Enhancement of astaxanthin production from Haematococcus pluvialis under autotrophic growth conditions by a sequential stress strategy

Abstract

The study of microalgal culture has been growing in recent decades, because the cellular structure of microalgae has diverse highly valuable metabolites that have attract attention of numerous companies and research groups. The pigment astaxanthin is considered one of the most powerful antioxidants in nature. The microalga Haematococcus pluvialis was proposed as one of the best natural astaxanthin sources, because it can accumulate high amount of the pigment. In this work, we studied different stress treatments on H. pluvialis growth cultures as well as astaxanthin production under autotrophic growth conditions. The results showed that extending nitrogen starvation before increasing radiation intensity up to 110?μmol photons m^?2 s^?1 during late the palmella cell phase incremented the astaxanthin concentration up to 2.7% of dry biomass with an efficient light energy utilization during the stress stage.     

Extraction of astaxanthin from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier

Conventional solvent extraction methods cannot attain high‐quality antioxidant extracts from microalgae and also require solvent recovery and posttreatment. In this study, we utilized environmental friendly supercritical carbon dioxide fluid extraction (SFE‐CO₂) techniques to obtain pigment (i.e. astaxanthin) from Haematococcus pluvialis. The effects of key operating parameters on the extraction efficiency of astaxanthin were investigated, giving an optimal condition of H. pluvialis weight, 6.5 g; CO₂‐flow rate, 6.0 NL/min; extraction time, 20 min; extraction pressure, 4500 psi; volume of ethanol modifier added, 9.23 mL/g; extraction temperature, 50°C; modifier composition, 99.5%. Under these optimum conditions, the astaxanthin yield was 73.9% (10.92 mg/g dry H. pluvialis powder) after eight cycle of extraction cycles. The saponification index (C_S/C₀, representing the ratio of astaxanthin concentration after and before the saponification procedures) of the extract could be increased from 1 to 12.78 by saponification with 3.5 M NaOH.     

Tagging of biomolecules with deuterated water (D2O) in commercially important microalgae

To understand the effect of any biomolecules in specific metabolic pathways in humans, bioavailability and for other basic understanding, stable isotopically-labelled biomolecules (preferably deuterated) is the fundamental pre-requisite. Production of deuterated biomolecules such as, astaxanthin, β-carotene, lutein, chlorophyll-a, and eicosapentaenoic acid (EPA, 20:5n-3) by metabolic tagging have been shown in commercially important microalgae, Haematococcus pluvialis and Phaeodactylum tricornutum. These microalgae were grown in appropriate optimized medium supplemented with 25 % (v/v) deuterated water. LC–MS analysis showed a maximum of 20, 25, 23, 24, and 27 % replacement of hydrogen by deuterium atoms respectively in astaxanthin, β-carotene, lutein, chlorophyll-a, and EPA. To our knowledge, this is the first report on the production of deuterated astaxanthin, chlorophyll-a and EPA by these microalgae.     

Role of media composition in biomass and astaxanthin production of Haematococcus pluvialis under two-stage cultivation

In the present study, the effects of four different culture media on the growth, astaxanthin production and morphology of Haematococcus pluvialis LUGU were studied under two-step cultivation. The interactions between astaxanthin synthesis and secondary messengers, reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) were also investigated. In the first green vegetative cell stage, maximal biomass productivity (86.54 mg L⁻¹ day⁻¹) was obtained in BBM medium. In the induction stage, the highest astaxanthin content (21.5 mg g⁻¹) occurred in BG-11 medium, which was higher than in any other media. The expressions of MAPK and astaxanthin biosynthetic genes in BG-11 were higher than in any other media, whereas the ROS content was lower. Biochemical and physiological analyses suggested that the ROS, MAPK and astaxanthin biosynthetic gene expression was involved in astaxanthin biosynthesis in H. pluvialis under different culture media conditions. This study proposes a two-step cultivation strategy to efficiently produce astaxanthin using microalgae.

     

Biological response of protists Haematococcus lacustris and Euglena gracilis to conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate

Improving the growth and pigment accumulation of microalgae by electrochemical approaches was considered a novel and promising method. In this research, we investigated the effect of conductive polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) dispersible in water on growth and pigment accumulation of Haematococcus lacustris and Euglena gracilis. The results revealed that effect of PEDOT:PSS was strongly cell-dependent and each cell type has its own peculiar response. For H. lacustris, the cell density in the 50 mg·l⁻¹ treatment group increased by 50·27%, and the astaxanthin yield in the 10 mg·l⁻¹ treatment group increased by 37·08%. However, under the high concentrations of PEDOT:PSS treatment, cell growth was significantly inhibited, and meanwhile, the smaller and more active zoospores were observed, which reflected the changes in cell life cycle and growth mode. Cell growth of E. gracilis in all the PEDOT:PSS treatment groups were notably inhibited. Chlorophyll a content in E. gracilis decreased while chlorophyll b content increased in response to the PEDOT:PSS treatment. The results laid a foundation for further development of electrochemical methods to promote microalgae growth and explore the interactions between conductive polymers and microalgae cells.     

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1–crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.     

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.     

Enhanced astaxanthin production from microalga,Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation

For efficient astaxanthin production from the culture of green microalga, Haematococcus pluvialis, a two-stage mixotrophic culture system was established with stepwise increased light irradiance. By perfusion process, high density biomass (2.47 g/L) was achieved during the vegetative stage due to no detrimental effect of inhibitory metabolites, which was 3.09 and 1.67 times higher than batch and fed-batch processes, respectively. During the induction stage, biomass and astaxanthin were subsequently produced to the very high level 12.3 g/L and 602 mg/L, under stepwise increased light irradiance (150–450 μE/m²/s), respectively. These results indicate that the combinatorial approach of perfusion culture during the vegetative stage and stepwise light irradiation during the induction stage is a promising strategy for the simultaneous production of high concentration of biomass and astaxanthin in microalgae including H. pluvialis.     

Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids

Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe²⁺ led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557–583] to propose that a cytochrome P450-dependent enzyme might be involved in the transformation of β-carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of β-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).     

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.

     

Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis

During light induction for astaxanthin formation in Haematococcus pluvialis, we substituted photoautotrophic induction for heterotrophic induction using acetate, both to prevent contamination by heterotrophs due to addition of organic carbon and to enhance carbon assimilation in the induced cells. Strong photoautotrophic induction was performed by N-deprivation of photoautotrophically grown Haematococcus cells followed by supplementation with bicarbonate (HCO₃^–) or CO₂. Bicarbonate-induced cells contained more astaxanthin than acetate-induced cells, and even further enhancement of astaxanthin accumulation was achieved by continuous CO₂ supply. The maximum astaxanthin content (77.2 mg g^–1 biomass, 3.4-fold higher than with heterotrophic induction) was obtained under conditions of 5% CO₂, yielding astaxanthin concentration and productivity of 175.7 mg l^–1 and 6.25 mg l^–1 day^–1, respectively. The results indicate that photoautotrophic induction is more effective than heterotrophic induction for astaxanthin synthesis in H. pluvialis.     

Astaxanthin biosynthesis enhanced by reactive oxygen species in the green algaHaematococcus pluvialis

The unicellular green algaHaematococcus pluvialis has recently attracted great interest due to its large amounts of ketocarotenoid astaxanthin, 3,3′-dihydroxy-β,β-carotene-4,4′-dione, widely used commercially as a source of pigment for aquaculture. In the life cycle ofH. pluvialis, astaxanthin biosynthesis is associated with a remarkable morphological change from green motile vegetative cells into red immotile cyst cells as the resting stage. In recent years we have studied this morphological process from two aspects: defining conditions governing astaxanthin biosynthesis and questioning the possible function of astaxanthin in protecting algal cells against environmental stress. Astaxanthin accumulation in cysts was induced by a variety of environmental conditions of oxidative stress caused by reactive oxygen species, intense light, drought, high salinity, and high temperature. In the adaptation to stress, abscisic acid induced by reactive oxygen species, would function as a hormone in algal morphogenesis from vegetative to cyst cells. Furthermore, measurements of bothin vitro andin vivo antioxidative activities of astaxanthin clearly demonstrated that tolerance to excessive reactive oxygen species is greater in astaxanthin-rich cysts than in astaxanthin-poor cysts or astaxanthin-less vegetative genesis and carotenogenesis, and the accumulated astaxanthin in cysts can function as a protective agent against oxidative stress damage. In this study, the physiological roles of astaxanthin in stress response and cell protection are reviewed.     

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.     

Outdoor cultivation of microalgae for carotenoid production: current state and perspectives

Microalgae are a major natural source for a vast array of valuable compounds, including a diversity of pigments, for which these photosynthetic microorganisms represent an almost exclusive biological resource. Yellow, orange, and red carotenoids have an industrial use in food products and cosmetics as vitamin supplements and health food products and as feed additives for poultry, livestock, fish, and crustaceans. The growing worldwide market value of carotenoids is projected to reach over US$1,000 million by the end of the decade. The nutraceutical boom has also integrated carotenoids mainly on the claim of their proven antioxidant properties. Recently established benefits in human health open new uses for some carotenoids, especially lutein, an effective agent for the prevention and treatment of a variety of degenerative diseases. Consumers’ demand for natural products favors development of pigments from biological sources, thus increasing opportunities for microalgae. The biotechnology of microalgae has gained considerable progress and relevance in recent decades, with carotenoid production representing one of its most successful domains. In this paper, we review the most relevant features of microalgal biotechnology related to the production of different carotenoids outdoors, with a main focus on β-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. We compare the current state of the corresponding production technologies, based on either open-pond systems or closed photobioreactors. The potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae is also discussed.     

Microalgae aquaculture feeds

Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the production of larvae and juvenile shell- and finfish, as well as for raising the zooplankton required for feeding of juvenile animals. The blue-green algaSpirulina is used in substantial amounts (over 100 t y^–1) as a fish and shrimp feed, and even larger markets can be projected if production costs could be reduced. Another potential large-scale application of microalgae is the cultivation ofHaematococcus for the production of the carotenoid astaxanthin, which gives salmon flesh its reddish color. In the long-term microalgae biomass high in lipids (omega-3 fatty acids) may be developed as substitutes for fish oil-based aquaculture feeds. In shrimp ponds the indigenous algal blooms supply a part of the dietary requirements of the animals, but it is difficult to maximize algal productivities. A separate algal production system could feed the shrimps and minimize the need for added feed. Bivalves feed essentially exclusively on marine microalgae throughout their life cycle. The development of cultivation technologies for such microalgae would allow the onshore production of these animals, with greatly improved product quality and safety.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

Phototrophic pigment production with microalgae: biological constraints and opportunities

There is increasing interest in naturally produced colorants, and microalgae represent a bio‐technologically interesting source due to their wide range of colored pigments, including chlorophylls (green), carotenoids (red, orange and yellow), and phycobiliproteins (red and blue). However, the concentration of these pigments, under optimal growth conditions, is often too low to make microalgal‐based pigment production economically feasible. In some Chlorophyta (green algae), specific process conditions such as oversaturating light intensities or a high salt concentration induce the overproduction of secondary carotenoids (β‐carotene in Dunaliella salina (Dunal) Teodoresco and astaxanthin in Haematococcus pluvialis (Flotow)). Overproduction of all other pigments (including lutein, fucoxanthin, and phycocyanin) requires modification in gene expression or enzyme activity, most likely combined with the creation of storage space outside of the photosystems. The success of such modification strategies depends on an adequate understanding of the metabolic pathways and the functional roles of all the pigments involved. In this review, the distribution of commercially interesting pigments across the most common microalgal groups, the roles of these pigments in vivo and their biosynthesis routes are reviewed, and constraints and opportunities for overproduction of both primary and secondary pigments are presented.     

Nutritional stress effects under different nitrogen sources on the genes in microalga Isochrysis zhangjiangensis and the assistance of Alteromonas macleodii in releasing the stress of amino acid deficiency

The expressions of nine nitrogen assimilation‐associated genes, NRT2, NAR1, NIA2, NIR, GLN2, GLSF, GSN1, GDH, and AAT2, in the microalga Isochrysis zhangjiangensis were investigated to unveil the effects of limitations of various nitrogen sources (NaNO₃, NH₄Cl, NaNO₂, and an amino acid mixture) on the microalgae. The results demonstrated that the NRT2, NAR1, GLN2, GSN1, and AAT2 genes were highly expressed in lipid‐rich microalgae under inorganic nitrogen‐deficient conditions and they decreased after nitrogen resupply. Significant increases in the expressions of NAR1, GLN2, and GLSF were found in nitrate‐depleted microalgae, whereas significant increases in the expressions of NRT2, NAR1, GLN2, and GSN1 were found in nitrite‐depleted microalgae. Significant increases in the expressions of only NRT2 and GSN1 were found in ammonium‐depleted microalgae (P < 0.05). Except for the NRT2, other genes were expressed at lower levels under amino acid‐deficient conditions compared with amino acid‐sufficient controls. The expression of the NIA2 gene decreased in nitrogen‐depleted microalgae regardless of the initial nitrogen source. However, the results of fatty acid analyses showed that the features of fatty acid profiles followed a similar mode, in which the percentage compositions of C16:0 and C18:1Δ⁹ increased in nitrogen‐depleted cells and that of C16:1Δ⁹, C18:3Δ^9,12,15, C18:4Δ^6,9,12,15, and C18:5Δ^3,6,9,12,15 decreased, regardless of the type of nitrogen source applied. It was also found that the epiphytic bacterium Alteromonas macleodii played a particularly important role in releasing microalgae from the stress of amino acid deficiency. These findings also provide a foundation for regulating microalgal lipid production through manipulation of the nitrogen assimilation‐associated genes.     

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.