Astaxanthin production by a green alga,Haematococcus pluvialis accompanied with morphological changes in acetate media

Two acetate containing media were developed for astaxanthin production by a green unicellular alga, Haematococcus pluvialis. The basal medium, a vegetative growth medium facilitated the algal cell growth, whereas the modified medium was likely to induce morphological changes with the formation of large cysts and bleached cells which seemed to consequently enhance the carotenoid biosynthesis. In the two-stage culture, the injection of ferrous ion with acetate into the basal medium on the fourth day, was greatly stimulative for both the algal cell growth and the astaxanthin formation at a high light intensity. In addition, carotenoid precursors, mevalonate and pyruvate were effective on the carotenoid formation in the modified medium. Pyruvate was an especially good carbon source both for the algal cell growth and the carotenoid synthesis.     

Astaxanthin production from the green alga Haematococcus pluvialis with different stress conditions

Summary Haematococcus pluvialis was induced to produce the astaxanthin pigment. A factorial design was carried out with three sodium acetate concentrations, 0.025, 0.05, 0.1 (g/l), and three NaCl concentrations (0.1, 0.2 and 0.4 %).The best conditions in algal culture for astaxanthin production were 0.2 % NaCl, 0.025 g/l sodium acetate and 0.05 g/l sodium acetate, each a 3.0, 1.83 and 1.78 % of astaxanthin, production in base to total dry weight, respectively. The higher astaxanthin production by bioreactor was 18.6 mg/l in the condition with 0.2 % NaCl.     

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.     

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%).     

Hyper-accumulation of astaxanthin in a green algaHaematococcus pluvialis at elevated temperatures

Summary When a green algaHaematococcus pluvialis was cultivated at 30°C, astaxanthin production was 3-fold more increased than at 20°C. With acetate supplementation to 30°C culture, the alga synthesized over 2-fold more carotenoid than without addition. Tiron, a radical scavenger, however, severely blocked the stimulated carotenogenesis, suggesting that endogenously generated active oxygen was responsible for the highly stimulated carotenogenesis. From these results, possible roles of the elevated cultivation temperatures for hyperaccumulation of astaxanthin were discussed.     

Enhanced production of free trans-astaxanthin by oxidative stress in the cultures of the green microalga Chlorococcum sp

Free trans-astaxanthin accumulated in the alga Chlorococcum sp. was markedly enhanced from 3.664 mg g⁻¹ cell dry weight to 5.724 mg g⁻¹ cell dry weight when the culture was supplemented with hydrogen peroxide (0.1 mM) under mixotrophic conditions of growth. After saponification, a total of 7.086 mg astaxanthin per g cell dry weight was achieved. Similarly, in heterotrophic cultures, the total astaxanthin content was increased from 1.034 mg g⁻¹ cell dry weight without H₂O₂ to 1.782 mg g⁻¹ cell dry weight with 0.1mM H₂O₂. Results indicate that hydrogen peroxide effectively induces the formation of free trans-astaxanthin in Chlorococcum sp.     

A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high‐value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark‐grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L^?1 day^?1) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark‐grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high‐light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L^?1 day^?1 by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark‐grown cells under photo‐induction conditions. Biotechnol. Bioeng. 2016;113: 2088–2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

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。     

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.     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production 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 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)     

印度块菌提取物抗氧化活性的研究

对印度块菌Tuber indicum子实体的提取物,包括55%乙醇粗提物(ECE)、石油醚提取物(PEF)和乙酸乙酯提取物(EAF)的清除DPPH自由基和羟基自由基能力、铁离子鳌合能力以及各提取物的总酚含量等进行了研究和测定,结果显示,3种提取物的清除自由基能力和铁离子鳌合能力具有显著差异(P0.05);ECE对DPPH自由基的清除活性最高,其EC50值为1.61g/L;EAF对羟基自由基及铁离子表现出较强的清除或螯合的能力,其EC50值分别为3.31g/L和0.70g/L;EAF的总酚含量(2.964mg GAE/g提取物)最高,其次是ECE,总酚含量为(2.618mg GAE/g提取物);PEF的清除自由基和铁离子鳌合能力较差,其总酚含量也最低(1.124mg GAE/g提取物);总酚含量与印度块菌提取物清除自由基以及鳌合铁离子的能力密切相关。     

Citrate, a possible precursor of astaxanthin in Phaffia rhodozyma: influence of varying levels of ammonium, phosphate and citrate in a chemically defined medium.

The influence of ammonium, phosphate and citrate on astaxanthin production by the yeast Phaffia rhodozyma was investigated. The astaxanthin content in cells and the final astaxanthin concentration increased upon reduction of ammonium from 61 mM to 12.9 mM (from 140 microg/g to 230 microg/g and 1.2 microg/ml to 2.3 microg/ml, respectively). Similarly, both the astaxanthin content and astaxanthin concentration increased by reducing phosphate from 4.8 mM to 0.65 mM (160 microg/g to 215 microg/g and 1.7 microg/ml to 2.4 microg/ml, respectively). Low concentrations of ammonium or phosphate also increased the fatty acid content in cells. By analogy with lipid synthesis in other oleaginous yeasts, an examination of the data for varying nitrogen and phosphate levels suggested that citrate could be the source of carbon for fatty acids and carotenoid synthesis. Supporting this possibility was the fact that supplementation of citrate in the medium at levels of 28 mM or higher notably increased the final pigment concentration and pigment content in cells. Increased carotenoid synthesis at low ammonium or phosphate levels, and stimulation by citrate were both paralleled by decreased protein synthesis. This suggested that restriction of protein synthesis could play an important role in carotenoid synthesis by P. rhodozyma.     

Enhanced Carotenoid Biosynthesis by Oxidative Stress in Acetate-Induced Cyst Cells of a Green Unicellular Alga, Haematococcus pluvialis

In a green alga, Haematococcus pluvialis, a morphological change of vegetative cells into cyst cells was rapidly induced by the addition of acetate or acetate plus Fe²⁺ to the vegetative growth phase. Accompanied by cyst formation, algal astaxanthin formation was more enhanced by the addition of acetate plus Fe²⁺ than by the addition of acetate alone. Encystment and enhanced carotenoid biosynthesis were inhibited by either actinomycin D or cycloheximide. However, after cyst formation was induced by the addition of acetate alone, carotenoid formation could be enhanced with the subsequent addition of Fe²⁺ even in the presence of the inhibitors. The Fe²⁺ -enhanced carotenogenesis was inhibited by potassium iodide, a scavenger for hydroxyl radical, suggesting that hydroxyl radical formed by an iron-catalyzed Fenton reaction may be required for enhanced carotenoid biosynthesis. Moreover, it was demonstrated that four active oxygen species, singlet oxygen, superoxide anion radical, hydrogen peroxide, and peroxy radical, were capable of replacing Fe²⁺ in its role in the enhanced carotenoid formation in the acetate-induced cyst. From these results, it was concluded that oxidative stress is involved in the posttranslational activation of carotenoid biosynthesis in acetate-induced cyst cells.     

Multistage operation of airlift photobioreactor for increased production of astaxanthin from Haematococcus pluvialis

An internally radiating photobioreactor was applied for the production of astaxanthin using the unicellular green alga Haematococcus pluvialis. The cellular morphology of H. pluvialis was significantly affected by the intensity of irradiance of the photobioreactor. Small green cells were widespread under lower light intensity, whereas big reddish cells were predominant under high light intensity. For these reasons, growth reflected by cell number or dry weight varied markedly with light conditions. Even under internal illumination of the photobioreactor, light penetration was significantly decreased as algal cells grew. Therefore, we employed a multistage process by gradually increasing the internal illuminations for astaxanthin production. Our results revealed that a multistage process might be essential to the successful operation of a photobioreactor for astaxnthin production using H. pluvialis.     

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.     

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。       

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10⁻³ M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress. Received: 26 January 2000 / Received revision: 5 April 2000 / Accepted: 1 May 2000     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Growth characteristics and corrinoid production of Methanosarcina barkeri on methanol-acetate medium

Methanosarcina barkeri strain Fusaro was grown on a mixed substrate medium of methanol and acetate. When 50 mM of acetate was added to the methanol basal medium (250 mM), the rates of methane production, methanol consumption, cell growth and corrinoid production were stimulated 3.2, 2.7, 3.5, and 2.4 times, respectively compared with those in methanol alone. Addition of acetate also has significant influence on corrinoid distribution decreasing the intracellular corrinoid content from 6.8 to 3.0 mg/g dry cell and increasing the extracellular corrinoid concentration from 4.0 to 5.4 mg/l. The carbon balance analysis for methanogenesis and cellular growth with or without acetate addition revealed that about 50% of the utilized acetate carbon might be incorporated in the cellular materials and the remaining might be oxidized to generate the electrons which stimulate the methanol reduction to methane, accelerating the metabolic activities of the methanogenesis from methanol consequently enhancing the rates of methane and corrinoid production, and cell growth.