Astaxanthin Production Research Articles

Effects of selenite on green microalga Haematococcus pluvialis: Bioaccumulation of selenium and enhancement of astaxanthin production

Algae are at a low trophic level and play a crucial role in aquatic food webs. They can uptake and accumulate the trace element selenium (Se), which can be either essential or toxic to algal growth depending on the dosage and species. Se toxicity and algae resistance varied across different organisms. In order to investigate the effects of Se on the unicellular green alga Haematococcus pluvialis, an important industrial resource for natural astaxanthin, the algal growth rate, chlorophyll content, and fluorescence parameters were derived from experimental treatment with different concentrations of selenite. The results showed that the EC50 for the algal growth rate was 24mg/L, and that a low dosage of selenite (3mg/L) may not hinder H. pluvialis cell growth, but selenite at levels higher than 13mg/L do restrain cell growth. Bioaccumulation experiments showed that H. pluvialis accumulated up to 646μg/g total Se and 380μg/g organic Se, dry weight. However, treatment with high concentrations of selenite significantly increased intracellular hydrogen peroxide levels, antioxidant enzyme activity, and the production of astaxanthin, suggesting that Se bioaccumulation might be toxic to H. pluvialis.

Biofilm cultivation of Haematococcus pluvialis enables a highly productive one-phase process for astaxanthin production using high light intensities

State-of-the-art commercial cultivation of Haematococcus pluvialis for astaxanthin production is exclusively based on two-phase processes, separating a vegetative “green” phase for cell multiplication from an astaxanthin producing “red” phase consisting of resting stages. We report the continuous co-production of H. pluvialis biomass and astaxanthin in a one-phase process at high light intensities. By means of a Twin-Layer biofilm photobioreactor, H. pluvialis was grown immobilized under illuminations up to 1015μmolphotonsm−2s−1 and supplementary CO2 in the range of 1% to 10%, reaching a maximal dry biomass productivity of 19.4gm−2day−1 and a final standing crop of 213gm−2 growth area after 16days of cultivation. During the cultivation period, astaxanthin productivity increased with increasing light intensity to values of 0.39gm−2day−1 and a final amount of 3.4gm−2, representing an astaxanthin content of about 2.5% in the dry biomass. Compared to a two-phase process using the same photobioreactor, the one-phase approach resulted in a similar total astaxanthin amount per growth area in half the cultivation period. As a one-phase process, it also simplifies cultivation of H. pluvialis compared to the established two phase suspension-based systems for astaxanthin production.

Intraspecific trait variation affecting astaxanthin productivity in two Haematococcus (Chlorophyceae) species

Microalgae are increasingly used as commercial sources of high-value compounds. However, the nature and genetic basis of variation in commercially relevant traits remain understudied. This study focuses on the green alga Haematococcus pluvialis, well-known for accumulating the carotenoid astaxanthin. We examined intra- and interspecific variation and correlations between six traits related to astaxanthin productivity among 30 natural isolates and cultivated strains of two Haematococcus species. Significant intraspecific genotypic variation was found for all traits assessed in both species (broad sense heritability estimates H2=0.48–0.89), resulting in a fifteen-fold variation in astaxanthin productivity between the poorest and the best-performing strain. The two species differed in five of the six traits. Cultivated strains had a lower astaxanthin productivity compared to natural isolates of H. pluvialis, possibly reflecting loss of photoprotective capacity during long-term cultivation. In general, trait correlations were weak yet stronger in H. rubicundus than in H. pluvialis. Most of the variation in overall astaxanthin productivity could be explained by the differences in post-stress traits. Our results reveal extensive trait variation among isolates of a commercially interesting microalga. We recommend natural strain panels as a valuable tool for cost-efficient trait mapping and to select targets for genetic engineering, marker-assisted strain selection or breeding aiming at the optimization of astaxanthin productivity.

Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis

Due to its superior antioxidant capabilities and higher activity than other carotenoids, astaxanthin is used widely in the nutraceutical and medicine industries. The most prolific natural producer of astaxanthin is the unicellular green microalga Haematococcus pluvialis. The correct identification of any contaminants in H. pluvialis cultures is both essential and nontrivial for several reasons. Firstly, while it is possible to distinguish the main microalgal contaminant Coelastrella sp. (in H. pluvialis cultures), in practice, it is frequently a daunting and error-prone task for personnel without extensive experience in the microscopic identification of algal species. Secondly, the undetected contaminants may decrease or stop production of astaxanthin. Lastly, the presence of other contaminants such as fungi can eventually infect and destroy the whole algae collection. In this study, high-resolution melting (HRM) analysis was developed to detect microalgal and fungal contamination. The developed diagnostic procedure allowed to distinguish pure H. pluvialis samples from cultures contaminated with low amounts (1.25 ng/ml) of microalgal DNA and fungal DNA (2.5 ng/ml). Such discrimination is not possible with the use of microscopy observations and allows fast and efficient collection testing.

Isolation and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis (Chlorophyceae) produced by dielectric barrier discharge plasma

:Haematococcus pluvialis has potential commercial application for its ability to accumulate astaxanthin under stress conditions. To improve astaxanthin productivity, nonthermal plasma can be used to produce mutant algal strains with enhanced production of astaxanthin. In this work, atmospheric pressure argon dielectric barrier discharge (DBD) was used for mutation induction. Several new algal strains with increased growth rate and higher astaxanthin accumulation were obtained. For analysis of the DBD-treated algal mutants, genetic variation was examined by random amplification of polymorphic DNA, and the pigment pattern in response to inhibitors (diphenylamine and nicotine) of carotenoid biosynthetic pathway was characterized by high-performance liquid chromatography. Furthermore, photosynthetic activity and gene expression were also examined by pulse amplitude-modulated fluorometry and quantitative real-time polymerase chain reaction, respectively. Our results confirmed that the enhanced astaxanthin accumulation in the mutants was associated with increased expression levels of lycopene cyclase, which is involved in the conversion of lycopene to β-carotene. As such, this work demonstrates a successful example for the effective application of DBD in the mutation breeding of astaxanthin-hyperproducing algal strains.

Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP)

In the present study, high light and nitrogen starvation with glucose-fed to the culture was found efficient to induce astaxanthin accumulation in Chlorella zofingiensis. Therefore, a two-step cultivation strategy including high biomass yield fermentation and outdoor induction with an energy-free RFP was conducted. During the fermentation, the highest cell density of 98.4gL−1 and astaxanthin yield of 73.3mgL−1 were achieved, which were higher than those so far reported in C. zofingiensis. During the outdoor induction, astaxanthin content was further increased by 1.5-fold leading to the highest astaxanthin productivity of 5.26mgL−1day−1 under an optimal dilution of 5-fold. Our work thus provided an effective two-step cultivation strategy for production of astaxanthin by C. zofingiensis.

Astaxanthin biosynthetic pathway: Molecular phylogenies and evolutionary behaviour of Crt genes in eubacteria

Astaxanthin (3, 3′-dihydroxy-β,β-carotene-4,4′-dione) is a high-value ketocarotenoid synthesized by several species of microalgae, plants, bacteria, and fungi and widely used as a potent antioxidant for human health and also a coloring agent. About six genes (CrtI, CrtL, CrtO, CrtW, CrtR and CrtZ) involve in the biosynthesis of astaxanthin. Phylogenetic analysis of astaxanthin biosynthetic pathway genes and their evolutionary rate variations has been studied among major phylogenetic groups of organisms such as plant, algae, and bacteria. The maximum likelihood (ML) algorithm was used in protein coding DNA sequences to infer the evolutionary relationship for the Crt genes among different eukaryotic and prokaryotic lineages. The phylogenetic analysis suggests that evolutionary pattern of Crt genes in eukaryotic and prokaryotic is characterized by even such as lateral gene transfer and gene duplication events. The specific amino acids present in the respective enzymes are the reason for their functional divergence. The dN values indicate that astaxanthin biosynthetic genes are more conserved in plant and algae than in any other bacterial phyla. This study provides the knowledge of individual Crt enzymes in different organisms that helps to increase the production of astaxanthin which is a biologically significant apocarotenoid that has various uses ranging from human health to cosmetic purposes.

Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

BackgroundRed yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed.ResultsXanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain.ConclusionsIn this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0556-x) contains supplementary material, which is available to authorized users.

The crosstalk between astaxanthin, fatty acids and reactive oxygen species in heterotrophic Chlorella zofingiensis

Chlorella zofingiensis can achieve ultrahigh cell density under heterotrophic growth conditions and has been considered as a promising producer of astaxanthin. Astaxanthin is predominantly esterified with fatty acids, in the form of either mono-ester or di-ester. The interrelationship between astaxanthin and fatty acids remains poorly understood. In the present study, we found that astaxanthin induction was accompanied by fatty acid accumulation in the glucose-fed heterotrophic C. zofingiensis cells. The presence of cerulenin, a specific inhibitor against fatty acid biosynthesis, led to a significant increase in the content of astaxanthin, di-ester in particular. When treated with sesamol, which is known to inhibit intracellular NADPH supply, a considerable decrease in astaxanthin was observed, together with a slight drop in fatty acids. Reactive oxygen species (ROS) level was found to correlate well with astaxanthin content in C. zofingiensis regardless of culture conditions. Collectively, these findings suggest that astaxanthin biosynthesis may compete with fatty acids for the carbon precursors and reducing power, and NADPH supply is important for astaxanthin accumulation, which will help us better understand astaxanthin biosynthesis in heterotrophic C. zofingiensis and benefit the future engineering for astaxanthin production.

Enhanced astaxanthin production from Haematococcus pluvialis using butylated hydroxyanisole

Haematococcus pluvialis is a promising natural source of high-value antioxidant astaxanthin under stress conditions. Biotic or abiotic elicitors are effective strategies for improving astaxanthin production in H. pluvialis. Butylated hydroxyanisole (BHA) was identified as an effective inducer for H. pluvialis LUGU. Under a treatment of 2mgL−1 BHA (BHA2), astaxanthin content reached a maximum of 29.03mgg−1 dry weight (DW) (2.03-fold of that in the control) after 12day of the mid-exponential growth phase. Subsequently, H. pluvialis LUGU was subjected to BHA2 at different growth phases because an appropriate time node for adding elicitors is vital for the entire production to succeed. As a result, the highest astaxanthin content (29.3mgg−1 DW) was obtained in cells on day 14 (BHA2 14) of the late-exponential growth phase. Furthermore, the samples treated with BHA2 14 and the control group were compared in terms of the transcriptional expression of seven carotenogenesis genes, fatty acid composition, and total accumulated astaxanthin. All selected genes exhibited up-regulated expression profiles, with chy, crtO, and bkt exhibiting higher maximum transcriptional levels than the rest. Oleic acid content increased 33.15-fold, with acp, fad, and kas expression being enhanced on the day when astaxanthin was produced rapidly.

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity.

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

A strategy for promoting astaxanthin accumulation in Haematococcus pluvialis by 1-aminocyclopropane-1-carboxylic acid application

The green algae Haematococcus pluvialis is a freshwater unicellular microalga belonging to Chlorophyceae. It is one of the best natural sources of astaxanthin, a secondary metabolite commonly used as an antioxidant and anti-inflammatory agent. Due to the importance of astaxanthin, various efforts have been made to increase its production. In this study, we attempted to develop a strategy for promoting astaxanthin accumulation in H. pluvialis using 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene (normally known as an aging hormone in plants). Our results demonstrated that ACC could enhance the growth of H. pluvialis, thereby promoting astaxanthin accumulation. Therefore, ACC has an indirect influence on astaxanthin production. We further verified the effect of ACC with a direct treatment of ethylene originated from banana peels. These results indicate that ethylene could be applied as an indirect method for enhancing growth and astaxanthin biosynthesis in H. pluvialis.

Effect of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid on different growth stages of Haematococcus pluvialis

In this study, we explored the effects of ACC on other stages of H. pluvialis. Interestingly, even though ACC displayed a dose-dependent effect on astaxanthin production, it is evident that astaxanthin production could be facilitated whenever the cells were treated at the early red stage. The transcriptional levels of BKT, CHY, SOD, and CAT genes supported enhanced astaxanthin biosynthesis upon ACC treatment at the early red stage. The combinatorial synergistic effect of ACC and light intensity was also confirmed. Finally, two-step application of ACC at the vegetative phase to increase biomass production and at the early-red stage to promote astaxanthin biosynthesis was proposed to maximize the efficiency of ACC treatment.

Complete genome sequence of astaxanthin-producing bacterium Altererythrobacter ishigakiensis NBRC 107699

Altererythrobacter ishigakiensis NBRC 107699 was isolated from marine sediment collected from a site on the coast of Ishigaki Island, Japan and deposited to the NITE Biological Resource Center. This strain is able to produce astaxanthin, which can be used as a food supplement. Here we describe the genome sequence and annotation, as well as the features of the organism. The genome of strain NBRC 107699 comprises 2,673,978bp and contains 2618 protein-coding genes (1966 with predicted functions), 42 tRNA genes and 3 rRNA genes. A. ishigakiensis NBRC 107699T encodes fifteen genes related to astaxanthin production, revealing its potential application in biotechnological industry. The genome sequence of A. ishigakiensis NBRC 107699 now provides the fundamental information for future studies.

Enhancement of astaxanthin production from Haematococcus pluvialis mutants by three-stage mutagenesis breeding

Haematococcus pluvialis was modified for higher astaxanthin production compatible with the superiorities of high biomass and high activity by three-stage mutagenesis breeding. UV irradiation mutants named UV11-4 made an increase on cell dry weight, but showed a longer growth circle than the wild type. On the basis of UV mutants, ethyl methane sulphonate (EMS) mutants E2-5 cut down the latent phase, brought forward and extended the logarithmic phase. The inhibitor diphenylamine (DPA) was employed to screen high-yield astaxanthin producer by the color change of colonies from green to red on solid medium. Via the contravariant cultivation, proliferation and transformation, the mutant DPA12-2 possessed an 1.7-fold astaxanthin production compared to the wild type, reaching 47.21±3.30mg/g dry cells.

Erratum: Water Science and Technology 72 (7): Development of a new wastewater treatment process for resource recovery of carotenoids, H. Sato, H. Nagare, T. N. C. Huynh and H. Komatsu, doi: 10.2166/wst.2015.330.

A new wastewater treatment process that involves coagulation, ozonation, and microalgae cultivation has been developed. Here, two challenges are discussed. The first was minimizing phosphorus removal during coagulation in order to maximize algal production. The second was to optimize microalgae cultivation; algal species that grow rapidly and produce valuable products are ideal for selection. Haematococcus pluvialis, which produces the carotenoid astaxanthin, was used. Growth rate, nutrient removal ability, and astaxanthin production of H. pluvialis in coagulated wastewater were investigated. After coagulation with chitosan, the turbidity and suspended solids decreased by 89% ± 8% and 73% ± 16%, respectively. The nitrogen and phosphorus contents of the supernatant remained at 86% ± 6% and 67% ± 24%, respectively. These results indicate that coagulation with chitosan can remove turbidity and SS while preserving nutrients. H. pluvialis grew well in the supernatant of coagulated wastewater. The astaxanthin yield from coagulated wastewater in which microalgae were cultured was 3.26 mg/L, and total phosphorus and nitrogen contents decreased 99% ± 1% and 90% ± 8% (Days 31-35), respectively.

Enhancing Photon Utilization Efficiency for Astaxanthin Production from Haematococcus lacustris Using a Split-Column Photobioreactor.

A split-column photobioreactor (SC-PBR), consisting of two bubble columns with different sizes, was developed to enhance the photon utilization efficiency in an astaxanthin production process from Haematococcus lacustris. Among the two columns, only the smaller column of SC-PBR was illuminated. Astaxanthin productivities and photon efficiencies of the SC-PBRs were compared with a standard bubble-column PBR (BC-PBR). Astaxanthin productivity of SC-PBR was improved by 28%, and the photon utilization efficiencies were 28-366% higher than the original BC-PBR. The results clearly show that the effective light regime of SC-PBR could enhance the production of astaxanthin.

Screening of Astaxanthin-Hyperproducing Haematococcus pluvialis Using Fourier Transform Infrared (FT-IR) and Raman Microspectroscopy.

Haematococcus pluvialis has promising applications owing to its ability to accumulate astaxanthin under stress conditions. In order to acquire higher astaxanthin productivity from H. pluvialis, it is critical not only to develop efficient mutagenesis techniques, but also to establish rapid and effective screening methods which are highly demanded in current research and application practice. In this work, we therefore attempted to develop a new approach to screening the astaxanthin-hyperproducing strains based on spectroscopic tools. Using Fourier transform infrared (FT-IR) and Raman microspectroscopy, we have achieved rapid and quantitative analysis of the algal cells in terms of astaxanthin, β-carotene, proteins, lipids, and carbohydrates. In particular, we have found that the ratio of the IR absorption band at 1740 cm-1 to the band at 1156 cm-1 can be utilized for identifying astaxanthin-hyperproducing strains. This work may therefore open a new avenue for developing high-throughput screening methods necessary for the microbial mutant breeding industry.

Production of the Marine Carotenoid Astaxanthin by Metabolically Engineered Corynebacterium glutamicum.

Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L−1·h−1 which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L−1, the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum.

Multiple optimization and statistical evaluation of astaxanthin production utilizing olive pomace

Solid State Fermentation (SSF) of olive pomace to astaxanthin pigment was accomplished within the frame of Box-Behnken and rotatable central composite experimental designs. Temperature, moisture content and pH for two astaxanthin producer yeasts, ATCC 24,202 (Xanthophyllomyces dendrorhous) and ATCC 24,259 (Sporidiobolus salmonicolor) were optimized at the first stage. 220.24±17.47 µg/gdp as maximum astaxanthin yield was obtained by ATCC 24,202 at 15.0 °C, 4.5 pH and 90.0% moisture content. Inoculation rate and incubation time factors of the second stage were optimized for yield and antioxidant capacity of the produced astaxanthin. Confirmation of the experimental and statistical evaluations concluded that astaxanthin production from olive pomace could be performed with a high efficient accurately.