Enhanced Astaxanthin Production in Escherichia coli via Morphology and Oxidative Stress Engineering.

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.

Accumulation of Astaxanthin Was Improved by the Nonmotile Cells of Haematococcus pluvialis.

The current commercial production of natural astaxanthin is mainly carried out using Haematococcus pluvialis vegetative cells in the "two-stage" batch mode. The motile vegetative cells are more sensitive to stress than nonmotile vegetative cells, thereby affecting the overall astaxanthin productivity in H. pluvialis cultures. In this study, we compared the differences between motile cells and nonmotile cells in astaxanthin productivity, morphological changes, the mortality rate, and the diameter of the formed cysts. The experimental design was achieved by two different types H. pluvialis cell under continuous light of 80 µmol photons m-2 s-1 for a 9-day induction period. The highest astaxanthin concentration of 48.42 ± 3.13 mg L-1 was obtained in the nonmotile cell cultures with the highest the productivity of 5.04 ± 0.15 mg L-1 day-1, which was significantly higher than that in the motile cell cultures. The microscopic examination of cell morphological showed a large number of photooxidative damaged cells occurring in the motile cell cultures, resulting in higher cell mortality rate (22.2 ± 3.97%) than nonmotile cell cultures (9.6 ± 0.63%). In addition, the analysis results of cell diameter statistics indicated that nonmotile cells were more conducive to the formation of large astaxanthin-rich cysts than motile cells. In conclusion, the works presented here suggest that the accumulation of astaxanthin was significantly improved by nonmotile cells of H. pluvialis, which provided a possibility of optimizing the existing H. pluvialis cultivation strategy for the industrial production.

Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids.

The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.

Purification and Identification of Astaxanthin and Its Novel Derivative Produced by Radio-tolerant Sphingomonas astaxanthinifaciens.

The red diketocarotenoid, astaxanthin, exhibits extraordinary health-promoting activities such as antioxidant, anti-inflammatory, antitumor, and immune booster, which may potentially protect against many degenerative diseases such as cancers, heart diseases, and exercise-induced fatigue. These numerous health benefits and consumer interest in natural products have therefore increased the market demand of astaxanthin as a nutraceutical and medicinal ingredient in food, aquaculture feed, and pharmaceutical industries. Consequently, many research efforts have been made to discover novel microbial sources with effective biotechnological production of astaxanthin. Using a rapid screening method based on 16S rRNA gene, and effective HPLC-Diode array-MS methods for carotenoids analysis, we isolated a novel astaxanthin-producing bacterium (strain TDMA-17T) that belongs to the family Sphingomonadaceae (Asker et al., FEMS Microbiol Lett 273:140-148, 2007).In this chapter, we provide a comprehensive description of the methods used for the analysis and identification of carotenoids produced by strain TDMA-17T. We will also describe the methods of isolation and identification for a novel bacterial carotenoid (an astaxanthin derivative), a major carotenoid that is produced by the novel strain. Finally, the identification methods of the novel strain will be summarized.

Rapid selection of astaxanthin-hyperproducing Haematococcus mutant via azide-based colorimetric assay combined with oil-based astaxanthin extraction.

The aim of this work was to develop a new approach for simple and high-throughput selection of astaxanthin-hyperproducing Haematococcus mutants through a sequential combination method of azide-based colorimetric assessment and oil-based astaxanthin quantification. Randomly mutagenized cells were spotted on solid culture medium containing 50 µM of sodium azide to accelerate the biosynthesis of astaxanthin. After 3 days, highly-induced mutants were preliminarily isolated by visual inspection and their astaxanthin accumulations were rapidly quantified by soybean oil-based extraction method. On the whole, the selected mutants showed reduced vegetative growth rates but eventually exhibited higher astaxanthin productions than the parental strain owing to their improved inductive growths. Among them, M13 showed 174.7 ±â€¯5.69 mg L-1 of the highest astaxanthin production, which is 1.59-times higher than that of wild-type. This wide-scope screening method expedites both upstream and downstream astaxanthin quantification, making it a useful tool for isolating microalgae with high astaxanthin production.

Biological interaction of newly synthesized astaxanthin-s-allyl cysteine biconjugate with Saccharomyces cerevisiae and mammalian α-glucosidase: In vitro kinetics and in silico docking analysis.

In humans, alpha-glucosidase activity is present in sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM). α-glucosidase is involved in the hydrolyses of disaccharide into monosaccharides and results in hyperglycemia. Subsequently chronic hyperglycemia induces oxidative stress and ultimately leads to the secondary complications of diabetes. Hence, identifying compounds with dual beneficial activity such as efficient antioxidant and α-glucosidase inhibition property has attracted the attention in recent years. Keeping these views, in the present study astaxanthin (AST; a natural antioxidant present in marine microalgae) was biconjugated with allyl sulfur amino acid such as s-allyl cysteine (SAC). The synthesized AST-SAC (with molecular weight of 883.28) was characterized using UV-visible spectrophotometer, ESI-MS, and NMR analysis. AST-SAC showed potent antioxidant property in vitro. AST-SAC inhibited Saccharomyces cerevisiae α-glucosidase (IC50 = 3.98 µM; Ki = 1 µM) and mammalian α-glucosidase [rat intestinal maltase (IC50 = 6.4 µM; Ki = 1.3 µM) and sucrase (IC50 = 1.6 µM; Ki = 0.18 µM)] enzyme activity in a dose-dependent manner. Kinetic analysis revealed that AST-SAC inhibited all the α-glucosidases in a competitive mode. In silico analysis determined the interaction of AST-SAC with the amino acids present in the active site of S. cerevisiae and human (MGAM and SI) α-glucosidases.

Optimum Production Conditions, Purification, Identification, and Antioxidant Activity of Violaxanthin from Microalga Eustigmatos cf. polyphem (Eustigmatophyceae).

Violaxanthin is a major xanthophyll pigment in the microalga Eustigmatos cf. polyphem, but the amount produced after propagation can vary depending upon culture conditions. In this study, the effects of cultivation time, nitrogen concentration, light intensity, and culture mode on violaxanthin production were investigated. The results showed that this microalga vigorously grew and maintained a high level of violaxanthin in the fed-batch culture, and the highest violaxanthin productivity of 1.10 ± 0.03 mg L-1 d-1 was obtained under low light illumination with 18 mM of initial nitrogen supply for ten days. Additionally, violaxanthin was purified from E. cf. polyphem by silica gel chromatography and preparative high-performance liquid chromatography (PHPLC), and identified with high-resolution mass spectrometry (HRMS). The antioxidant activity of the purified violaxanthin was evaluated by three tests in vitro: reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical assays. The strongest inhibition of purified violaxanthin occurred during the scavenging of ABTS⁺ radicals, with EC50 of 15.25 µg mL-1. In conclusion, this is the first report to investigate the effects of different culture conditions on violaxanthin accumulation in E. cf. polyphem and provide a novel source for the production of violaxanthin that can be used for food and pharmaceutical applications.

Carotenoids of Microalgae Used in Food Industry and Medicine.

BACKGROUND: Since the industrial revolution, the consumption of processed food increased dramatically. During processing, food material loses many of its natural properties. OBJECTIVE: The simple restoration of the original properties of the processed food as well as fortification require food supplementation with compounds prepared chemically or of natural origin. The observations that natural food additives are safer and better accepted by consumers than synthetic ones have strongly increased the demand for natural compounds. Because some of them have only a low abundance or are even rare, their market price can be very high. This is the case for most carotenoids of natural origin to which this review is dedicated. The increasing demand for food additives of natural origin contributes to an accelerated depletion of traditional natural resources already threatened by intensive agriculture and pollution. To overcome these difficulties and satisfy the demand, alternative sources for natural carotenoids have to be found. In this context, photosynthetic microalgae present a very high potential because they contain carotenoids and are able to produce particular carotenoids under stress. Their potential also resides in the fact that only ten thousands of microalgal strains have been described while hundred thousands of species are predicted to exist. Carotenoids have been known for ages for their antioxidant and coloring properties, and a large body of evidence has been accumulated about their health potential. CONCLUSION: This review summarizes both the medicinal and food industry applications of microalgae with emphasis on the former. In addition, traditional and alternative microalgal sources used for industrial carotenoid extraction, the chemical and physical properties, the biosynthesis and the localization of carotenoids in algae are also briefly discussed.

Optimizing gas transfer to improve growth rate of Haematococcus pluvialis in a raceway pond with chute and oscillating baffles.

Up-down chute and oscillating (UCO) baffles were used to generate vortex and oscillating flow field to improve growth rate of Haematococcus pluvialis in a raceway pond. Effects of gas flow rate, solution velocity, and solution depth on solution mass transfer coefficient and mixing time were evaluated using online pH and dissolved oxygen probes. Mass transfer coefficient increased by 1.3 times and mixing time decreased by 33% when UCO baffles were used in the H. pluvialis solution, resulting in an 18% increase in biomass yield with 2% CO2. The H. pluvialis biomass yield further increased to 1.5g/L, and astaxanthin composition accumulated to 29.7mg/L under relatively higher light intensity and salinity.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid.

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a ß-carotene hydroxylase and a ß-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the ß-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

Effect of red cyst cell inoculation and iron(II) supplementation on autotrophic astaxanthin production by Haematococcus pluvialis under outdoor summer conditions.

The negative effect of heat stress on the autotrophic astaxanthin production by Haematococcus pluvialis has been observed during outdoor culture in summer. Under the summer conditions, the proliferation of vegetative cells was highly halted in the green stage and the inducibility in the biosynthesis of astaxanthin was partly hindered in the red stage. Herein, under outdoor summer conditions in which variations of the diurnal temperature occur, heat-stress-driven inefficient vegetative growth of H. pluvialis was highly improved by inoculating the red cyst cells; thereby, maintaining relatively moderate intracellular carotenoid levels in the green stage. Subsequently, a remarkably enhanced astaxanthin titer was successfully obtained by supplementing 50 µM iron(II) to induce the heat stress-driven Haber-Weiss reaction in the red stage. As a result, the productivity of astaxanthin in the cells cultured under summer temperature conditions (23.4-33.5 °C) using the two methods of red cell (cyst) inoculation and the iron(Fe(2+)) supplementation was increased by 147% up to 5.53 mg/L day compared with that of the cells cultured under spring temperature conditions (17.5-27.3 °C). Our technical solutions will definitely improve the annual natural astaxanthin productivity in H. pluvialis in locations confronted by hot summer weather, particularly in large-scale closed photobioreactor systems.

Induced carotenoid accumulation in Dunaliella salina and Tetraselmis suecica by plant hormones and UV-C radiation.

Carotenoids prevent different degenerative diseases and improve human health. Microalgae are commercially exploited for carotenoids, including astaxanthin and ß-carotene. Two commercially important microalgae, Dunaliella salina and Tetraselmis suecica, were treated with plant hormones salicylic acid (SA) and methyl jasmonate (MJ), or by UV-C radiation (T. suecica only) and a combination thereof. Significant increases in total carotenoids were found for D. salina and T. suecica after treatment with MJ (10 µmol/L) and SA (70-250 µmol/L), respectively. T. suecica also had significant increases in total carotenoids following UV-C radiation compared to control cultures. Among the carotenoids, lutein was the highest induced carotenoid. A combination of these two treatments also showed a significant increase in total carotenoids and lutein for T. suecica, when compared to controls. Plant hormones and UV-C radiation may be useful tools for increasing carotenoid accumulation in green microalgae although the responses are species- and dose-specific and should be trialed in medium to large scale to explore commercial production.

Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation

Background To study the relationship between intracellular anabolism and astaxanthin production, the influence of intracellular protein and fatty acids on astaxanthin production by four mutant Phaffia rhodozyma strains and their variations was investigated in this research. Results First, the content of astaxanthin in cells showed a reverse fluctuation in contrast to that of protein during the whole fermentation process. Moreover, compared with the three other strains, the astaxanthin-overproducing mutant strain of the yeast P. rhodozyma, called JMU-MVP14, had the highest specific productivity of astaxanthin as 6.8 mg/g, whereas its intracellular protein and fatty acid contents were the lowest. In addition, as a kind of sugar metabolic product, ethanol was only produced by P. rhodozyma JMU-VDL668 and JMU-7B12 during fermentation. Conclusions The results indicated that the accumulation of ethanol, intracellular protein, and fatty acids had competition effects on astaxanthin synthesis. This condition may explain why the P. rhodozyma strains JMU-VDL668 and JMU-7B12 achieved relatively lower astaxanthin production (1.7 and 1.2 mg/L) than the other two strains JMU-MVP14 and JMU-17W (20.4 and 3.9 mg/L).

Mesoflavibacter aestuarii sp. nov., a zeaxanthin-producing marine bacterium isolated from seawater.

An orange, rod-shaped, Gram-reaction-negative, aerobic and gliding bacterial strain devoid of flagella, designated strain KYW614(T), was isolated from seawater collected from Gwangyang Bay, Republic of Korea. Zeaxanthin was the major carotenoid pigment produced and flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain KYW614(T) belonged to the family Flavobacteriaceae and it was most closely related to Mesoflavibacter zeaxanthinifaciens TD-ZX30(T) (96.5%, sequence similarity). The predominant cellular fatty acids of strain KYW614(T) were iso-C(15 : 1) G (10.5%), summed feature 3 (C(16 : 1)ω7c/C(16 : 1)ω6c; 10.0%), iso-C(15 : 0) (9.5%), C(15 : 0) (7.5%) and iso-C(17 : 0) 3-OH (7.4%). MK-6 was the only isoprenoid quinone and the DNA G+C content was 32.6 mol%. Data from a polyphasic taxonomic study suggested that the isolate represents a novel species in the genus Mesoflavibacter, for which the name Mesoflavibacter aestuarii sp. nov. is proposed. The type strain is KYW614(T) ( = KCTC 32269(T) = JCM 19524(T)).

Astaxanthin: sources, extraction, stability, biological activities and its commercial applications--a review.

There is currently much interest in biological active compounds derived from natural resources, especially compounds that can efficiently act on molecular targets, which are involved in various diseases. Astaxanthin (3,3'-dihydroxy-ß, ß'-carotene-4,4'-dione) is a xanthophyll carotenoid, contained in Haematococcus pluvialis, Chlorella zofingiensis, Chlorococcum, and Phaffia rhodozyma. It accumulates up to 3.8% on the dry weight basis in H. pluvialis. Our recent published data on astaxanthin extraction, analysis, stability studies, and its biological activities results were added to this review paper. Based on our results and current literature, astaxanthin showed potential biological activity in in vitro and in vivo models. These studies emphasize the influence of astaxanthin and its beneficial effects on the metabolism in animals and humans. Bioavailability of astaxanthin in animals was enhanced after feeding Haematococcus biomass as a source of astaxanthin. Astaxanthin, used as a nutritional supplement, antioxidant and anticancer agent, prevents diabetes, cardiovascular diseases, and neurodegenerative disorders, and also stimulates immunization. Astaxanthin products are used for commercial applications in the dosage forms as tablets, capsules, syrups, oils, soft gels, creams, biomass and granulated powders. Astaxanthin patent applications are available in food, feed and nutraceutical applications. The current review provides up-to-date information on astaxanthin sources, extraction, analysis, stability, biological activities, health benefits and special attention paid to its commercial applications.

Aquibacter zeaxanthinifaciens gen. nov., sp. nov., a zeaxanthin-producing bacterium of the family Flavobacteriaceae isolated from surface seawater, and emended descriptions of the genera Aestuariibaculum and Gaetbulibacter.

A Gram-stain-negative, strictly aerobic, rod-shaped, non-flagellated, non-spore-forming and gliding marine bacterium, designated strain CC-AMZ-304(T), was isolated from coastal surface seawater near Taichung harbour, Taiwan. Strain CC-AMZ-304(T) predominantly synthesized zeaxanthin and thus formed yellow colonies on marine agar. The novel strain showed an unstable phylogenetic position, although sharing high pairwise 16S rRNA gene sequence similarities of 95.9-94.9, 95.7 and 95.1-93.9 % with Gaetbulibacter species (n = 4), Aestuariibaculum suncheonense SC17(T) and Bizionia species (n = 7), respectively. The polar lipid profile of strain CC-AMZ-304(T) consisted of phosphatidylethanolamine, five unidentified lipids, one unidentified phospholipid, two unidentified aminolipids and one unidentified glycolipid. The major (>5 % of the total) fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, iso-C15 : 0 3-OH and C15 : 1ω5c. The DNA G+C content was 36.0 mol%. Menaquinone-6 (MK-6) was the sole respiratory quinone and the major polyamine was triamine sym-homospermidine. Phylogenetic distinctiveness, unique polar lipid composition, presence of significant amounts of branched hydroxyl fatty acids (iso-C17 : 0 3-OH and iso-C15 : 0 3-OH) and a low amount of anteiso-C15 : 0, and several additional distinguishing biochemical features clearly discriminated strain CC-AMZ-304(T) from the type species of the genera Aestuariibaculum and Gaetbulibacter. Thus, based on data from the present polyphasic study, strain CC-AMZ-304(T) is considered to represent a novel species of a new genus within the family Flavobacteriaceae, for which the name Aquibacter zeaxanthinifaciens gen. nov., sp. nov. is proposed; the type strain of Aquibacter zeaxanthinifaciens is CC-AMZ-304(T) ( = JCM 18557(T) = BCRC 80463(T)). Emended descriptions of the genera Aestuariibaculum and Gaetbulibacter are also proposed.

Importance of bacillithiol in the oxidative stress response of Staphylococcus aureus.

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.

Carotenoids, versatile components of oxygenic photosynthesis.

Carotenoids (CARs) are a group of pigments that perform several important physiological functions in all kingdoms of living organisms. CARs serve as protective agents, which are essential structural components of photosynthetic complexes and membranes, and they play an important role in the light harvesting mechanism of photosynthesizing plants and cyanobacteria. The protection against reactive oxygen species, realized by quenching of singlet oxygen and the excited states of photosensitizing molecules, as well as by the scavenging of free radicals, is one of the main biological functions of CARs. X-ray crystallographic localization of CARs revealed that they are present at functionally and structurally important sites of both the PSI and PSII reaction centers. Characterization of a CAR-less cyanobacterial mutant revealed that while the absence of CARs prevents the formation of PSII complexes, it does not abolish the assembly and function of PSI. CAR molecules assist in the formation of protein subunits of the photosynthetic complexes by gluing together their protein components. In addition to their aforementioned indispensable functions, CARs have a substantial role in the formation and maintenance of proper cellular architecture, and potentially also in the protection of the translational machinery under stress conditions.

Production, characterization, and antioxidant activity of fucoxanthin from the marine diatom Odontella aurita.

The production, characterization, and antioxidant capacity of the carotenoid fucoxanthin from the marine diatom Odontella aurita were investigated. The results showed that low light and nitrogen-replete culture medium enhanced the biosynthesis of fucoxanthin. The maximum biomass concentration of 6.36 g L-1 and maximum fucoxanthin concentration of 18.47 mg g-1 were obtained in cultures grown in a bubble column photobioreactor (Ø 3.0 cm inner diameter), resulting in a fucoxanthin volumetric productivity of 7.96 mg L-1 day-1. A slight reduction in biomass production was observed in the scaling up of O. aurita culture in a flat plate photobioreactor, yet yielded a comparable fucoxanthin volumetric productivity. A rapid method was developed for extraction and purification of fucoxanthin. The purified fucoxanthin was identified as all-trans-fucoxanthin, which exhibited strong antioxidant properties, with the effective concentration for 50% scavenging (EC50) of 1,1-dihpenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical being 0.14 and 0.03 mg mL-1, respectively. Our results suggested that O. aurita can be a natural source of fucoxanthin for human health and nutrition.

Development of thin-film photo-bioreactor and its application to outdoor culture of microalgae.

Photosynthetic microalgae have received much attention as a microbial source of diverse useful biomaterials through CO(2) fixation and various types of photo-bioreactors have been developed for efficient microalgal cultivation. Herein, we developed a novel thin-film photo-bioreactor, which was made of cast polypropylene film, considering outdoor mass cultivation. To develop optimal design of photo-bioreactor, we tested performance of three shapes of thin-film photo-bioreactors (flat, horizontal and vertical tubular shapes) and various parts in the bioreactor. Collectively, vertical tubular bioreactor with H/D ratio 6:1 and cylindrical stainless steel spargers showed the most outstanding performance. Furthermore, the photo-bioreactor was successfully applied to the cultivation of other microalgae such as Chlamydomonas reinhardtii and Chlorella vulgaris. The scalability of photo-bioreactor was confirmed by gradually increasing culture volume from 4 to 25 L and the biomass productivity of each reactor was quite consistent (0.05-0.07 g/L/day) during the cultivation of H. pluvialis under indoor and outdoor conditions. Especially, we also achieved dry cell weight of 4.64 g/L and astaxanthin yield of 218.16 mg/L through long-term cultivation (100 days) under outdoor condition in 15 L photo-bioreactor using Haematococcus pluvialis, which means that the astaxanthin yield from outdoor cultivation is equal or superior to that obtained from controlled indoor condition. Therefore, these results indicate that we can apply this approach to development of optimal photo-bioreactor for the large-scale culture of microalgae and production of useful biomaterials under outdoor condition.