) astaxanthin accumulation and also exhibited higher lycopene cyclase activity.     

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.     

雨生红球藻中虾青素的研究与应用

雨生红球藻是单细胞微藻,其中的虾青素具有抗氧化、抗肿瘤、预防心脑血管疾病等多种生物活性,在食品、医药、保健品、化妆品及养殖业有诸多用途。概述了雨生红球藻虾青素含量影响因素,雨生红球藻培养方法、虾青素的提取方法及其应用领域等最新研究成果,为虾青素的开发利用提供帮助。     

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis1

Cells of the green microalga Haematococcus pluvialis were inducedto accumulate the ketocarotenoid pigment, astaxanthin. Thisinduction was achieved by the application of the following environmentalconditions: light intensity (170 µmol m~^–2s^–1),phosphate starvation and salt stress (NaCl 0.8%). These conditionsretarded cell growth as reflected by a decrease in cell divisionrate, but led to an increase in astaxanthin content per cell.Accumulation of astaxanthin required nitrogen and was associatedwith a change in the cell stage from biflagellate vegetativegreen cells to non-motile and large resting cells. It is suggestedthat environmental or nutritional stresses, which interferewith cell division, trigger the accumulation of astaxanthin.Indeed, when a specific inhibitor of cell division was applied,a massive accumulation of astaxanthin occurred. ¹ Contribution No. 55 of The Microalgal Biotechnology Laboratory (Received April 22, 1991; Accepted August 6, 1991)     

Optimization of culture medium for growth of Haematococcus pluvialis

A central composite rotatable design was used to examine the effects of five components of the medium on the growth of Haematococcus pluvialis in batch culture. The medium components considered were: sodium acetate,potassium nitrate, major elements, trace elements and vitamins. Within the range of the concentrations tested, a moderate concentration of the major elements significantly enhanced algal growth, both in terms of specific growth rate and cell dry weight, whereas the vitamins had no significant effect. Based on the response surface contour plots and the results of numerical analyses, the optimal nutrient concentrations for growth in terms of specific growth rate were 0.51 g L^-1 sodium acetate, 0.25 g L^-1 potassium nitrate, 0.63 mL L^-1 of the major element stock solution and 0.2 mL L^-1 of the trace element stock solution. The optimal nutrient concentrations for biomass production were 1.64 g L^-1 sodium acetate, 0.37 g L^-1potassium nitrate, 2.52 mL L^-1 of the major element stock solution and 0.03 mL L^-1 of the trace element stock solution. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Response surface methodology to supercritical carbon dioxide extraction of astaxanthin from Haematococcus pluvialis

Experimental design was used to investigate the effect of operating temperature (40-80 degrees C), operating pressure (30-50 MPa), and extraction time (1-4h) of supercritical carbon dioxide (SC-CO2) extraction on astaxanthin yields and the extract antioxidant activity (IC50). The ranges of the factors investigated were 40-80 degrees C for the operating temperature (X1), 30-50 MPa for the operating pressure (X2), and 1-4h for the extraction time (X3). The statistical analysis of the experiment indicated that pressure, extraction time, and the interaction between temperature and pressure (X1X2) had significant effect on astaxanthin yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.924 and 0.846 for astaxanthin yield and antioxidant activity, respectively. The optimal condition for astaxanthin yield within the experimental range of the variables studied was at 70 degrees C, 50 MPa, and 4h. At this condition, the predicted amount of astaxanthin extracted was 23.04 mg/g (2.3 wt% or 83.78% recovery). For the effect of experimental extraction conditions on antioxidant activity, IC50 was used as an index, which is the concentration that gives a 50% reduction in the absorbance of the ABTS free radical. The analysis of the results showed that the interaction between the operating temperature and operating pressure (X1X2) was the only significant factor affecting the extract antioxidant activity. The statistical model gave the minimum point for antioxidant activity at 67 degrees C, 40.3 MPa, and 1.86 h of extraction, at which the value for 1/IC50 was 0.39 l/mg (or IC50 was 2.57 mg/l).     

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。     

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.     

Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis

The green unicellular alga, Haematococcus pluvialis has two antioxidative mechanisms against environmental oxidative stress: antioxidative enzymes in vegetative cells and the antioxidative ketocarotenoid, astaxanthin, in cyst cells. We added a reagent that generates superoxide anion radicals (O₂ ⁻), methyl viologen, to mature and immature cysts of H. pluvialis. Tolerance to methyl viologen was higher in mature than in immature cysts. Mature (astaxanthin-rich) cysts showed high antioxidant activity against O₂ ⁻ in permeabilized cells, but not in astaxanthin-free cell extracts, while immature (astaxanthin-poor) cysts had very low antioxidant activities against O₂ ⁻ in both. The results suggested that astaxanthin accumulated in the cyst cells functions as an antioxidant against excessive oxidative stress. The same levels of antioxidant activities against O₂ ⁻ in both permeabilized cells and cell extracts from vegetative cells suggested the presence of antioxidative enzymes (superoxide dismutase). Received: 13 January 1997 / Received revision: 26 February 1997 / Accepted: 27 March 1997     

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.     

雨生红球藻中IPP异构酶基因的系统发育分析

通过系统进化树的构建对IPP异构酶的系统发育进行分析研究。结果表明,不同来源的IPP异构酶基因均是单系分支,并且各个分支有着不同进化模式;似然比分析结果发现,绿藻来源的IPP异构酶有9．8％的氨基酸位点经受了正选择的压力,其基因的进化模式不同于高等植物和细菌中的IPP异构酶基因。     

Induction of astaxanthin accumulation by nitrogen and magnesium deficiencies in Haematococcus pluvialis

Haematococcus pluvialis was cultured under N– and Mg+2-deficient conditions with two light intensities: 40 and 230 mol m² s–1. Highest astaxanthin concentration, 49.5 g·ml–1, was obtained when high light was applied under N-deficient conditions. N-deficiency has a greater effect than high light intensity on astaxanthin synthesis by exerting a stronger blocking effect on cell division. The effect of high light was synergetic with the other stress conditions in stimulating the synthesis of astaxanthin. Mg+2 deficiency also stimulated the synthesis of astaxanthin but produced lower concentrations: 7 and 26 g·ml–1 for low and high light intensities respectively. When both N and Mg+2 were absent from the culture media the concentration of astaxanthin was lower than with N-deficiency alone but higher than with Mg+2-deficiency. © Rapid Science Ltd. 1998     

Hyperspectral imaging techniques for the characterization of Haematococcus pluvialis (Chlorophyceae)

A hyperspectral imaging camera was combined with a bright‐field microscope to investigate the intracellular distribution of pigments in cells of the green microalga Haematococcus pluvialis, a synonym for H. lacustris (Chlorophyceae). We applied multivariate curve resolution to the hyperspectral image data to estimate the pigment contents in culture and revealed that the predicted values were consistent with actual measurements obtained from extracted pigments. Because it was possible to estimate pigment contents in every pixel, the intracellular distribution of the pigments was investigated during various life‐cycle stages. Astaxanthin was localized specifically at the eyespot of zoospores in early culture stages. Then, it became widely distributed in cells, but subsequently localized differently than the chl. Integrated with our recently developed image‐processing program “HaematoCalMorph,” the hyperspectral imaging system was useful for monitoring intracellular distributions of pigments during culture as well as for studying cellular responses under various conditions.     

雨生红球藻(Haematococcus pluvialis)的光谱特性研究

实验测定了雨生红球藻不同生长阶段的色素组成,吸收光谱,荧光光谱,并对其进行了分析。结果表明,用490nm波长激发时,雨生红球藻在绿色细胞阶段存在710nm和730nm附近的长波长荧光发射峰,而在红色细胞阶段仅存在730nm的长波长荧光发射峰,预示着雨生红球藻不同生长阶段在PSⅠ结构,组成,及其色素蛋白的排布等方面有很大差异。     

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.