Astaxanthin Production Research Articles

Comparative transcriptome analysis of a long-time span two-step culture process reveals a potential mechanism for astaxanthin and biomass hyper-accumulation in Haematococcus pluvialis JNU35

BackgroundAmong all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process.ResultsIn the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine–urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass.ConclusionsTo the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, β-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.

Astaxanthin production by Xanthophyllomyces dendrorhous growing on a low cost substrate

The aim of the study was to evaluate the new approaches for the reduction of food wastes, promoting industrial by-products and food waste recovery. The possibility, starting from fruit and vegetable waste (FVW), of producing astaxanthin from Xanthophyllomyces dendrorhous, in fermentative process using a batch mode was evaluated and the effect of light intensity on carotenoid biosynthesis from the yeast, has been investigated. Astaxanthin is a red oxygenated carotenoid valuable in different industries such as aquaculture, nutraceutical, pharmaceutical, and food. The results obtained in this study highlight that the growth and carotenogenesis of X. dendrorhous on FVW and on the synthetic medium, used as reference, were not suggestively different, neither for yeast growth, being 6.2 g/l and 5.3 g/l, nor for astaxanthin production, resulting 410 μg/g and 355 μg/g, respectively. This preliminary research study and show the chance for reducing waste and pollution from food and agro-industrial wastes discussing the opportunity of producing astaxanthin from X. dendrorhous using low-cost substrates.

Acidic cultivation of Haematococcus pluvialis for improved astaxanthin production in the presence of a lethal fungus

An acidic cultivation strategy was developed to prevent contamination of a lethal fungus Paraphysoderma sedebokerensis in Haematococcus pluvialis culture for astaxanthin production. Instead of generally used neutral pH, an acidic condition (pH 4) was applied to the cultivation, resulting in a significant inhibition of the fungal contamination. This could be ascribed to the acidity-associated denaturation of a surface protein of P. sedebokerensis, which plays an important role in recognition of H. pluvialis. Stress relief strategies including stepwise light irradiation and naturally occurring nitrogen deficiency were employed in the induction stage to minimize the reduction of astaxanthin production caused by acidic pH. Accordingly, an astaxanthin titer of 84.8 mg L−1 was obtained, which is 141-fold of that from the completely contaminated culture and double of that without the stress relief methods. This strategy provides a persistent contamination control method that can be used for practical astaxanthin production by H. pluvialis.

Differences between Motile and Nonmotile Cells of Haematococcus pluvialis in the Production of Astaxanthin at Different Light Intensities.

Haematococcus pluvialis, as the best natural resource of astaxanthin, is widely used in nutraceuticals, aquaculture, and cosmetic industries. The purpose of this work was to compare the differences in astaxanthin accumulation between motile and nonmotile cells of H. pluvialis and to determine the relationship between the two cells and astaxanthin production. The experiment design was achieved by two different types of H. pluvialis cell and three different light intensities for an eight day induction period. The astaxanthin concentrations in nonmotile cell cultures were significantly increased compared to motile cell cultures. The increase of astaxanthin was closely associated with the enlargement of cell size, and the nonmotile cells were more conducive to the formation of large astaxanthin-rich cysts than motile cells. The cyst enlargement and astaxanthin accumulation of H. pluvialis were both affected by light intensity, and a general trend was that the higher the light intensity, the larger the cysts formed, and the larger the quantity of astaxanthin accumulated. In addition, the relatively low cell mortality rate in the nonmotile cell cultures indicated that the nonmotile cells have a stronger tolerance to photooxidative stress. We suggest that applying nonmotile cells as the major cell type of H. pluvialis to the induction period may help to enhance the content of astaxanthin and the stability of astaxanthin production.

High-throughput label-free molecular fingerprinting flow cytometry.

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm-1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.

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 (86.54mgL-1day-1) was obtained in BBM medium. In the induction stage, the highest astaxanthin content (21.5mgg-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.

Direct extraction of astaxanthin from the microalgae Haematococcus pluvialis using liquid-liquid chromatography.

The microalgae Haematococcus pluvialis (H. pluvialis) is used for biotechnological production of the red carotenoid astaxanthin. Astaxanthin synthesis involves the formation of a rigid cell wall that impedes direct astaxanthin extraction into a solvent. During the subsequent downstream processing, the algal broth is harvested by centrifugation, dried and mechanically disrupted; finally, astaxanthin is extracted with supercritical CO2. In this study, an alternative extraction process was established, using a liquid–liquid chromatographic column to directly extract astaxanthin from the fermentation broth into a solvent. To achieve this, germination of H. pluvialis cyst cells was initiated, resulting in the release of flagellated zoospores into the fermentation broth. It was shown that astaxanthin could be extracted from the zoospores directly from the algal broth using different solvents; with ethyl acetate, yields reaching 85% were achieved in a shake-flask extraction. Using a liquid–liquid chromatographic column, astaxanthin concentrations reaching 500 mg L−1 were obtained, corresponding to eightfold concentration of the astaxanthin content in the fermentation broth. The mechanical cell disruption, drying and extraction with supercritical CO2 in the conventional astaxanthin production can be replaced by a direct astaxanthin extraction process, using a liquid–liquid chromatographic column. This allows direct astaxanthin extraction at the site of H. pluvialis production.

Bioremediation of minkery wastewater and astaxanthin production by <i>Haematococcus pluvialis</i>

A two-stage system was designed for culturing and induction processes of H. pluvialis. H. pluvialis was cultivated in minkery wastewater and compared with the conventional Bold's basal medium, and grew better in diluted (1.5%) minkery wastewater, yielding a biomass production of 906.3 ± 34.0 mg L−1. Total nitrogen and total phosphorus were also removed successfully. In the following induction stage, nitrogen-deprived vegetative cells were exposed to high light intensity for astaxanthin production, and the resultant production was 39.72 ± 1.69 mg L−1. Employing the diluted wastewater, a mixotrophic induction strategy was also tested by using a series of acetate and NaCl concentrations. The findings indicated that the optimal combination for astaxanthin production was 38.14 mM acetate and 0.58% (w/v) NaCl. And the optimal astaxanthin concentration was 67.95 ± 3.93 mg L−1 after a 12-day induction period. This study concludes that H. pluvialis offers a potential opportunity for treating minkery wastewater and producing high-value astaxanthin.

Influence of inoculum size, CO2 concentration and LEDs on the growth of green microalgae Haematococcus pluvialis Flotow

Green microalgae Haematococcus pluvialis is best known for astaxanthin production. The cultivation of H. pluvialis involves two main phases, namely green vegetative stage and cyst stage with astaxanthin accumulation. In fact, the growth of H. pluvialis in the vegetative stage is one of the most important parts in the entire cultivation process. The aim of the study is to investigate the influence of temperature, inoculum size, CO2 concentration and light emitting diodes (LEDs) on the specific growth rate, cell density and dry weight of H. pluvialis in the vegetative stage. Results indicated that the temperature from 25 to 280C and the inoculum size from 3×104 cells.ml-1 were suitable for the growth of the studied strain. Illumination with red light LEDs (630 nm) at 80 μE.m-2.s-1, the highest specific growth rate (μ) was 0.197 day-1 and the maximal density was of 2.87×105 cells.ml-1. A concentration of 5% CO2 (v/v) was the optimal dose for the growth of this strain. Under the condition of both 5% CO2 and illumination with red light LEDs at 80 μE.m-2.s-1, the specific growth rate was 0.242 day-1 and the cell density was 4.28×105cells.ml-1. Illumination with only blue LEDs (430 nm) at 120 μE.m-2.s-1 stimulated the astaxanthin accumulation with a maximum content at 2.36 μg.ml-1.

Enhancing Astaxanthin Accumulation in Haematococcus pluvialis by Coupled Light Intensity and Nitrogen Starvation in Column Photobioreactors.

Natural astaxanthin mainly derives from a microalgae producer, Haematococcus pluvialis. The induction of nitrogen starvation and high light intensity is particularly significant for boosting astaxanthin production. However, the different responses to light intensity and nitrogen starvation needed to be analyzed for biomass growth and astaxanthin accumulation. The results showed that the highest level of astaxanthin production was achieved in nitrogen starvation, and was 1.64 times higher than the control group at 11 days. With regard to the optimization of light intensity utilization, it was at 200 μmo/m²/s under nitrogen starvation that the highest astaxanthin productivity per light intensity was achieved. In addition, both high light intensity and a nitrogen source had significant effects on multiple indicators. For example, high light intensity had a greater significant effect than a nitrogen source on biomass dry weight, astaxanthin yield and astaxanthin productivity; in contrast, nitrogen starvation was more beneficial for enhancing astaxanthin content per dry weight biomass. The data indicate that high light intensity synergizes with nitrogen starvation to stimulate the biosynthesis of astaxanthin.

Astaxanthin Production by Newly Isolated Rhodosporidium toruloides: Optimization of Medium Compositions by Response Surface Methodology

Astaxanthin is a valuable carotenoid pigment, which has been extensively used in various industries. In this study, Rhodosporidium toruloides was first used as a new microbial source for producing natural astaxanthin. Various carbon, nitrogen, and mineral sources were evaluated for their effect on astaxanthin production of R. toruloides. Response surface methodology (RSM) was then used to optimize the medium compositions for maximizing the astaxanthin concentration. Among the examined nutrients, glucose, peptone, and KH2PO4 were the most efficient carbon, nitrogen, and mineral source for astaxanthin production, respectively. Through RSM, a maximum astaxanthin concentration of 927.11 µg l-1 was obtained by using Hansen broth containing 83.74 g l-1 glucose, 20.01 g l-1 peptone, and 6.19 g l-1 KH2PO4. This study suggested that R. toruloides is a promising candidate to produce natural astaxanthin.

Design scenario analysis for porous substrate photobioreactor assemblies

For commercial-scale production of microalgae, in the recent years, biofilm bioreactors have been proven to solve some of major technical problems in this field. Among different approaches, porous substrate photobioreactor (PSBR) is one of the most promising bioreactor types. However, before the actual construction of such a system and application, various design parameters need to be determined. For this purpose, mathematical modeling is considered to be an efficient tool. In the present study, a model for estimating design parameters of PSBRs is proposed. The proposed model estimates the effects of different design parameters on the productivity of PSBR systems based on (1) global solar irradiance data, (2) geographical location of the simulated system, and (3) empirical functions describing the relationship between light intensity on the module surface and module productivity, derived from experimental data. In the present study, to demonstrate the capacity of the proposed model, production of Halochlorella rubescens using a PSBR (Twin-Layer technology) was modeled for various scenarios. Also, simulated production of astaxanthin using Haematococcus pluvialis was performed. The results demonstrated the ability of the proposed model to estimate various design parameters for PSBR systems under various conditions. Based on the prediction made by the proposed model, these parameters can be individually optimized for different geographical locations and/or applications.

Genome and Transcriptome Sequencing of the Astaxanthin-Producing Green Microalga, Haematococcus pluvialis.

Haematococcus pluvialis is a freshwater species of Chlorophyta, family Haematococcaceae. It is well known for its capacity to synthesize high amounts of astaxanthin, which is a strong antioxidant that has been utilized in aquaculture and cosmetics. To improve astaxanthin yield and to establish genetic resources for H. pluvialis, we performed whole-genome sequencing, assembly, and annotation of this green microalga. A total of 83.1 Gb of raw reads were sequenced. After filtering the raw reads, we subsequently generated a draft assembly with a genome size of 669.0 Mb, a scaffold N50 of 288.6 kb, and predicted 18,545 genes. We also established a robust phylogenetic tree from 14 representative algae species. With additional transcriptome data, we revealed some novel potential genes that are involved in the synthesis, accumulation, and regulation of astaxanthin production. In addition, we generated an isoform-level reference transcriptome set of 18,483 transcripts with high confidence. Alternative splicing analysis demonstrated that intron retention is the most frequent mode. In summary, we report the first draft genome of H. pluvialis. These genomic resources along with transcriptomic data provide a solid foundation for the discovery of the genetic basis for theoretical and commercial astaxanthin enrichment.

Identification and Morphological-Physiological Characterization of Astaxanthin Producer Strains of Haematococcus pluvialis from the Black Sea Region

The molecular genetic, morphometric, and physiological-biochemical characteristics of three isolates of the carotenogenic microalga Haematococcus sp. obtained in different regions of the Caucasus and Crimea were studied. According to the results of light microscopy and PCR analysis of 18s rDNA gene fragments, all isolates were identified as Haematococcus pluvialis (clade Chlorogonia, order Volvocales, class Chlorophyceae). It is shown that the strains isolated in the mountainous and valley areas of the Black Sea region differ in important characteristics relevant for mass cultivation: cell size, growth rate, stress tolerance, mass fraction of total carotenoids and astaxanthin in dry matter, and their yield per liter of the start culture. The H. pluvialis isolates most promising for further research and the development of a cultivation technology for the Black Sea regions of the Caucasus and Crimea were strains IMBR-3 (Sevastopol) and IMBR-1 (Adler), which were characterized by the highest astaxanthin production (16.77 ± 1.32 and 17.12 ± 0.72 mg/(L day), respectively).

Tight nylon 6,6 market could persist

Dynamic flux analysis methods have been widely used for deciphering complex metabolic fluxes transients. However, many of them require frequent experimental measurements and are ineffective in dealing with under-determined metabolic reaction networks. In this study, we addressed these challenges by (i) integrating a macroscale kinetic model with its dynamic metabolic flux model to enable flux simulation over the entire time course for batch operation, and (ii) constructing a single-level mixed-integer quadratic program (MIQP) to automatically identify the shortest metabolic pathways from substrate inflow to biosynthesis of biomass and desired bioproducts. To demonstrate the advantages of the proposed framework, a X. dendrorhous fermentation process for astaxanthin production was utilised as the case study. It is found that the current framework is able to efficiently identify essential pathways and reduce the size of the original metabolic network by 70% with negligible computational cost. Furthermore, the modelling consistency, robustness, and limitation of this framework were thoroughly investigated. This research provides a new avenue for efficient in-silico design, analysis, and gene knockout of microbial strains for bioproduct synthesis.

Optimizing the localization of astaxanthin enzymes for improved productivity

BackgroundOne important metabolic engineering strategy is to localize the enzymes close to their substrates for improved catalytic efficiency. However, localization configurations become more complex the greater the number of enzymes and substrates is involved. Indeed, optimizing synthetic pathways by localizing multiple enzymes remains a challenge. Terpenes are one of the most valuable and abundant natural product groups. Phytoene, lycopene and β-carotene serve as common intermediates for the synthesis of many carotenoids and derivative compounds, which are hydrophobic long-chain terpenoids, insoluble in water and usually accumulate in membrane compartments.ResultsWhile β-ionone synthesis by β-carotene cleavage dioxygenase PhCCD1 and astaxanthin synthesis by β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) differ in complexity (single and multiple step pathways), the productivity of both pathways benefited from controlling enzyme localization to the E. coli cell membrane via a GlpF protein fusion. Especially, the astaxanthin synthesis pathway comprises both CrtW and CrtZ, which perform four interchangeable reactions initiated from β-carotene. Up to four localization strategies of CrtW and CrtZ were exhaustively discussed in this work, and the optimal positioning strategy was achieved. CrtW and CrtZ were linked using a flexible linker and localized to the membrane via a GlpF protein fusion. Enzymes in the optimal localization configuration allowed a 215.4% astaxanthin production increase.ConclusionsThis work exploits a localization situation involving membrane-bound substrates, intermediates and multiple enzymes for the first time, and provides a workable positioning strategy to solve problems in similar circumstances.

Accurate quantification of astaxanthin from Haematococcus pluvialis using DMSO extraction and lipase-catalyzed hydrolysis pretreatment

Haematococcus pluvialis accumulates high contents of astaxanthin and is considered as an important source of natural astaxanthin production. Fast and accurate quantification of astaxanthin is needed to evaluate the quality of microalgal sources for astaxanthin extraction and to monitor the H. pluvialis cultivation process. In the present study, a less-invasive method for astaxanthin accurate quantification was developed. Dimethyl sulfoxide (DMSO) was used as an extraction solvent to improve the membrane permeability of H. pluvialis. Cholesterol esterase was used to hydrolyze astaxanthin esters via incubation at 36 °C for 30 min. The detection of astaxanthin after hydrolysis was achieved by high performance liquid chromatography (HPLC) within 10 min using a C18 column. The astaxanthin content measurements after enzymatic pretreatment were 1.5–1.7 times higher than that measured after saponified pretreatment. DMSO solvent extraction combined with lipase-catalyzed hydrolysis followed by HPLC detection is proposed for the accurate quantification of astaxanthin in H. pluvialis.

Process Simulation and Techno Economic Analysis of Astaxanthin Production from Agro-Industrial Wastes

In this study, process simulation of solid state fermentation processes is used to model and evaluate the economic viability of industrial-scale facilities that produce powdered astaxanthin (PAX) and an astaxanthin oil mixture (AXO) from wheat bran (WB) and olive pomace (OP) respectively. The proposed process flowsheets are analyzed and the results from the techno-economic and sensitivity analysis are presented. Simulation results indicate that the profitability of the two processes did not change much when capacity was increased beyond 10,000 kg per batch that was the base case for study. Through a sensitivity analysis, the effect of batch size, raw material cost, labor cost and product selling price on process economics was investigated. For the base case capacity, the total capital investment for the PAX and AXO plants were estimated at $22,308,000 and $13,489,000 respectively. The payback times of PAX and AXO processes were calculated as 4.92 and 7.19 years respectively. The capacities of 4799.91 kg/batch and 6815.15 kg/batch were calculated as the break-even points for PAX and AXO productions. The techno-economic analysis demonstrated that producing AX from agro-industrial wastes is a feasible and promising technology.

Rice Bran or Apple Pomace? Comparative Data Analysis of Astaxanthin Bioproduction

Modeling and optimization of high value-added astaxanthin pigment bioproduction statistically by Sporidiobolus salmonicolor ATCC 24259 from two substantial wastes, rice bran (RB) and apple pomace (AP) was aimed in this study. The experimental data was obtained at constant inoculum rate (2%) and particle size (0.85 mm) for both wastes by conducting 17 runs, which were generated by Box-Behnken design. 33.41 µg astaxanthin gRB- and 77.31 µg astaxanthin gAP- were produced as the maximum amount at the end of fermentation period, 10 days. Apple pomace was concluded as the optimized waste for the production of astaxanthin based upon the highest yield. Predicted response results of response surface methodology (RSM) and radial basis function-neural network (RBF-NN) were compared in order to evaluate the accuracy of two methodologies on non-linear behavior of the astaxanthin bioproduction. RBF-NN became prominent with its well-suited to apple pomace fermentation system by resulting in quite low 0.8495, root mean square error (RMSE), 0.3349, mean absolute error (MAE), and 0.9985, correlation coefficient (CC) as best measures of a model performance.

Technical-economic Prefeasibility Study of Astaxanthin Production System from�H. pluvialMicroalgae in Colombia

Background: Nowadays, microalgae has been considered as source of valuable products such as astaxanthin, which is a carotenoid with great commercial potential as antioxidant widely used in many industries such as pharmaceutical, cosmetics and health supplement. Objectives: This work was focused on technical-economic pre-feasibility study of hybrid system for cultivation, harvesting and extraction of astaxanthin from H. pluvialis microalgae in five Colombian cities (Barranquilla, Cartagena, Santa Marta, Barrancabermeja and Cucuta). Methods/Analysis: A hybrid system was purposed based on flat panel + open pond Photo Bioreactor (PBR), in which cell growth is inhibited by increasing radiation. Additional stages as centrifugation, filtration, and drying, milling and supercritical extraction were also implemented. An economic prefeasibility study was applied in order to determine the suitability of locations in terms of NPV, IRR and PP. Findings: It was found that the most feasible location for astaxanthin production is Santa Marta with NPV, IRR and PP of € 5,529,203, 50% and 1.9 years, respectively. Novelty/Improvement: These results suggested that astaxanthin can satisfy national demand of antioxidants by producing it from H. pluvialis microalgae. Keywords: Antioxidant, Astaxanthin, H. pluvialis, Microalgae, Technical-economic Prefeasibility