Pluvialis Cells Research Articles

Continuously tunable separation of light-induced Haematococcus pluvialis using an ultrastretchable, sheath-flow-assisted elasto-inertial microchannel

BackgroundA proportion of Haematococcus pluvialis under the light stress can effectively conduct astaxanthin biosynthesis, leading to the increase in cell size. Although the size is a critical indicator for identifying the astaxanthin-rich H. pluvialis cells, the cut-off size to be separated varies from sample to sample. ResultsHere, we report an ultrastretchable, straight elasto-inertial microchannel with tunable separation threshold to continuously separate the light-induced H. pluvialis cells by size. The symmetrical sheath flows confine the particles to the channel sidewalls, and large particles can cross the interface of viscoelastic fluids to the equilibrium position at the channel centerline. By stretching the microfluidic chip, the medium-sized particles can gradually migrate to the channel centerline in the narrower and longer channel, bringing the tunable separation threshold. Results show that the separation performance of the ultrastretchable microfluidic device is affected by total flow rate, flow rate ratio of sheath to sample, polyethylene oxide (PEO) solution configuration. Lastly, size-tunable separation of light-induced H. pluvialis cells is demonstrated. SignificanceTo the best of our knowledge, this is the first report on cell migration in co-flow configurations in the ultra-stretchable microfluidics. Separation of H. pluvialis is not only a relevant end application in harvesting the astaxanthin-rich species, but the separated populations of highly productive microalgal cells will open a venue for cellular directed evolution.

Comparative Proteome Profiling of Extracellular Vesicles from Three Growth Phases of Haematococcus pluvialis under High Light and Sodium Acetate Stresses.

Extracellular vesicles (EVs) are nano-sized particles involved in intercellular communications that intrinsically possess many attributes as a modern drug delivery platform. Haematococcus pluvialis-derived EVs (HpEVs) can be potentially exploited as a high-value-added bioproduct during astaxanthin production. The encapsulation of HpEV cargo is a crucial key for the determination of their biological functions and therapeutic potentials. However, little is known about the composition of HpEVs, limiting insights into their biological properties and application characteristics. This study examined the protein composition of HpEVs from three growth phases of H. pluvialis grown under high light (350 µmol·m-2·s-1) and sodium acetate (45 mM) stresses. A total of 2038 proteins were identified, the majority of which were associated with biological processes including signal transduction, cell proliferation, cell metabolism, and the cell response to stress. Comparative analysis indicated that H. pluvialis cells sort variant proteins into HpEVs at different physiological states. It was revealed that HpEVs from the early growth stage of H. pluvialis contain more proteins associated with cellular functions involved in primary metabolite, cell division, and cellular energy metabolism, while HpEVs from the late growth stage of H. pluvialis were enriched in proteins involved in cell wall synthesis and secondary metabolism. This is the first study to report and compare the protein composition of HpEVs from different growth stages of H. pluvialis, providing important information on the development and production of functional microalgal-derived EVs.

Mitochondrial alternative oxidase pathway accelerates non-motile cell germination by enhancing respiratory carbon metabolism and maintaining redox poise in Haematococcus pluvialis

Low efficiency of the cultivation process is a major obstacle in the commercial production of Haematococcus pluvialis. Germination of red, non-motile cells is an efficient strategy for rapid acquisition of zoospores. However, the regulatory mechanisms associated with germination remain unexplored. In the present study, it was confirmed that the mitochondrial alternative oxidase (AOX) pathway accelerates H. pluvialis cell germination, and the regulatory mechanisms were clarified. When the AOX pathway was inhibited, the transcriptomic and metabonomic data revealed a downregulation in respiratory carbon metabolism and nucleotide synthesis due to NADH accumulation. This observation suggested that AOX promoted the rapid consumption of NADH, which accelerated carbohydrate and lipid catabolism, thereby producing carbon skeletons for DNA replication through respiratory metabolism. Moreover, AOX could potentially enhance germination by disturbing the abscisic acid signaling pathway. These findings provide novel insights for developing industrial cultivation models based on red-cell-germination for achieving rapid proliferation of H. pluvialis.

Extracellular vesicles involved in growth regulation and metabolic modulation in Haematococcus pluvialis

BackgroundMicroalgae-derived extracellular vesicles (EVs), which transfer their cargos to the extracellular environment to affect recipient cells, play important roles in microalgal growth and environmental adaptation. And, they are also considered as sustainable and renewable bioresources of delivery nanocarrier for bioactive molecules and/or artificial drug molecules. However, their molecular composition and functions remain poorly understood.ResultsIn this study, isolation, characterization, and functional verification of Haematococcus pluvialis-derived EVs (HpEVs) were performed. The results indicated that HpEVs with typical EV morphology and size were secreted by H. pluvialis cells during the whole period of growth and accumulated in the culture medium. Cellular uptake of HpEVs by H. pluvialis was confirmed, and their roles in regulation of growth and various physiological processes of the recipient cells were also characterized. The short-term inhibition of HpEV secretion results in the accumulation of functional cellular components of HpEVs, thereby altering the biological response of these cells at the molecular level. Meanwhile, continuously inhibiting the secretion of HpEVs negatively influenced growth, and fatty acid and astaxanthin accumulation in H. pluvialis. Small RNA high-throughput sequencing was further performed to determine the miRNA cargoes and compelling details in HpEVs in depth. Comparative analysis revealed commonalities and differences in miRNA species and expression levels in three stages of HpEVs. A total of 163 mature miRNAs were identified with a few unique miRNAs reveal the highest expression levels, and miRNA expression profile of the HpEVs exhibited a clear stage-specific pattern. Moreover, a total of 12 differentially expressed miRNAs were identified and their target genes were classified to cell cycle control, lipid transport and metabolism, secondary metabolites biosynthesis and so on.ConclusionIt was therefore proposed that cargos of HpEVs, including miRNA constituents, were suggested potential roles in modulate cell physiological state of H. pluvialis. To summarize, this work uncovers the intercellular communication and metabolism regulation functions of HpEVs.

On-Chip Photoacoustics-Activated Cell Sorting (PA-ACS) for Label-Free and High-Throughput Detection and Screening of Microalgal Cells.

Microalgae play a crucial role in global carbon cycling as they convert carbon dioxide into various valuable macromolecules. Among them, Haematococcus pluvialis (H. pluvialis) is the richest natural source of astaxanthin (AXT), which is a valuable antioxidant, anti-inflammatory, and antiapoptosis agent. These benefits make AXT highly commercially valuable in pharmaceuticals, cosmetics, and nutritional industries. However, intrinsic genetic characteristics and extrinsic cultivation conditions influence biomass gains, leading to low productivity and extraction as the main techno-economic bottlenecks in this industry. Thus, detecting AXT in H. pluvialis is essential to determine the influence of multiple parameters on biocompound accumulation, enabling optimization of cultivation and enrichment of AXT-rich H. pluvialis cells. This work developed an opto-acousto-fluidic microplatform for detection, analysis, and sorting of microalgae. Via label-free monitoring and extraction of sample-induced ultrasonic signals, a photoacoustic microscopic system was proposed to provide a full-field visualization of AXT's content and distribution inside H. pluvialis cells. When employed as on-chip image-based flow cytometry, our microplatform can also offer high-throughput measurements of intracellular AXT in real time, which demonstrates similar results to conventional spectrophotometry methods and further reveals the heterogeneity of AXT content at the single-cell level. In addition, a solenoid valve-pump dual-mode cell sorter was integrated for effective sorting of cells with a maximum working frequency of 0.77 Hz, reducing the fluid response time by 50% in rising and 40-fold in recovery. The H. pluvialis cells which have more AXT accumulation (>30 μm in diameter) were 4.38-fold enriched with almost no dead empty and small green cells. According to the results, automated and reliable photoacoustics-activated cell sorting (PA-ACS) can screen AXT-rich cells and remove impurities at the terminal stage of cultivation, thereby increasing the effectiveness and purity of AXT extraction. The proposed system can be further adopted to enrich strains and mutants for the production of biofuels or other rare organic substances such as β-carotene and lutein.

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

Ultrasonic Treatment Enhanced Astaxanthin Production of Haematococcus pluvialis.

In this study, effects of ultrasonic treatment on Haematococcus pluvialis (H. pluvialis) were investigated. It has been confirmed that the ultrasonic stimulation acted as stress resources in the red cyst stage H. pluvialis cells containing astaxanthin, resulting in additional astaxanthin production. With the increase in production of astaxanthin, the average diameter of H. pluvialis cells increased accordingly. In addition, to determine how ultrasonic stimulation had an effect on the further biosynthesis of astaxanthin, genes related to astaxanthin synthesis and cellular ROS level were measured. As a result, it was confirmed that astaxanthin biosynthesis related genes and cellular ROS levels were increased, and thus ultrasonic stimulation acts as an oxidative stimulus. These results support the notion on the effect of the ultrasonic treatment, and we believe our novel approach based on the ultrasonic treatment would help to enhance the astaxanthin production from H. pluvialis.

Extraction of Valuable Biomolecules from the Microalga Haematococcus pluvialis Assisted by Electrotechnologies.

The freshwater microalga Haematococcus pluvialis is well known as the cell factory for natural astaxanthin, which composes up to 4-7% of its total dry weight. The bioaccumulation of astaxanthin in H. pluvialis cysts seems to be a very complex process that depends on different stress conditions during its cultivation. The red cysts of H. pluvialis develop thick and rigid cell walls under stress growing conditions. Thus, the biomolecule extraction requires general cell disruption technologies to reach a high recovery rate. This short review provides an analysis of the different steps in H. pluvialis's up and downstream processing including cultivation and harvesting of biomass, cell disruption, extraction and purification techniques. Useful information on the structure of H. pluvialis's cells, biomolecular composition and properties and the bioactivity of astaxanthin is collected. Special emphasis is given to the recent progress in application of different electrotechnologies during the growth stages and for assistance of the recovery of different biomolecules from H. pluvialis.

Optimization of cell wall breaking process of the microalgae Haematococcus pluvialis.

Haematococcus pluvialis can synthesize and accumulate astaxanthin, and is regarded as the organism with the highest content of astaxanthin in the current nature. However, only when Haematococcus pluvialis cell wall is broken can the astaxanthin be effectively extracted. Therefore, the breaking of the hard cell wall of Haematococcus pluvialis becomes the key difficulty hindering the extraction of astaxanthin. In this study, the cell wall was broken using enzyme digestion and mechanical action and the response surface methodology was used to optimize the cell wall breaking process. Taking the cell wall breaking rate of Haematococcus pluvialis as the index, the single factor experiment was used to calculate the effects of solid-liquid ratio, enzyme type and dosage, digestion temperature, digestion pH and hydrolysis time on the cell wall breaking rate. When the ultrasonic power is 175 W and the ultrasonic time is 2 min, the highest wall breaking rate is 68.8%. The high pass tissue grinder has the best wall breaking efficiency of 70.3% in 6 min and 70 Hz. In combination with single factor analysis and response surface analysis, the optimum enzyme digestion conditions were determined as follows: the ratio of material to liquid was 1:150, the amount of enzyme was 1%, the ratio of Pectinase to cellulase was 1:1, and the optimum hydrolysis conditions were as follows: hydrolysis time 5 h, hydrolysis temperature 55 °C, pH 5.5; and the amount of enzyme was 1%. The theoretical wall breaking rate is 77.2%, the actual extraction rate is 75.4%, and the relative error is 2.2%. In conclusion, the method of using combined enzyme to break the wall of Haematococcus pluvialis is simple, mild, green and efficient. It can be used in the processing of broken wall.

Development and validation of reliable astaxanthin quantification from natural sources.

Astaxanthin derived from natural sources occurs in the form of various esters and stereomers, which complicates its quantitative and qualitative analysis. To simplify and standardize astaxanthin measurement with high precision, an enzymolysis-based astaxanthin quantification method was developed to hydrolyze astaxanthin esters and determine free astaxanthin in all its diastereomeric forms. Astaxanthin standards and differently processed Haematococcus pluvialis biomass were investigated. Linear correlation of standards of all-E-astaxanthin was observed in a measurement range between extract concentrations of 1.0 μg/mL and 11.2 μg/mL with a coefficient of variation below 5%. The diastereomers 9Z-, and 13Z-astaxanthin, and two di-Z-forms were detected. In contrast to the measurement of standards, the observed measurement range was extended to 30 μg/mL in extracts from H. pluvialis. The nature of the sample had to be taken into account for measurement, as cell, respectively, sample composition altered the optimal concentration for astaxanthin determination. The measurement precision of all-E-astaxanthin quantification in dried H. pluvialis biomass (1.2-1.8 mg dried biomass per sample) was calculated with a coefficient of variation of maximum 1.1%, whereas it was below 10% regarding the diastereomers. Complete enzymolysis was performed with 1.0 to 2.0 units of cholesterol esterase in the presence of various solvents with up to 2.0 mg biomass (dry weight). The method was compared with other astaxanthin determination approaches in which astaxanthin is converted to acetone in a further step before measurement. The developed method resulted in a higher total astaxanthin recovery but lower selectivity of the diastereomers. The reliability of photometric astaxanthin estimations was assessed by comparing them with the developed chromatographic method. At later stages in the cell cycle of H. pluvialis, all methods yielded similar results (down to 0.1% deviation), but photometry lost precision at earlier stages (up to 31.5% deviation). To optimize sample storage, the shelf life of astaxanthin-containing samples was investigated. Temperatures below -20°C, excluding oxygen, and storing intact H. pluvialis cells instead of dried or disrupted biomass reduced astaxanthin degradation.

A strategy for interfering with the formation of thick cell walls in Haematococcus pluvialis by down-regulating the mannan synthesis pathway

The challenges associated with effective cell wall disruption remain an important bottleneck that has restricted efforts to extract astaxanthin from Haematococcus pluvialis. Here, available transcriptomic data and an Agrobacterium tumefaciens-mediated transformation system were used to establish an H. pluvialis strain in which the key cell wall formation-related enzyme α-1,6-mannosyltransferase (HpOCH1) was downregulated in an effort to thin cell walls and thereby simplify the astaxanthin extraction process. The cell wall remodeling activity observed in these HpOch1 knockdown H. pluvialis cells resulted in dramatic reductions in the mannan organization and protective ability of the established cell walls. The cell fragmentation rate increased by 58% in HpOch1− group relative to the control group. Critically, astaxanthin synthesis was not altered in the HpOch1 knockdown cells. Overall, this study highlights a novel technical approach to artificial cell wall thinning, offering a foundation for further efforts to more effectively leverage the astaxanthin resources of H. pluvialis.

Impact of sodium pyrophosphate and static magnetic field on Haematococcus pluvialis: enhancement of astaxanthin accumulation, PAL, and antioxidant enzyme activities.

The Haematococcus pluvialis microalga is known as a main source of astaxanthin with a strong antioxidant capacity and low growth rate. The induction of growth and astaxanthin content was established in H. pluvialis alga using astatic magnetic field (SMF) and tetrasodium pyrophosphate (NaPP) as an inhibitor of isopentenyl pyrophosphate (precursor of astaxanthin biosynthesis) translocator between cytosol to plastid. NaPP (0.3mM), SMF (4 mT), and their combinations were applied to the H. pluvialis cell culture. Results showed chlorophyll a and b were induced in H. pluvialis by SMF treatment, but didn't change significantly under NaPP. Astaxanthin content enhanced under NaPP, SMF, and their combination, and the highest astaxanthin content was obtained under NaPP after 21days (late of stationary phase) of culture. A significant increase in total phenol and flavonoid contents, and activities of phenylalanine ammonia-lyase (PAL) and DPPH were observed under both NaPP and SMF treatments. In contrast to NaPP, SMF decreased H2O2 content, which was associated with more activity of SOD and CAT enzymes. These results revealed that NaPP and SMF might stimulate both phenol and astaxanthin biosynthesis pathways by impacting the activity of enzymes, and inhibition of IPP translocation by NaPP didn't affect astaxanthin biosynthesis at the late growth phase of H. pluvialis.

Oxidative stress facilitates infection of the unicellular alga Haematococcus pluvialis by the fungus Paraphysoderma sedebokerense

BackgroundThe green microalga Haematococcus pluvialis is used as a cell factory for producing astaxanthin, the high-value carotenoid with multiple biological functions. However, H. pluvialis is prone to the infection by a parasitic fungus Paraphysoderma sedebokerense, which is the most devastating threat to the mass culture of H. pluvialis all over the world. Through dissecting the mechanisms underlying the infection process, effective measures could be developed to mitigate the pathogen threatening for the natural astaxanthin industry. By far, understanding about the interaction between the algal host and fungal pathogen remains very limited.ResultsWe observed that there were heat-stable substances with small molecular weight produced during the infection process and enhanced the susceptibility of H. pluvialis cells to the pathogen. The infection ratio increased from 10.2% (for the algal cells treated with the BG11 medium as the control) to 52.9% (for the algal cells treated with supernatant contained such substances) on the second day post-infection, indicating the yet unknown substances in the supernatant stimulated the parasitism process. Systematic approaches including multi-omics, biochemical and imaging analysis were deployed to uncover the identity of the metabolites and the underlying mechanisms. Two metabolites, 3-hydroxyanthranilic acid and hordenine were identified and proved to stimulate the infection via driving oxidative stress to the algal cells. These metabolites generated hydroxyl radicals to disrupt the subcellular components of the algal cells and to make the algal cells more susceptible to the infection. Based on these findings, a biosafe and environment-friendly antioxidant butylated hydroxyanisole (BHA) was selected to inhibit the fungal infection, which completely abolished the infection at 12 ppm. By applying 7 ppm BHA every 2 days to the algal cell culture infected with P. sedebokerense in the 100 L open raceway ponds, the biomass of H. pluvialis reached 0.448 g/L, which was comparable to that of the control (0.473 g/L).ConclusionsThis study provides for the first time, a framework to dissect the functions of secondary metabolites in the interaction between the unicellular alga H. pluvialis and its fungal parasite, indicating that oxidative degradation is a strategy used for the fungal infest. Eliminating the oxidative burst through adding antioxidant BHA could be an effective measure to reduce parasitic infection in H. pluvialis mass culture.Graphical

Acquisition of desiccation tolerance in Haematococcus pluvialis requires photosynthesis and coincides with lipid and astaxanthin accumulation

The unicellular Chlorophyte Haematococcus pluvialis synthesizes astaxanthin and lipids in response to various stress factors. This species colonises temporary water bodies, leaving it vulnerable to desiccation, but mechanisms of desiccation tolerance (DT) in H. pluvialis have not yet been explored. We found that astaxanthin-rich red-coloured H. pluvialis cells tolerated desiccation sensu stricto, but green-coloured cells did not. Mechanisms of DT were elucidated by studying cells under conditions that permitted or prevented attainment of DT. Cellular changes associated with attainment of DT included major reorganisation of cell ultra-structure, such as reduction of chloroplast area and synthesis of astaxanthin-rich lipid bodies. Such changes were prevented in cells desiccated in the dark, or in the light, by blocking electron transfer out of photosystem II. Processes occurring during acclimation to desiccation under low light overlapped with those under high light without desiccation, a treatment that also led to DT, e.g. fatty acids, lipid bodies, astaxanthin, α-tocopherol and glutathione accumulated under both treatments. Furthermore, exposing cells grown in liquid medium to a water-saturated atmosphere without any dehydration also enabled attainment of DT, but at a slower rate compared to when cells were dehydrated. We conclude that atmospheric exposure alone triggered acclimation to desiccation, and photosynthesis powered this process, but neither high light stress nor dehydration are strictly necessary.

Enhancement of Astaxanthin and Fatty Acid Production in Haematococcus pluvialis Using Strigolactone

Improving the production rate of high-value nutraceutical compounds, such as astaxanthin and polyunsaturated fatty acids (PUFAs), is important for the commercialization of Haematococcus pluvialis biorefineries. Here, the effects of a phytohormone, strigolactone analog rac-GR24, on cell growth and astaxanthin and fatty acid biosynthesis in H. pluvialis were investigated. Four concentrations (2, 4, 6, and 8 µM) of rac-GR24 were initially added during 30 days of photoautotrophic cultivation. The addition of rac-GR24 improved cell number density and chlorophyll concentration in H. pluvialis cultures compared to the control; the optimal concentration was 8 µM. Despite a slightly reduced astaxanthin content of 30-d-old cyst cells, the astaxanthin production (26.1 ± 1.7 mg/L) improved by 21% compared to the rac-GR24-free control (21.6 ± 1.5 mg/L), owing to improved biomass production. Notably, at the highest dosage of 8 µM rac-GR24, the total fatty acid content of the treated H. pluvialis cells (899.8 pg/cell) was higher than that of the untreated cells (762.5 pg/cell), resulting in a significant increase in the total fatty acid production (361.6 ± 48.0 mg/L; 61% improvement over the control). The ratio of PUFAs, such as linoleic (C18:2) and linolenic (C18:3) acids, among total fatty acids was high (41.5–44.6% w/w) regardless of the rac-GR24 dose.

A Novel Bifunctional Wax Ester Synthase Involved in Early Triacylglycerol Accumulation in Unicellular Green Microalga Haematococcus pluvialis Under High Light Stress.

The bulk of neutral lipids, including astaxanthin esters and triacylglycerols (TAGs), are accumulated in the green microalga Haematococcus pluvialis under high light (HL) stress. In this study, a novel bifunctional wax ester synthase (WS) gene was cloned from H. pluvialis upon HL stress. The overexpression of HpWS restored the biosynthesis of wax esters and TAGs in neutral lipid-deficient yeast mutant Saccharomyces cerevisiae H1246 fed with C18 alcohol and C18:1/C18:3 fatty acids, respectively. Under HL stress, HpWS was substantially upregulated at the transcript level, prior to that of the type I diacylglycerol:acyl-CoA acyltransferase encoding gene (HpDGAT1). HpDGAT1 is the major TAG synthase in H. pluvialis. In addition, the application of xanthohumol (a DGAT1/2 inhibitor) in the H. pluvialis cells did not completely eliminate the TAG biosynthesis under HL stress at 24 h. These results indicated that HpWS may contribute to the accumulation of TAGs in H. pluvialis at the early stage under HL stress. In addition, the overexpression of HpWS in Chlamydomonas reinhardtii bkt5, which is engineered to produce free astaxanthin, enhanced the production of TAGs and astaxanthin. Our findings broaden the understanding of TAG biosynthesis in microalgae and provide a new molecular target for genetic manipulation in biotechnological applications.

An improved method for sensitive quantification of isoprenoid diphosphates in the astaxanthin-accumulating Haematococcus pluvialis

A sensitive method has been established to simultaneously determine the concentrations of isopentenyl pyrophosphate (IPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in H. pluvialis under different environments. This method increased the extraction efficiency of isoprenoid diphosphates through releasing isoprenoid diphosphates using Tissue Lyser. This is the first report on the efficient extraction method of metabolites in H. pluvialis cells, being suitable for all algae and plants with thick cell wall. The concentrations of isoprenoid diphosphates were measured on poroshell EC-C18 column by UHPLC-MS/MS with the LODs of 0.015, 0.027, 0.022 and 0.076 pmol for DMAPP, GPP, FPP and GGPP, respectively. It is the most sensitive method for the determination of isoprenoid diphosphates in any sample to date. Using this method, the profile of isoprenoid diphosphates was analyzed and cisoid isomers of FPP and GGPP, (Z, Z)-FPP and (Z, Z, Z-GGPP) were found firstly in H. pluvialis.

Determinants of astaxanthin industrial-scale production under stress caused by light photoperiod management of Haematococcus pluvialis cultivation

Haematococcus pluvialis (Chlorophyta) is a microalga with the greatest capacity to generate pure natural astaxanthin with powerful antioxidant properties, through special cysts, in response to disruptions caused by stress conditions. This research tested the design of a prototype for applying artificial light photoperiods controlled by light-emitting diodes on an industrial scale for generating forced stress in H. pluvialis cells during two seasons of the year (winter and spring 2019) in the Coquimbo region in Chile. Three different culture structures were used for the four stages of the alga production cycle. Two containers, A and B, were used for the first and second culture stages, while a raceway (large pool) was used for the third and fourth culture stages. Experiments with four different photoperiods (PP) that represent hours of light:darkness were conducted in two trials (spring 16:8 and winter 18:6) and two for control (spring 11:13 and winter 10:14). In the experiment, an exponential increase of β-carotenoid was achieved, used in human and animal food for its health properties and as a natural colorant in the salmonids industry. Biomass and astaxanthin production under forced stress were measured with physical and chemical variables such as light intensity, temperature, pH, and dissolved oxygen. Results show that the spring culture showed a considerable increase of cysts and, therefore, of astaxanthin reservoirs, reaching a pigment production density of 276 g m -3 , with the consequent increase in density of up to 22% more than the control PP.

Enhancement of astaxanthin production in Haematococcus pluvialis using zinc oxide nanoparticles

Today, there is a great interest in using astaxanthin due to its potential health advantages. Application of different types of nanoparticles (NPs) as stress agents to enhance astaxanthin production in Haematococcus pluvialis, a microalgae strain, has been reported in the literature. In this study, the effect of different concentrations of zinc oxide (ZnO) NPs on the enhancement of astaxanthin production in H. pluvialis was investigated. First, ZnO NPs were synthesized from zinc nitrate as the precursor and sodium hydroxide (chemical method), and peel extract of pomegranate (green method) as reducing agents. To study the cell viability and stimulate the astaxanthin production, H. pluvialis cells were exposed to the different concentrations (i.e. 50, 100, 200, and 400 μg.ml−1) of ZnO NPs. The synthesized powders were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) methods. The characterization results showed that the pure ZnO NPs were successfully synthesized via both methods with uniform particle size distribution. But, the average particle size of the green synthesized ZnO NPs (about 30 nm) was smaller than that of the chemically synthesized ones (about 80 nm). Maximum astaxanthin production (~ 20 mg.g−1 of dry biomass of H. pluvialis) was achieved at 100 μg.ml−1 of green synthesized ZnO NPs exposure to the H. pluvialis in comparison with the control culture after 15 days. However, ZnO NPs concentration above 200 μg.ml−1 was toxic to the microalgae. From these results, it can be concluded that a specific amount of ZnO NPs could be considered as a worthy candidate for the enhancement of astaxanthin production in H. pluvialis.

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.