Haematococcus Pluvialis Cells Research Articles

Influence of light absorption rate on the astaxanthin production by the microalga Haematococcus pluvialis during nitrogen starvation

Large amounts of astaxanthin (about 4% DW) can be produced under nitrogen starvation of Haematococcus pluvialis in photobioreactors (PBRs) exposed to high light conditions to induce a light stress. However, in PBR, the large biomass concentration usually achieved leads to strong light attenuation conditions, which makes complex the analysis of this “light stress”. This study aims to elucidate the role of light transfer in astaxanthin cell content and productivity from the microalga Haematococcus pluvialis during nitrogen starvation. Haematococcus pluvialis was cultivated in a flat-panel PBR in a batch mode with sudden nitrogen starvation conditions and an incident photon flux density (PFD) of 250 µmolhν m−2 s−1. Different initial biomass concentrations ({C}_{{x}_{0}}) were evaluated, 0.21, 0.52, 1.39 and 2.21 kg m−3. As a result, spectral mass absorption cross-sections of Haematococcus pluvialis were measured at different times during nitrogen starvation, and were used to relate the mean rate of photon absorption (MRPA) to the astaxanthin productivity. A minimum initial MRPA of 7000 ± 500 µmolhν kgx−1 s−1 was found necessary to trigger large accumulation of astaxanthin in Haematococcus pluvialis cells (up to 3.21% DW) during nitrogen starvation conditions. The results also demonstrated the link between the MRPA and the daily astaxanthin productivity of Haematococcus pluvialis cultures, introducing then the MRPA as a physical quantity of interest for a rational optimization of the light culture conditions in PBRs.Graphical

Multispectral Imaging Flow Cytometry with Spatially and Spectrally Resolving Snapshot-Mosaic Cameras for the Characterization and Classification of Bioparticles.

In the development and optimization of biotechnological cultivation processes the continuous monitoring through the acquisition and interpretation of spectral and morphological properties of bioparticles are challenging. There is therefore a need for the parallel acquisition and interpretation of spatially and spectrally resolved measurements with which particles can be characterized and classified in-flow with high throughput. Therefore, in this paper we investigated the scientific and technological connectivity of standard imaging flow cytometry (IFC) with filter-on-chip based spatially and spectrally resolving snapshot-mosaic cameras for photonic sensing and control in a smart and innovative microfluidic device. For the investigations presented here we used the microalgae Haematococcus pluvialis (HP). These microalgae are used commercially to produce the antioxidant keto-carotenoid astaxanthin. Therefore, HP is relevant to practically demonstrate the usability of the developed system for Multispectral Imaging Flow Cytometry (MIFC) platform. The extension of standard IFC with snapshot-mosaic cameras and multivariate data processing is an innovative approach for the in-flow characterization and derived classification of bioparticles. Finally, the multispectral data acquisition and the therefore developed methodology is generalizable and enables further applications far beyond the here characterized population of HP cells.

E/Z isomerization of astaxanthin and its monoesters in vitro under the exposure to light or heat and in overilluminated Haematococcus pluvialis cells

Thermo- and photoisomerization of astaxanthin was investigated in a model system (solutions in methanol and chloroform), and the dynamics of astaxanthin isomers and esters content was analyzed in Haematococcus pluvialis green algal cells exposed to factors inducing astaxanthin accumulation. In both systems, the astaxanthin isomerization process seems to be defined by a) the action of light (or heat), and b) the dielectric constant of the surrounding medium. Upon heating, the accumulation of Z-isomers occurred in a model system during the entire incubation period. For the first 5 h of illumination, both Z-isomers accumulated in the solutions up to 5%, and then their content decreased. The accumulated amount of the Z-isomers in the cells of H. pluvialis was found to reach 42% of the total content of astaxanthin initially, and then it decreased during the experiment. The results lead to a conclusion that both cultivation of H. pluvialis culture in specific conditions and heat treatment of the resulting extracts from it might be efficient for obtaining large amounts of economically useful astaxanthin Z-isomer.

Quantitative detection on metabolites of Haematococcus pluvialis by terahertz spectroscopy

Stressed Haematococcus Pluvialis cells would accumulate astaxanthin with the changes of lipid, pigment, and starch, which recently has been widely studied. Real-time dynamic monitoring of these components will have important guiding significance for screening high-yield and high-quality strains in the industry. Terahertz spectroscopy is not only suitable for measuring large flux samples, but also widely used for quantitative analysis of multicomponent mixtures. To investigate the potential of using terahertz spectroscopy to monitor and quantify metabolites in the algae, terahertz spectrum of Haematococcus Pluvialis under nitrogen starvation were captured and examined against those standard reagents of β-carotene, astaxanthin, and starch. The correlation between intensities of the characteristic peaks and the metabolite contents which were quantified by chemical analysis was established, and the results showed the correlation coefficient were higher than 0.92, indicating a relatively accurate performance to predict the metabolites concentration. Furthermore, to identify meaningful spectra range for quantification, the partial least squares combined with intensities of the interval characteristic peaks were established, where it demonstrated an accurate model with high prediction correlation coefficients (greater than0.98), low root-mean-square error of prediction (RMSEP), and high residual predictive deviation. The results approve the feasibility of using terahertz spectroscopy for synchronous detection on metabolites concentration in the microalgae production process.

Three step flow focusing enables image-based discrimination and sorting of late stage 1 Haematococcus pluvialis cells.

Label-free and gentle separation of cell stages with desired target properties from mixed stage populations are a major research task in modern biotechnological cultivation process and optimization of micro algae. The reported microfluidic sorter system (MSS) allows the subsequent investigation of separated subpopulations. The implementation of a viability preserving MSS is shown for separation of late stage 1 Haematococcus pluvialis (HP) cells form a mixed stage population. The MSS combines a three-step flow focusing unit for aligning the cells in single file transportation mode at the center of the microfluidic channel with a pure hydrodynamic sorter structure for cell sorting. Lateral displacement of the cells into one of the two outlet channels is generated by piezo-actuated pump chambers. In-line decision making for sorting is based on a user-definable set of image features and properties. The reported MSS significantly increased the purity of target cells in the sorted population (94%) in comparison to the initial mixed stage population (19%).

The influence of Rose Bengal photosensitizer on the productivity and pigment composition of Haematococcus pluvialis

The stimulation of astaxanthin synthesis in Haematococcus pluvialis cells under high-intensity light in the combined action of Rose Bengal photosensitizer has been established. It was revealed that when Rose Bengal was used as an additional inducer of carotenogenesis in high-intensity light, the dry weight of the Haematococcus and the diameter of the cells in suspension increase compared to the action of only high-intensity light on algae cells. The observed increases in dry weight are significant and reach 40 % excess over control. We suggest that the increase in dry weight, Haematococcus cell diameter and astaxanthin yield when H. pluvialis photosensitizer is added to the incubation medium against the background of high-intensity light is not associated with increased stress (since the used concentrations of Rose Bengal are too small to induce photo-oxidative stress), but the signaling properties of the generated Rose Bengal singlet oxygen, which may be the primary agent in the transduction of a signal that triggers increased synthesis of astaxanthin in H. pluvialis cells.

Chlororespiration protects the photosynthetic apparatus against photoinhibition by alleviating inhibition of photodamaged-PSII repair in Haematococcus pluvialis at the green motile stage

During outdoor mass cultivation, Haematococcus pluvialis cells, especially at the green motile stage, are frequently exposed to high light. However, the photoprotective mechanisms in H. pluvialis have not been well characterized. This study aimed to confirm the photoprotection of chlororespiration in H. pluvialis at the green motile stage. Under high light, inhibition of plastid terminal oxidase (PTOX), a key enzyme of chlororespiration, increased the relative variable fluorescence at the I-step (VI), causing a decrease in the plastoquinone (PQ) pool, which further led to photosystem II (PSII) acceptor side over-reduction. This condition disturbed the balance between light absorption and energy utilization, causing the over-excitation of PSII reaction centers, which was indicated by the fact that the inhibition of PTOX increased the relative variable fluorescence at the J-step (VJ) and the light absorption flux per active reaction center (ABS/RC), but decreased the electron transport flux per active reaction center (ETo/RC) in H. pluvialis. The over-reduction of the PSII acceptor side and the over-excitation of PSII reaction centers inevitably caused more severe photoinhibition as shown by a decrease in Fv/Fm (maximal photochemical efficiency of PSII). Furthermore, the level of photoinhibition was not changed when the PTOX was inhibited in the presence of the photodamaged-PSII repair inhibitor chloramphenicol, indicating that the inhibition of PTOX did not accelerate the photodamage to PSII directly. The above results suggest that the chlororespiration pathway protects H. pluvialis at the green motile stage against photoinhibition by decreasing the inhibition of the photodamaged-PSII repair mechanism.

Astaxanthin accumulation in Haematococcus pluvialis cells induced by nitrogen deficiency and high light intensity

It was established that nitrogen deficiency initiated at the resting phase of Haematococcus pluvialis (H. pluvialis) is not an effective stress factor in combination with high light intensity to induce the accumulation of astaxanthin in algae cells. The amounts of astaxanthin in H. pluvialis cells in the absence of nitrogen were lower compared to those when complete Rudic medium was used, regardless of the light intensity. This fact indicates the need for a certain amount of nitrogen in the medium for the functioning of the astaxanthin synthesis system. In addition, the low yield of astaxanthin in the experimental variants cultivated on incomplete Rudic medium is associated with the insufficient amount of nitrogen for its biosynthesis and also with a decrease in the expression level of the PSY gene encoding a key enzyme of the carotenoid biosynthesis chain – phytoene synthase.

Incorporation of Magnetic Nanoparticles into Protoplasts of Microalgae Haematococcus pluvialis: A Tool for Biotechnological Applications.

Intensive research on the use of magnetic nanoparticles for biotechnological applications of microalgae biomass guided the development of proper treatment to successfully incorporate them into these single-cell microorganisms. Protoplasts, as cells lacking a cell wall, are extensively used in plant/microalgae genetic manipulation as well as various biotechnological applications. In this work, a detailed study on the formation of protoplasts from Haematococcus pluvialis with the use of enzymatic and mechanical procedures was performed. The optimization of several parameters affecting the formation of protoplasmic cells and cell recovery was investigated. In the enzymatic treatment, a solution of cellulase was studied at different time points of incubation, whereas in the mechanical treatment, glass beads vortexing was used. Mechanical treatment gave better results in comparison to the enzymatic one. Concerning the cell recovery, after the protoplast formation, it was found to be similar in both methods used; cell viability was not investigated. To enhance the protoplast cell wall reconstruction, different “recovery media” with an organic source of carbon or nitrogen were used. Cell morphology during all treatments was evaluated by electron microscopy. The optimal conditions found for protoplast formation and cell reconstruction were successfully used to produce Haematococcus pluvialis cells with magnetic properties.

Hydrothermal synthesis of novel two-dimensional α-quartz nanoplates and their applications in energy-saving, high-efficiency, microalgal biorefineries

A facile template-free hydrothermal method was successfully developed for the controlled synthesis of ultrathin α-quartz nanoplates (NPLs) for the first time. Analyses of the α-quartz NPLs revealed the characteristic anisotropic nanostructures of highly crystalline α-quartz with an average lateral size and thickness of 1.14 ± 0.32 μm and 7.7 ± 0.6 nm, respectively. Importantly, efficient extraction of highly valuable chemicals such as astaxanthin (ATX) from the microalgal cells of Haematococcus pluvialis (H. pluvialis) was accomplished by lacerating the cell walls using the ultrathin α-quartz NPLs under mild ultrasonication. The incorporation of α-quartz NPLs enhanced the ATX extraction efficiency significantly (99%, 18.0 ± 0.6 mg ATX/g cell) when coupled with 5 min of ultrasonication. The dosage of α-quartz NPLs (800.0 mg/L) for maximum ATX extraction was reduced substantially to only 8% of the nanomaterial dose used in the extraction controls. The enhanced extraction efficiency and dosage were explained by the role of the structural anisotropy of α-quartz NPLs in multiple phase separation-extraction processes. This work provides a novel, facile, and economical route for the synthesis of uniform ultrathin nanoplates, offering a new material for the highly efficient harvesting of chemical products from various microalgal biorefinery processes.

Effect of plant growth regulators on growth and lipid accumulation of microalgal Haematococcus pluvialis Flotow in two-stage culture

Haematococcus pluvialis cells were cultured in aerated liquid Bold’s Basal medium in two-stage (initial stage during in 7 weeks for increased biomass growth and second stage during in 3 weeks for increased lipid accumulation) with different volumes 250 mL, 10 L, and 1,000 L. With a volume of 250 mL, the medium was supplied with benzyl adenine (BA), indole-3-acetic acid (IAA) or gibberellic acid (GA3) at concentration from 0.1 - 0.2 mg/L in initial stage and IAA or GA3 at concentration from 0.1 - 0.2 mg/L in second stage. After 10 weeks of culture, results showed that supplement of 0.1 mg/L BA in initial stage and 0.125 mg/L IAA in second stage increased cell density, and microalgal cells had green color with a spherical shape. On the contrary, supplement of 0.15 mg/L IAA in initial stage and 0.175 mg/L GA3 in second stage increased lipid accumulation, and microalgal cells had red color with a spherical shape. With a volume of 10 L, the medium was supplied with 0.1 mg/L BA in initial stage, and treated with separation or combination from 2 - 3 of these factors (nitrogen starvation, 0.5% NaCl, 4.98 mg/L FeSO4) were applied in second stage. The result showed that the cultures was treated with nitrogen starvation increased dry biomass and biofuel, but treated with 4.98 mg/L FeSO4 only increased biofuel. With a volume of 1,000 L, microalgal cells were cultured in BB liquid medium in initial stage, and treated with 4.98 mg/L FeSO4 increased fresh 78.67 mg/mL and dry biomass 2.05 mg/L and total lipid content 28.24 %/ DW.

Antioxidant enzymes and the mitochondrial alternative oxidase pathway play important roles in chilling tolerance of Haematococcus pluvialis at the green motile stage

During outdoor large-scale cultivation, Haematococcus pluvialis cells, especially at the green motile stage, are frequently exposed to chilling stress in winter. However, the physiological responses and adaptation mechanisms to chilling in H. pluvialis have not been characterized. In order to better understand the chilling tolerance mechanisms of H. pluvialis in the green motile stage, the responses of photosynthetic characteristics and photoprotective mechanisms to chilling were investigated. Chilling stress significantly decreased the activities of key enzymes (ribulose-1,5-bisphosphate carboxylase/oxygenase and NADP glyceraldehyde-3-phosphate dehydrogenase) in photosynthetic carbon assimilation, which would cause the accumulation of excess reducing equivalents and an imbalance of light absorption and energy utilization, leading to more severe photoinhibition. After chilling treatment, there was no induction of cyclic electron flow, non-photochemical quenching, fluorescence emission, or catalase, indicating these factors do not protect H. pluvialis from photoinhibition. However, chilling significantly enhanced the activities of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase), which efficiently dissipated excess electrons generated by photosynthetic linear electron flow (LEF). The malate-oxaloacetate shuttle was activated and the mitochondrial alternative oxidase (AOX) pathway was significantly up-regulated after chilling, indicating the transport of excess reducing equivalents generated by photosynthetic LEF to cytosol and mitochondria and oxidization by the AOX pathway, the mitochondrial non-phosphorylating pathway. As a result, the LEF was not inhibited by negative feedback and the H. pluvialis cells were well protected under chilling stress in the light, with no change in the effective quantum yield of PSII (ΦPSII) and the yield of non-regulated energy dissipation of PSII (ΦNO). The results indicate that antioxidant enzymes and the mitochondrial AOX pathway play important roles in protecting H. pluvialis at the green motile stage against chilling stress.

The pigment composition of <i>Haematococcus pluvialis</i> microalgae cells upon cultivated on biogenic-deficient media

The accumulation of carotenoids (carotenogenesis) in the cells of Haematococcus pluvialis (strain IBCE H-17) upon cultivation in nutrient media deficient in nutrient elements (N and K + P) is shown. K and P deficiency in nutrient medium led to accumulation of astaxanthin, β-carotene, anteraxanthin, zeaxanthin and violaxanthin after 18 days of cultivation, while the amount of neoxanthin and lutein was at the control level. Carotenogenesis under such conditions was accompanied by high levels of chlorophyll (a + b). The use of nutrient medium deficient in N led to the accumulation of astaxanthin and, to a lesser extent, β-carotene in haematococcus cells, while lutein, anteraxanthin, zeaxanthin, violaxanthin and neoxanthin levels were lower or the same as control. N deficiency caused the decrease in amount of chlorophyll (a + b). Nitrogen deficiency was 4.3 times more effective than control medium and 1.8 times more effective than combined potassium and phosphorus deficiency in promoting astaxanthin accumulation.

The Effect of the Solvent Nature and Lighting on Isomerization and Oxidative Degradation of Astaxanthin

Astaxanthin and its esters, which are classified as secondary carotenoids, are synthesized under stress conditions in Haematococcus pluvialis cells that undergo transformation from vegetative cells to hematocysts. Stress factors can affect not only the rate and the direction of synthesis, but also the conformational changes in the pigment molecule, causing conversion of trans-isomers to cis-isomers. The aim of this study was to evaluate the effects of different factors (the polarity of the medium and light) on the process of trans–cis isomerization of astaxanthin. Investigation of the formation of 9-cis- and 13-cis-isomers from all-trans astaxanthin solutions at 50°C showed that medium polarity solvents stabilized cis-isomers more efficiently than highly polar solvents. Nonpolar solvents did not contribute to the stabilization of cis-isomers. The presence of π-bonds in the solvent molecule also stimulated isomerization. Analysis of the effects of the solvent refractive index on polarizability of astaxanthin revealed differences between the dependencies for polar and nonpolar solvents. Supposedly, solvents of medium polarity facilitate the process of trans–cis astaxanthin isomerization by decreasing the energy of the torque and stabilizing the cis-conformation of the molecule due to diminished dipole–dipole repulsion between the methyl groups in the polyene chain and the ionone ring. Exposure of astaxanthin solutions to light at 25°C induced formation of cis-isomers, which were more abundant within the first 4 h of exposure than after 6 h or later. Exposure to irradiation for longer periods caused astaxanthin decomposition and formation of degradation and oxidation products.

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.

A semi-continuous cultivation method for Haematococcus pluvialis from non-motile cells to motile cells

Non-motile cells of Haematococcus pluvialis grow slowly, whereas motile cells grow fast and divide frequently. Cultivation from non-motile cells to motile cells of H. pluvialis was implemented to promote semi-continuous production. When old cultures which consist of non-motile cells were inoculated in fresh medium with an inoculation amount less than 15%, zoospores were produced in the non-motile cells and developed into motile cells, as the concentration of astaxanthin inducer in the medium was below the threshold value. This process was accomplished within 3 days after inoculation. Furthermore, enhancing KNO3 content to 1200 mg L−1 or reducing light intensity to 20 μmol photons m−2 s−1 could increase growth during the late culturing period of H. pluvialis and postpone the next round of transformation from motile cells to non-motile cells. A semi-continuous cultivation method for H. pluvialis from non-motile cells to motile cells is proposed in order to regulate the life cycle and promote industrial production. This cultivation mode shortens the inoculum cultivation stage and simplifies the production process of H. pluvialis, showing considerable commercial potential.

Production of stable food-grade microencapsulated astaxanthin by vibrating nozzle technology.

Astaxanthin is a carotenoid known for its strong antioxidant and health-promoting characteristics, but it is also highly degradable and thus unsuited for several applications. We developed a sustainable method for the extraction and the production of stable astaxanthin microencapsulates. Nearly 2% astaxanthin was extracted by high-pressure homogenization of dried Haematococcus pluvialis cells in soybean oil. Astaxanthin-enriched oil was encapsulated in alginate and low-methoxyl pectin by Ca2+-mediated vibrating-nozzle extrusion technology. The 3% pectin microbeads resulted the best compromise between sphericity and oil retention upon drying. We monitored the stability of these astaxanthin beads under four different conditions of light, temperature and oxygen exposition. After 52weeks, the microbeads showed a total-astaxanthin retention of 94.1±4.1% (+4°C/-light/+O2), 83.1±3.2% (RT/-light/-O2), 38.3±2.2% (RT/-light/+O2), and 57.0±0.4% (RT/+light/+O2), with different degradation kinetics. Refrigeration, therefore, resulted the optimal storage condition to preserve astaxanthin stability.

Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back.

The engagement of different photoprotective mechanisms in the cells of the carotenogenic astaxanthin-accumulating chlorophyte Haematococcus pluvialis (i) under favorable conditions, (ii) in the course of stress-induced haematocyst formation and (iii) during recovery from the stress was studied. To this end, we followed the changes in primary photochemistry, electron flow at the acceptor side of photosystem II, and non-photochemical quenching (NPQ) using PAM chlorophyll fluorimetry. A general trend recorded in the stressed cells undergoing transition to haematocysts (and reversed during recovery from the stress) was a gradual reduction of the photosynthetic apparatus accompanied by down-regulation of energy-dependent photoprotective mechanisms such as NPQ, along with the accumulation of astaxanthin. On this background, a transient up-regulation of the photosynthetic activity was detected at the intermediated stages (20-50h of the stress exposure) of haematocyst formation. This phenomenon was tentatively related with the peak of metabolic activity found earlier in the forming haematocysts. The role of secondary carotenogenesis coupled with a reversible transition from 'active' (energy-dependent) to 'passive' photoprotective mechanisms in the extremely high stress tolerance of carotenogenic phototrophs is discussed.

Systemic Statistical Optimization of Astaxanthin Inducing Methods in Haematococcus pluvialis cells -Statistical Optimization of Astaxanthin Production in Haematococcus

The production of astaxanthin in the microalga Haematococcus pluvialis has been investigated using a sequential methodology based on the application of two types of statistical designs. The employed preliminary experiment was a fractional factorial design <TEX>$2^6$</TEX> in which the factors studied were: excessive irradiance and nitrate starvation, phosphate deficiency, acetate supplementation, salt stress, and elevated temperature. The experimental results indicate that the amount of astaxanthin accumulation in the cells can be enhanced by excessive irradiance and nitrate starvation whereas the other factors tested did not yield any enhancement. In the subsequent experiment, a central composite design was applied with four variables, light intensity, nitrate, phosphate, and acetate, at five levels each. The optimal conditions for the highest astaxanthin production were found to be <TEX>$1040{\mu}E/(m^2{\cdot}s)$</TEX> light intensity, 0.04 g/L nitrate, 0.31 g/L phosphate, 0.05 g/L acetate concentration.

Carotenogênese em células de Haematococcus pluvialis induzidas pelos estresses luminoso e nutricional

O objetivo deste trabalho foi avaliar as respostas das células de Haematococcus pluvialis ao processo de indução à carotenogênese, sob estresse luminoso e nutricional. As células foram aclimatadas durante 15 dias em meio WC, com aeração com ar atmosférico sintético filtrado e fluxo de 100 mL min-1, intensidade luminosa de 50 µmol fótons m-2 s-1, fotoperíodo de 12 horas e temperatura de 23ºC. Foram comparados dois tratamentos: cultivo nas condições descritas, mas com aumento da intensidade luminosa para 350 µmol fótons m-2 s-1 ; e cultivo nas mesmas condições do tratamento anterior, mas com aeração contendo 4% de CO2. Os tratamentos foram conduzidos em triplicata, durante dez dias. Com a adição de CO2 e o incremento da iluminação, observou-se aumento da razão carotenoides/clorofila e da biomassa celular. As células cessaram a divisão no segundo dia de estresse, quando o nitrato se tornou limitante, e aumentaram significativamente seu biovolume. A excreção de carbono orgânico e a concentração de astaxantina aumentam em resposta à adição de CO2. O estresse por intensidade luminosa, aliado à adição de CO2, otimiza a carotenogênese em H. pluvialis e aumenta a produção de astaxantina.