Accumulation Of Secondary Carotenoids Research Articles

Multimodal non-invasive probing of stress-induced carotenogenesis in the cells of microalga Bracteacoccus aggregatus.

Microalgae are the richest source of natural carotenoids-accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis-gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging.

Inhibition of antioxidant enzyme activities enhances carotenogenesis in microalga Dactylococcus dissociatus MT1.

Microalgal biotechnology has become a promising field of research for the production of valuable, sustainable and environmentally friendly byproducts, especially for carotenoids. Bulk accumulation of secondary carotenoids in microalgae are mostly induced by oxidative stress of cells. In this research, we investigated the effects of antioxidant enzyme activity inhibition on carotenogenesis in a microalga Dactylococcus dissociatus MT1. The activities of four major antioxidant enzyme families, namely superoxide dismutase (SOD), catalases (CAT), glutathione peroxydases (GPX) and ascorbate perxodases (APX), were inhibited by relevant inhibitors during the stressed cultivation of D. dissociatus to observe the effects on carotenogensis. A 91% decrease in activity was observed for CAT, comparing with controls without any inhibitors added, followed by 65%, 61%, and 47% for the enzymes SOD, APX, and GPX, respectively. Concomitantly, it was found that this partial inhibition had substantial influences on the accumulation of carotenoids, with the highest production levels obtained in CAT inhibition conditions and an increase of 2.6 times of carotenoid concentration observed, comparing with control cultivation conditions. We conclude that the modulation of antioxidant enzyme activities could lead to the overproduction of carotenoids in this microalgal cell culture, and we expect that this novel approach of optimizing carotenogenesis processes for D. dissociatus cell cultures could be transferrable to other cell culture systems and might have an important impact on the carotenoid production industry.

Tetraedron minimum, First Reported Member of Hydrodictyaceae to Accumulate Secondary Carotenoids.

We isolated a novel strain of the microalga Tetraedron minimum in Iceland from a terrestrial habitat. During long-term cultivation, a dish culture turned orange, indicating the presence of secondary pigments. Thus, we characterized T. minimum for growth and possible carotenoid production in different inorganic media. In a lab-scale photobioreactor, we confirmed that nitrogen starvation in combination with salt stress triggered a secondary carotenoid accumulation. The development of the pigment composition and the antioxidant capacity of the extracts was analyzed throughout the cultivations. The final secondary carotenoid composition was, on average, 61.1% astaxanthin and 38.9% adonixanthin. Moreover, the cells accumulated approx. 83.1% unsaturated fatty acids. This work presents the first report of the formation of secondary carotenoids within the family Hydrodictyaceae (Sphaeropleales, Chlorophyta).

Ultrastructure of the green alga Dunaliella salina strain CCAP19/18 (Chlorophyta) as investigated by quick-freeze deep-etch electron microscopy

The single-celled green alga Dunaliella salina is a model system for studies on stress biology, in particular regarding secondary carotenoid accumulation. Under non-stress conditions the cells are green, but under abiotic stress the cells turn orange, because they switch their metabolism and accumulate β-Carotene in globules in the chloroplast. For the first time, Quick-freeze deep-etch electron microscopy was used to visualize cellular structures in green and orange cells of D. salina strain CCAP19/18. This allowed us to present an in-depth analysis of the cellular ultrastructure describing and comparing the features of the two cell types. Our images illustrate the presence of a pericellular matrix for this strain of D. salina. The pericellular matrix was spongy and strands of unknown material anchored it into the plasma membrane. The cytoplasm contained a variety of vesicles, vacuoles, and acidocalcisomes. We could show for strain CCAP19/18 that cytoplasmic lipid bodies were often in close proximity to and sometimes in contact with the outer chloroplast envelope membrane and with the endoplasmic reticulum. Major visible differences between green and orange cells were in the chloroplast: the orange cells have greatly reduced amounts of thylakoid membranes and greatly increased numbers of β-Carotene globules. We showed that the β-Carotene globules often made point contacts with thylakoid membranes, and frequently laid side-by-side along the thylakoid membrane surface, providing support to studies that indicated exchange of molecules between β-Carotene globules and thylakoid membranes. A novel finding was the β-Carotene globule duplets, suggesting intermediate stages in β-Carotene globule morphogenesis. Overall, the β-Carotene globules appear to be similar to plant plastoglobuli. We provide this description of the cellular ultrastructure features as a resource in the context of the recent publication of the genome of D. salina strain CCAP19/18 to expand on the knowledge regarding this novel reference strain.

Effects of 2-azahypoxanthine on extracellular terpene accumulations by the green microalga Botryococcus braunii, race B

The B race of the green microalga Botryococcus braunii accumulates large amounts of triterpene hydrocarbons that are attractive as a source for biofuels. The alga, however, exhibits rather slow growth and this characteristic has hindered its practical application for biofuel production. In order to test a phytohormone-like compound that may regulate the growth or hydrocarbon production of this alga, the effects of 2-azahypoxanthine (AHX) on algal biomass and terpene contents in the extracellular matrix (ECM) in the Showa strain of B. braunii were investigated. The biomass of the Showa strain increased with AHX supplementation under static culture conditions although such effects were not observed in cultures with aeration or agitation. AHX supplementation significantly induced the accumulation of secondary carotenoids, including species-specific tetramethylsqualene (TMS)-conjugated carotenoids, botryoxanthin A and braunixanthin 1. Conversely, the addition of AHX showed a tendency to slightly lower the triterpene hydrocarbon (botryococcenes) content in the ECM. These metabolic changes in tri- and tetraterpene accumulation could be caused by a reduction in nitrogen uptake from the culture medium due to AHX addition. Thus, there is a possibility that AHX could be used as a reagent to modulate terpene biosynthesis in the B race of B. braunii.

Morphology and Phylogenetic Position of the Freshwater Green Microalgae Chlorochytrium (Chlorophyceae) and Scotinosphaera (Scotinosphaerales, ord. nov., Ulvophyceae).

The green algal family Chlorochytriaceae comprises relatively large coccoid algae with secondarily thickened cell walls. Despite its morphological distinctness, the family remained molecularly uncharacterized. In this study, we investigated the morphology and phylogenetic position of 16 strains determined as members of two Chlorochytriaceae genera, Chlorochytrium and Scotinosphaera. The phylogenetic reconstructions were based on the analyses of two data sets, including a broad, concatenated alignment of small subunit rDNA and rbcL sequences, and a 10-gene alignment of 32 selected taxa. All analyses revealed the distant relation of the two genera, segregated in two different classes: Chlorophyceae and Ulvophyceae. Chlorochytrium strains were inferred in two distinct clades of the Stephanosphaerinia clade within the Chlorophyceae. Whereas clade A morphologically fits the description of Chlorochytrium, the strains of clade B coincide with the circumscription of the genus Neospongiococcum. The Scotinosphaera strains formed a distinct and highly divergent clade within the Ulvophyceae, warranting the recognition of a new order, Scotinosphaerales. Morphologically, the order is characterized by large cells bearing local cell wall thickenings, pyrenoid matrix dissected by numerous anastomosing cytoplasmatic channels, sporogenesis comprising the accumulation of secondary carotenoids in the cell periphery and almost simultaneous cytokinesis. The close relationship of the Scotinosphaerales with other early diverging ulvophycean orders enforces the notion that nonmotile unicellular freshwater organisms have played an important role in the early diversification of the Ulvophyceae.

Carotenoid production and change of photosynthetic functions in Scenedesmus sp. exposed to nitrogen limitation and acetate treatment

Under stress conditions, some microalgae up-regulate certain biosynthetic pathways, leading to the accumulation of specific compounds. For example, changing nutrient composition can induce stress in algae’s physiological activities, which may trigger an intense increase in carotenoid production. In this study, the change of photosynthetic functions and carotenoid production in the green microalga Scenedesmus sp. was investigated when algal cultures were exposed to conditions including limited nitrogen content with the addition of sodium acetate. Microalgal cultures were treated for 18 days under higher irradiance conditions. We observed a decrease of chlorophyll content induced concomitantly with a decline of photosystem II and I photochemistry. At the same time, an important increase in carotenoid content was detected. By using high-performance liquid chromatographic analysis, we found that the secondary carotenoids astaxanthin and canthaxanthin were accumulated compared to controls. During the process of carotenoid accumulation, chlorophyll degradation was found in addition to a strong decrease in photosynthetic electron transport. Such changes may be associated with the structural reorganization of the photosynthetic apparatus and can be a useful indicator of secondary carotenoid accumulation in algal cultures.

THE ACCUMULATION OF ASTAXANTHIN AND THE RESPONSE OF PHOTOSYNTHETIC ACTIVITY IN <I>SCENEDESMUS OBLIQUUS</I>

The accumulation of astaxanthin in Scenedesmus obliquus under stress conditions was analyzed, and the responses of Photosynthetic activity and morphological change of algal cells were observed in the study. Under the temperature of 30 degrees C and illumination of 180 mu mol/m(2) . s, the content of chlorophyll fell to 4.20 mg/L from initial 5.59 mg/L while the content of carotenoid rose up from 0.25 mg/L to 0.44 mg/L in 48 hours. The composition of individual carotenoid was isolated and identified by HPLC/MS analysis. The results showed cells accumulated secondary carotenoids such as echinenone, adonixanthin, canthaxanthin adonirubin and 3'-hydroxyechinenone and so on, the ketocarotenoid astaxanthin (3, 3'-dihydroxy-beta, beta-carotene-4, 4'-dione) was found as a final product for the synthesis of secondary carotenoid. With the accumulation of secondary carotenoids, the algal coenobium composed of 4 or 8 cells was split up into single or two cells, and the shape of cells changed into swollen and irregular contrast to their initial state. The photosynthetic activity was also influenced by the stress conditions. The photosynthetic rate decreased about 50% in the first 3 hours, and then went up from 19.54 mu mol O-2/mg Chla/h to 34.29 mu mol O-2/mg Chla/h in the next 9 hours. From 12 hours to 48 hours, the photosynthetic rate experienced a dramatically drop and reduced to nearly 5.21 mu mol O-2/mg Chla/h. The respiration rate of algal cells showed an inverse trend, which increased from 18.24 mu mol O-2/mg Chla/h to 60.37 mu mol O-2/mg Chla/h in the first 24 hours although there was a fluctuation in this course, then it decreased to 38.40 mu mol O-2/Mg Chla/h in the next 24 hours which was still more higher than that of the control group. The change of chlorophyll fluorescence tended to be similar to that of photosynthetic rate and decreased by 63.9%. These results indicated that the S. obliquus cells could biosynthesize astaxanthin by induced conditions. The accumulation of secondary carotenoids led the changes of contents ratio for chlorophyll to carotenoid. The light inhibition, enhanced respiration rate and the damage to PS II were all responses to the stress, which also yielded more metabolism products and reactive oxygen species which further engendered the biosynthesis of secondary carotenoids. Simultaneously, stress conditions inhibited the cell division and led the changes of cell morphology. The regulating of photosynthetic activity and carotenoids accumulation were both the phsioligical mechanism for algal cells to resist the inclement environmental conditions.

Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae)

Many unicellular green algae can become yellow or red in various natural habitats due to mass accumulation of a secondary carotenoid, such as lutein, or astaxanthin. The accumulation of secondary carotenoids is generally thought to be a survival strategy of the algae under photo-oxidative stress or other adverse environmental conditions. The physiological role of the carotenoids in stress response is less well understood at the subcellular or molecular level. In this study, a stable astaxanthin overproduction mutant (MT 2877) was isolated by chemical mutagenesis of a wild type (WT) of the green microalga Haematococcus pluvialis Flotow NIES-144. MT 2877 was identical to the WT with respect to morphology, pigment composition, and growth kinetics during the early vegetative stage of the life cycle. However, it had the ability to synthesize and accumulate about twice the astaxanthin content of the WT under high light, or under high light in the presence of excess amounts of ferrous sulphate and sodium acetate. Under stress, the mutant exhibited higher photosynthetic activities than the WT, based on considerably higher chlorophyll fluorescence induction, chlorophyll autofluorescence intensities, and oxygen evolution rates. Cell mortality caused by stress was reduced by half in the mutant culture compared with the WT. Enhanced protection of the mutant against stress is attributed to its accelerated carotenogenesis and accumulation of astaxanthin. Our results suggest that MT 2877, or other astaxanthin overproduction Haematococcus mutants, may offer dual benefits, as compared with the wild type, by increasing cellular astaxanthin content while reducing cell mortality during stress-induced carotenogenesis.

Photosynthetic acclimation and photoprotective mechanism of Haematococcus pluvialis (Chlorophyceae) during the accumulation of secondary carotenoids at elevated irradiation

B.S. Qiu and Y. Li. 2006. Photosynthetic acclimation and photoprotective mechanism of Haematococcus pluvialis (Chlorophyceae) during the accumulation of secondary carotenoids at elevated irradiation. Phycologia 45: 117–126. DOI: 10.2216/04-99.1The photosynthetic acclimation of Haematococcus pluvialis Flotow to elevated irradiance and its photoprotective mechanisms were investigated. High light caused a remarkable increment in carotenoid content per cell. Cellular and volumetric chlorophyll contents were significantly increased after four days of high light treatment. Net photosynthesis of high light treated cells was decreased but their dark respiration was increased during the accumulation of secondary carotenoids (SC). The inactivation of reaction centers was observed in high light treated cells, and their normalized complementary area and turnover number were higher than those under low light. The PS II activity of red cells from high light was decreased by 17% compared with green cells from low light but their PS I activity was significantly increased. The K-step could not be observed in the fluorescence transients of red cells, indicating that the oxygen-evolving complex was not affected during SC accumulation. Haematococcus pluvialis could protect itself against strong irradiance through the D1 protein repair cycle and the xanthophyll cycle. The D1 protein repair cycle was the most important protective mechanism in H. pluvialis and its operation could alleviate photoinhibition by 49% in green cells and by 53~55% in red cells. The xanthophyll cycle could contribute to the protection of green cells subjected to strong irradiance but its role was negligible in red cells. Fv /Fo values were decreased in green cells and red cells by 45% and 32~34% respectively after 2.5 hours of photoinhibitory treatment. However, this may not necessarily indicate that the accumulated SC in red cells might play a photoprotective role.

β-carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis

Recent findings on the localization in Haematococcus pluvialis of anearly and a late enzyme in astaxanthin synthesis have suggested thesequential involvement of two compartments, the chloroplast (site of earlysynthesis) and the cytoplasmic lipid vesicles. Here the intermediatetransported across the chloroplast envelope into the cytoplasm is elucidatedby means of the inhibitors diphenylamine (DPA) and2-(4-chlorophenylthio)-triethylamine (CPTA). Based on the amounts of theintermediates accumulated upon inhibitor treatment and their occurrencein the lipid vesicle fractions, it is concluded that the transport takes placeafter β-ionone ring cyclization. A non-vesicle mechanism of β-iononecarotenoid transport from the chloroplast towards the cytoplasmic lipidvesicles via carotenoid binding proteins is suggested.

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

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

Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae).

The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated.

Accumulation of secondary carotenoids in flagellates of Haematococcus pluvialis (Chlorophyta) is accompanied by an increase in per unit chlorophyll productivity of photosynthesis

Flagellates of Haematococcus pluvialis accumulate secondary carotenoids (mainly astaxanthin and its esters) under nitrogen deficiency in combination with exposure to stronger light. Our cultivation scheme allows the characterization of metabolic activities during the accumulation process without interference by formation of the resting state (aplanospores). The highest rate of secondary carotenoid biosynthesis (0.046 h−1) was observed almost simultaneously with the highest rates of cell division (0.027 h−1) and of chlorophyll biosynthesis (0.015 h−1) after 1.5 days of exposure to nitrogen limitation and higher irradiance. Photosynthetic activity was studied by oxygen evolution and chlorophyll fluorescence measurements; changes in protein components of the photosynthetic apparatus were quantified by gel electrophoresis and immunoblot analysis. Accumulation of secondary carotenoids was accompanied by an increase in photosynthetic productivity expressed on a unit chlorophyll basis. Changes in the photosynthetic apparatus occurring mainly during the first 2 days of exposure to nitrogen limitation and stronger light can be interpreted as acclimation to the increased cultivation irradiance. Photoprotective action of secondary carotenoids as a ‘sunshade’ was demonstrated in red flagellates exhibiting a lower blue-light-induced decrease in photosystem II efficiency as compared with red actinic light.

Accumulation of secondary carotenoids in flagellates of Haematococcus pluvialis (Chlorophyta) is accompanied by an increase in per unit chlorophyll productivity of photosynthesis

Flagellates of Haematococcus pluvialis accumulate secondary carotenoids (mainly astaxanthin and its esters) under nitrogen deficiency in combination with exposure to stronger light. Our cultivation scheme allows the characterization of metabolic activities during the accumulation process without interference by formation of the resting state (aplanospores). The highest rate of secondary carotenoid biosynthesis (0.046 h−1) was observed almost simultaneously with the highest rates of cell division (0.027 h−1) and of chlorophyll biosynthesis (0.015 h−1) after 1.5 days of exposure to nitrogen limitation and higher irradiance. Photosynthetic activity was studied by oxygen evolution and chlorophyll fluorescence measurements; changes in protein components of the photosynthetic apparatus were quantified by gel electrophoresis and immunoblot analysis. Accumulation of secondary carotenoids was accompanied by an increase in photosynthetic productivity expressed on a unit chlorophyll basis. Changes in the photosynthetic apparatus occurring mainly during the first 2 days of exposure to nitrogen limitation and stronger light can be interpreted as acclimation to the increased cultivation irradiance. Photoprotective action of secondary carotenoids as a ‘sunshade’ was demonstrated in red flagellates exhibiting a lower blue-light-induced decrease in photosystem II efficiency as compared with red actinic light.

SECONDARY CAROTENOID ACCUMULATION IN SCENEDESMUS KOMAREKII (CHLOROPHYCEAE, CHLOROPHYTA)

As Scenedesmus komarekii Hegewald was cultured under high light intensity and nitrogen limitation, the color of cells progressed from green to brown, and finally through orange to brick red. The secondary carotenoids astaxanthin and canthaxanthin, as well as apolar carotenoids, were detected in the brown and orange cells. These carotenoids were contained in lipoidal globules that were first formed at the periphery of the cell and progressively propagated toward its inside, eventually filling most of it. The chloroplast was single and parietal in the green cells. As the cells turned brown, the chloroplast divided into several small lobes and was pushed toward the interior by the accumulating lipoidal globules. Sometimes the outer layer of the wall of the brown cell developed one or two diametrically opposed swellings. Once the cells became orange or red, neither lipoidal globules nor any major organelles were distinguishable. The cell wall in the orange cells became thick because of the formation of electron‐dense granules between its outer and inner layers. The mode of the secondary carotenoid accumulation in S. komarekii differs from that of Haematococcus, an alga well known for its ability to accumulate secondary carotenoids, but resembles that of “Chlorella”zofingiensis (=Mychonastes zofingiensis (Dönz) Kalina et Punčochářová).

Secondary carotenoid accumulation in flagellates of the green alga Haematococcus lacustris

The combination of nitrogen deprivation and increased cultivation light intensity resulted in the synthesis of secondary carotenoids in flagellates of Haematococcus lacustris. The pigment pattern was characterized by high-pressure liquid chromatography analysis during the accumulation period and in response to inhibitors of carotenoid biosynthesis (diphenylamine, norflurazon and tetcyclacis). Diphenylamine treatment resulted in (i) a decrease in ketocarotenoids and (ii) an accumulation of b-carotene and zeaxanthin due to inhibition of the bcarotene oxygenase. Our results indicate that astaxanthin synthesis in H. lacustris follows the biosynthetic pathway elucidated in the marine bacterium Agrobacterium aurantiacum.

Secondary carotenoid accumulation in flagellates of the green alga Haematococcus lacustris

The combination of nitrogen deprivation and increased cultivation light intensity resulted in the synthesis of secondary carotenoids in flagellates of Haematococcus lacustris. The pigment pattern was characterized by high-pressure liquid chromatography analysis during the accumulation period and in response to inhibitors of carotenoid biosynthesis (diphenylamine, norflurazon and tetcyclacis). Diphenylamine treatment resulted in (i) a decrease in ketocarotenoids and (ii) an accumulation of β-carotene and zeaxanthin due to inhibition of the β-carotene oxygenase. Our results indicate that astaxanthin synthesis in H. lacustris follows the biosynthetic pathway elucidated in the marine bacterium Agrobacterium aurantiacum.

Composition and accumulation of secondary carotenoids in Chlorococcum sp.

A locally isolated Chlorococcum sp. could accumulate astaxanthin and its esters as secondary carotenoids. The secondary carotenoids could reach a concentration of 5.2 mg g−1 d. wt, and were located in the cytoplasm and chloroplast as globules. Cells grew best at pH 8.0 and 30 °C, at which the growth rate was about 0.066 h−1. Acidic condition (pH 5.5 and 6.5) and slightly elevated temperature (35 °C) enhanced the cellular accumulation of astaxanthin. Outdoor studies indicated that Chlorococcum sp. grew well in a tubular photobioreactor. In medium containing 2 mM and 10 mM NH4CI, the cellular contents of total secondary carotenoids and astaxanthin reached similar levels (5.0 mg g−1 d. wt and 2.0 mg g−1 d. wt, respectively) in the 15 days of cultivation, while the yield of total secondary carotenoids and astaxanthin in 10 mM NH4CI were higher (45 mg L−1 and 18 mg L−1, respectively). The advantages of tolerance to high temperature and extreme pH values, relative fast growth rate and ease of cultivation in outdoor system suggest that Chlorococcum sp. could be a potential candidate for mass production of secondary carotenoids in particular astaxanthin.

Enhanced accumulation of secondary carotenoids in a mutant of the green alga, Chlorococcum sp.

A colour mutant of the unicellular green alga Chlorococcum sp. was obtained by visual colour detection method on plates with medium containing sodium azide. The growth of the mutant MA-1 was more susceptible to azide compared with the wild type, whereas the total secondary carotenoid (SC)synthesis was more resistant to the inhibitor. The azide concentration that inhibited SC formation by 50% (I50) was ten times higher than that required for the wild type. The mutant was stable over several consecutive subculturings in the absence of azide. The indoor and outdoor studies showed that the mutant could synthesise more than 2-fold of the total SC and astaxanthin of the wild type. The mutant MA-1 could be a natural source of SC and astaxanthin.