Green Microalga Haematococcus Pluvialis Research Articles

Melatonin enhances astaxanthin accumulation in the green microalga Haematococcus pluvialis by mechanisms possibly related to abiotic stress tolerance

Melatonin (N‑acetyl‑5‑methoxytryptamine) is widely regarded as an important messenger in higher plants and mammals in their resistance to various biotic and abiotic stresses. However, the role of melatonin in microalgae has been rarely investigated. In this study, melatonin was first used to promote astaxanthin biosynthesis under limited nitrogen and high light conditions. The interactions between melatonin and the secondary messengers, namely, nitric oxide (NO) and salicylic acid (SA), during the stress response were also investigated. Moreover, fatty acid biosynthesis was explored. Finally, the expression levels of astaxanthin biosynthesis genes in Haematococcus pluvialis LUGU supplemented with melatonin were simultaneously monitored through quantitative real-time PCR. The astaxanthin content of the microalgae increased 2.36-fold after treatment with 10 μM of melatonin. The maximal astaxanthin content achieved was 31.32 mg g−1. The increased NO and SA production caused by melatonin occurred in parallel with the up-regulation of the expression of astaxanthin biosynthesis genes and the enhancement in astaxanthin biosynthesis in H. pluvialis LUGU. The analysis of fatty acid composition showed that melatonin stimulated the production of C16:0 (palmitic acid); thus, the percentage of astaxanthin esters was higher than that observed in the control algae. Furthermore, the inhibitors, namely, carboxy-PTIO, paclobutrazol, and N‑acetyl‑l‑cysteine, significantly suppressed the levels of secondary messengers. Meanwhile, the role of melatonin in mediating the rise in astaxanthin content was prevented when the SA- and NO-dependent pathways were inhibited. These findings indicate that melatonin plays a physiological role in direct and indirect responses to abiotic stresses in H. pluvialis.

Sequencing and Characterization of Novel PII Signaling Protein Gene in Microalga Haematococcus pluvialis.

The PII signaling protein is a key protein for controlling nitrogen assimilatory reactions in most organisms, but little information is reported on PII proteins of green microalga Haematococcus pluvialis. Since H. pluvialis cells can produce a large amount of astaxanthin upon nitrogen starvation, its PII protein may represent an important factor on elevated production of Haematococcus astaxanthin. This study identified and isolated the coding gene (HpGLB1) from this microalga. The full-length of HpGLB1 was 1222 bp, including 621 bp coding sequence (CDS), 103 bp 5′ untranslated region (5′ UTR), and 498 bp 3′ untranslated region (3′ UTR). The CDS could encode a protein with 206 amino acids (HpPII). Its calculated molecular weight (Mw) was 22.4 kDa and the theoretical isoelectric point was 9.53. When H. pluvialis cells were exposed to nitrogen starvation, the HpGLB1 expression was increased 2.46 times in 48 h, concomitant with the raise of astaxanthin content. This study also used phylogenetic analysis to prove that HpPII was homogeneous to the PII proteins of other green microalgae. The results formed a fundamental basis for the future study on HpPII, for its potential physiological function in Haematococcus astaxanthin biosysthesis.

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.

A β-carotene ketolase gene (bkt1) promoter regulated by sodium acetate and light in a model green microalga Chlamydomonas reinhardtii

Many microalgae could accumulate high level of astaxanthin, especially a green microalga Haematococcus pluvialis. β-carotene ketolase gene (bkt) is a key enzyme for astaxanthin biosynthesis in Haematococcus pluvialis, and astaxanthin accumulation could be induced effectively by abiotic stresses such as high concentration of sodium acetate and high light. In Haematococcus there are 3 bkt gene copies, among them, the transcription level of bkt1 was obviously correlated with high light and sodium acetate. Thus, the function of bkt1 promoter played a key role in understanding the regulatory mechanisms of astaxanthin biosynthesis. A TATA-box and cis-acting elements associated with high light and stress-related responses were detected in bkt1 promoter in previously studies. However, detailed information about cis-acting regulatory elements of this important promoter is still unavailable since a reliable genetic manipulation technique is not available yet. To identify the functions of cis-elements in bkt1 promoter, 8 constructs contained different length of bkt1 promoter were successfully introduced into a model microalga Chlamydomonas reinhardtii and different transformants were obtained with zeomycin resistant selection. Exposed to high light or sodium acetate, differentially increased ble transcripts and protein production in transformants indicated that these cis-elements respond to high light and sodium acetate. Moreover, we found that TATA-box in bkt1 promoter is essential for the promoter function and ble expression was correlated with cis-acting elements in bkt1 promoter. Our results shed lights on regulation mechanisms of the key genes from Haematococcus for astaxanthin biosynthesis in Chlamydomonas reinhardtii.

Inducible expression of Haematococcus oil globule protein in the diatom Phaeodactylum tricornutum: Association with lipid droplets and enhancement of TAG accumulation under nitrogen starvation

Microalgae accumulate the storage lipids triacylglycerols (TAG) in lipid droplets (LDs) in response to environmental cues. Mechanistic insights into LD biogenesis in microalgal cells hold great promise for the manipulation of TAG production. In this report, we demonstrate the heterologous expression of a major LD protein of the green microalga Haematococcus pluvialis in the diatom Phaeodactylum tricornutum. The N-terminus of Haematococcus oil globule protein (HOGP) was fused with EGFP and expressed under the control of the strong light-inducible fcpA gene promoter and the novel nitrogen-starvation-inducible DGAT1 gene promoter. Fatty acid feeding enabled fast monitoring of LDs formation under nitrogen-replete conditions and detection of a fluorescent EGFP-HOGP signal as ring-like structures adjacent to the periphery of LDs. During the prolonged nitrogen starvation, EGFP-HOGP was expressed only under the control of the DGAT1 promoter and was similarly associated with LDs. Furthermore, expression of HOGP under nitrogen starvation enhanced TAG accumulation. The tools and approaches described in this report open the door for characterizing endogenous LD proteins in P. tricornutum. The results of this study indicate the significant biotechnological potential of such LD-associated protein genes in the metabolic engineering of microalgae for enhanced oil productivity.

The chlorophyll a fluorescence induction curve in the green microalga Haematococcus pluvialis: further insight into the nature of the P-S-M fluctuation and its relationship with the "low-wave" phenomenon at steady-state.

Chlorophyll fluorescence is an information-rich signal which provides an access to the management of light absorbed by PSII. A good example of this is the succession of fast fluorescence fluctuations during light-induced photosynthetic induction after dark-adaptation. During this period, the fluorescence trace exhibits several inflexion points: O-J-I-P-S-M-T. Whereas the OJIP part of this kinetics has been the subject of many studies, the processes that underly the PSMT transient are less understood. Here, we report an analysis of the PSMT phase in the green microalga Haematococcus pluvialis in terms of electron acceptors and light use by photochemistry, fluorescence and non-photochemical quenching (NPQ). We identify additional sub-phases between P and S delimited by an inflexion point, that we name Q, found in the second time scale. The P-Q phase expresses a transient photochemical quenching specifically due to alternative electron transport to oxygen. During the transition from Q to S, the NPQ increases and then relaxes during the S-M phase in about 1min. It is suggested that this transient NPQ observed during induction is a high energy state quenching (qE) dependent on the alternative electron transport to molecular oxygen. We further show that this NPQ is of the same nature than the NPQ, known as the low-wave phenomenon, which is transiently observed after a saturating light pulse given at steady-state. In both cases, the NPQ is oxygen-dependent. This NPQ is observed at external pH 6.0, but not at pH 7.5, which seems correlated with faster saturation of the PQ pool at pH 6.0.

Rapid screening astaxanthin-hyperproducing Haematococcus pluvialis mutants through near-infrared spectroscopy.

The unicellular freshwater green microalga Haematococcus pluvialis is the richest source of natural astaxanthin. Since accumulation of astaxanthin differs significantly among various algal strains at different stages, it is therefore critical to develop an effective high-throughput assay for rapid screening astaxanthin-hyperproducing strains. In the present study, near-infrared spectroscopy (NIRS) in combination with biochemical assay was employed for evaluation of the wide-type H.Pluvialis strains. The partial least squares (PLS) models of total biomass, astaxanthin content and astaxanthin expressed as a percentage of dry weight (DW) were developed with the R(2) values as 0·959, 0·982 and 0·952, the prediction correlation factor (r) values as 0·979, 0·988 and 0·966, and the residual predictive deviation (RPD) values as 4·88, 6·22 and 3·86, respectively. Furthermore, the PLS models were employed to evaluate H.pluvialis mutants, with the r values as 0·973, 0·983 and 0·976, and the RPD values as 3·45, 7·59 and 4·07, respectively. This work thus demonstrates that NIRS is an easy, fast and non-invasive approach that can be applied in high-throughput screening of astaxanthin-hyperproducing algal mutants. Haematococcus pluvialis has potential application for its ability to accumulate natural antioxidant astaxanthin. In this study, we initiated the application of near-infrared spectroscopy (NIRS) in the analysis of total biomass and astaxanthin content of different mutant strains, demonstrating that NIRS can be very useful in the screening of axataxanthin-hyperproducing mutant strains.

A new flagellated dispersion stage in Paraphysoderma sedebokerense, a pathogen of Haematococcus pluvialis.

The blastocladialean fungus Paraphysoderma sedebokerense Boussiba, Zarka and James is a devastating pathogen of the commercially valuable green microalga Haematococcus pluvialis, a natural source of the carotenoid pigment astaxanthin. First identified in commercial Haematococcus cultivation facilities, P. sedebokerense is hypothesised to have a complex life cycle that switches between a vegetative and a resting phase depending on favourable or unfavourable growth conditions. Rather unusually for blastocladialean fungi, P. sedebokerense was described as lacking flagellated zoospores and only propagating via aplanosporic amoeboid cells. However, during repeated microscopic observation of P. sedebokerense cultivated in optimal conditions, we detected fast-swimming, transiently uniflagellated zoospores which rapidly transform into infectious amoeboid swarmers, the existence of which suggests a closer than previously thought relatedness of P. sedebokerense to its sister genera Physoderma and Urophlyctis. Additionally, we found some morphological and physiological differences between amoeboid swarmers and discuss hypotheses about their significance. These amoeboid and flagellated propagules are key to the dissemination of P. sedebokerense and are probably also the life stages most vulnerable to adverse environmental conditions. They are therefore a prime target for the development of disease management protocols in industrial cultivation facilities, a goal which requires a detailed understanding of their physiology.Electronic supplementary materialThe online version of this article (doi:10.1007/s10811-015-0700-8) contains supplementary material, which is available to authorized users.

Evaluation of hepatoprotective and antioxidant activity of astaxanthin and astaxanthin esters from microalga-Haematococcus pluvialis.

Effect of isolated astaxanthin (ASX) and astaxanthin esters (ASXEs) from green microalga-Haematococcus pluvialis on hepatotoxicity and antioxidant activity against carbon tetrachloride (CCl4) induced toxicity in rats was compared with synthetic astaxanthin (SASX). ASX, ASXEs, and SASX, all dissolved in olive oil, fed to rats with 100 and 250μg/kg b.w for 14days. They were evaluated for their hepatoprotective and antioxidant activity by measuring appropriate enzymes. Among the treated groups, the SGPT, SGOT and ALP levels were decreased by 2, 2.4, and 1.5 fold in ASXEs treated group at 250μg/Kg b.w. when compared to toxin group. Further, antioxidant enzymes catalase, glutathione, superoxide dismutase and lipid peroxidase levels were estimated in treated groups, their levels were reduced by 30-50% in the toxin group, however these levels restored by 136.95 and 238.48% in ASXEs treated group at 250μg/kg. The lipid peroxidation was restored by 5.2 and 2.8 fold in ASXEs and ASX treated groups at 250μg/kg. The total protein, albumin and bilirubin contents were decreased in toxin group, whereas normalized in ASXEs treated group. These results indicates that ASX and ASXEs have better hepatoprotection and antioxidant activity, thereforecan be used in pharmaceutical and nutraceutical applications and also extended to use as food colorant.

Molecular mechanisms of the coordination between astaxanthin and fatty acid biosynthesis in Haematococcus pluvialis (Chlorophyceae).

Astaxanthin, a red ketocarotenoid with strong antioxidant activity and high commercial value, possesses important physiological functions in astaxanthin-producing microalgae. The green microalga Haematococcus pluvialis accumulates up to 4% fatty acid-esterified astaxanthin (by dry weight), and is used as a model species for exploring astaxanthin biosynthesis in unicellular photosynthetic organisms. Although coordination of astaxanthin and fatty acid biosynthesis in a stoichiometric fashion was observed in H.pluvialis, the interaction mechanism is unclear. Here we dissected the molecular mechanism underlying coordination between the two pathways in H.pluvialis. Our results eliminated possible coordination of this inter-dependence at the transcriptional level, and showed that this interaction was feedback-coordinated at the metabolite level. In vivo and in vitro experiments indicated that astaxanthin esterification drove the formation and accumulation of astaxanthin. We further showed that both free astaxanthin biosynthesis and esterification occurred in the endoplasmic reticulum, and that certain diacylglycerol acyltransferases may be the candidate enzymes catalyzing astaxanthin esterification. A model of astaxanthin biosynthesis in H.pluvialis was subsequently proposed. These findings provide further insights into astaxanthin biosynthesis in H.pluvialis.

Hyperspectral imaging techniques for the characterization of Haematococcus pluvialis (Chlorophyceae).

A hyperspectral imaging camera was combined with a bright-field microscope to investigate the intracellular distribution of pigments in cells of the green microalga Haematococcus pluvialis, a synonym for H.lacustris (Chlorophyceae). We applied multivariate curve resolution to the hyperspectral image data to estimate the pigment contents in culture and revealed that the predicted values were consistent with actual measurements obtained from extracted pigments. Because it was possible to estimate pigment contents in every pixel, the intracellular distribution of the pigments was investigated during various life-cycle stages. Astaxanthin was localized specifically at the eyespot of zoospores in early culture stages. Then, it became widely distributed in cells, but subsequently localized differently than the chl. Integrated with our recently developed image-processing program "HaematoCalMorph," the hyperspectral imaging system was useful for monitoring intracellular distributions of pigments during culture as well as for studying cellular responses under various conditions.

Image-Based Monitoring System for Green Algal Haematococcus pluvialis (Chlorophyceae) Cells during Culture

The green microalga Haematococcus pluvialis accumulates the red pigment astaxanthin accompanied by morphological changes under stress conditions, including nutrient depletion, continuous light and high temperature. To investigate the physiological state of the algal cells, we developed the digital image-processing software called HaematoCalMorph. The software automatically outputs 25 single-cell measurements of cell morphology and pigments based on color, bright-field microscopic images. Compared with manual inspection, the output values of cell shape were reliable and reproducible. The estimated pigment content fits the values calculated by conventional methods. Using a random forests classifier, we were able to distinguish flagellated cells from immotile cells and detect their transient appearance in culture. By performing principal components analysis, we also successfully monitored time-dependent morphological and colorimetric changes in culture. Thus, combined with multivariate statistical techniques, the software proves useful for studying cellular responses to various conditions as well as for monitoring population dynamics in culture.

Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale

The freshwater green microalga Haematococcus pluvialis is the richest source of natural astaxanthin. Astaxanthin is a high-value red carotenoid pigment commonly used in the food, feed and cosmetics industries due to its well-known antioxidant, anti-inflammatory and antitumour properties. This study assesses the environmental impacts associated with the production of natural astaxanthin from H. pluvialis at both lab and pilot scale. Closed airlift photobioreactors with artificial illumination, typically used for the production of high-value products to avoid contamination risks and allow controlled lighting conditions, were considered. The study extends from the production of the different inputs to the system to microalgal production, harvesting and further extraction of the carotenoid. The life cycle assessment was performed following the ISO 14040 and ten impact categories were considered in the study: abiotic depletion, acidification, eutrophication, global warming, ozone layer depletion, human toxicity, freshwater aquatic ecotoxicity, marine aquatic ecotoxicity, terrestrial ecotoxicity and photochemical oxidant formation. According to the results, electricity requirements represented the major contributor to the environmental burdens among the activities involved in the production of astaxanthin. For the lab-scale process, the air supply and the production of chemicals and lab materials were also significant contributors in several categories. In the pilot-scale production, the relative environmental impacts were greatly reduced, partially due to changes implemented in the system as a result of lab-scale environmental assessment. However, the production of electricity still dominated the impacts in all categories, particularly due to the cultivation stage. For this reason, a sensitivity assessment was proposed in order to identify alternative photobioreactor configurations for astaxanthin production. Two of the evaluated options, based on the use of sunlight instead of artificial illumination, presented significant reductions of impact. However, the improvements observed in these cases were limited by the decrease in biomass productivity associated with sunlight culture systems. Therefore, a two flat-panel photobioreactor system with artificial illumination is proposed as a suitable option, allowing reductions between 62% and 79% of the impact depending on the considered category.

Effect of nitrate concentration on growth of green microalga Haematococcus pluvialis under laboratory conditions

Astaxanthin is a pigment that belongs to the family of carotenoids and it is one of the major carotenoids supplied in aquaculture. In addition to its role in the coloration of aquatic animals, this pigment possesses several important bioactivities, including antioxidation, enhancement of immune response on anticancer activities. Although astaxanthin can be synthesized by plants, bacteria, a few fungi, and green algae, Haematococcus pluvialis is the current best source of natural astaxanthin. Although the feasibility of continuous production of astaxanthin by vegetative motile cells has been recently demonstrated, the most common production process consists in separating the biomass production phase culture and the astaxanthin accumulation phase one. In addition to light and temperature, nutrients (including N) play important role in the cell division and in the accumulation of astaxanthin of H. pluvialis. This work aimed to investigate effects of nitrate concentration on growth of microalga H. pluvialis. The highest cell density was 1.74 × 106 cells/mL after the 36th days of cultivation at RM medium contained nitrate concentration of 876 mg/L. At this concentration, the ratio of vegetative cells was higher than other nitrate concentrations. Chlorophyll a and astaxanthin contents yielded 2081 and 1053 µg/L, respectively. In addition, protein content tended to decrease during cultivation process at all tested nitrate concentrations.

From genetic improvement to commercial-scale mass culture of a Chilean strain of the green microalga Haematococcus pluvialis with enhanced productivity of the red ketocarotenoid astaxanthin

Astaxanthin is a red ketocarotenoid, widely used as a natural red colourant in marine fish aquaculture and poultry and, recently, as an antioxidant supplement for humans and animals. The green microalga Haematococcus pluvialis is one of the richest natural sources of this pigment. However, its slow growth rate and complex life cycle make mass culture difficult for commercial purposes. The aims of this research were (i) to standardize and apply a genetic improvement programme to a Chilean strain of H. pluvialis in order to improve its carotenogenic capacity and (ii) to evaluate the performance of a selected mutant strain in commercial-sized (125 000 L) open ponds in the north of Chile. Haematococcus pluvialis strain 114 was mutated by ethyl methanesulfonate. The level of mutagen dose (exposure time and concentration) was one that induced at least 90 % mortality. Surviving colonies were screened for resistance to the carotenoid biosynthesis inhibitor diphenylamine (25 µM). Resistant mutants were grown in a 30-mL volume for 30 days, after which the total carotenoid content was determined by spectrophotometry. Tens of mutants with improved carotenogenic capacity compared with the wild-type strain were isolated by the application of these standardized protocols. Some mutants exhibited curious morphological features such as spontaneous release of astaxanthin and loss of flagella. One of the mutants was grown outdoors in commercial-sized open ponds of 125 000 L in the north of Chile. Grown under similar conditions, the mutant strain accumulated 30 % more astaxanthin than the wild-type strain on a per dry weight basis and 72 % more on a per culture volume basis. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of H. pluvialis and that improved carotenogenic capacity is maintained when the volume of the cultures is scaled up to a commercial size.

Effect of initial biomass density on growth and astaxanthin production of Haematococcus pluvialis in an outdoor photobioreactor

Initial biomass density (IBD) is an important factor that affects the viability and productivity of microalgae particularly when sunlight is used for photosynthesis. In this paper, the effect of IBD on photosynthesis, growth, and astaxanthin production of the green microalga Haematococcus pluvialis during the astaxanthin induction stage was studied in a glass column photobioreactor during different seasons. Of seven IBDs, i.e., 0.1, 0.5, 0.8, 1.5, 2.7, 3.5, and 5.0 g L−1 tested, 0.8 g L−1 IBD was optimal and resulted in the highest astaxanthin productivity of 17.1 mg L−1 day−1. Severe photoinhibition of photosynthesis occurred at low IBD (e.g., 0.1 g L−1) cultures, especially in the winter, and severe light limitation to individual cells in high IBD cultures (>2.7 g L−1) were responsible for reduced astaxanthin production. This was the first report quantitatively assessing IBD as the key limiting factor for astaxanthin production in H. pluvialis outdoor cultivation.

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

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

A two-stage solar photobioreactor for cultivation of microalgae based on solar concentrators

A novel two-stage experimental photobioreactor (PBR) with a total volume of 450 L and based uniquely on solar concentrators—linear Fresnel lenses—has been constructed and tested. Daily courses of irradiance, and also its distribution inside cultivation tubes, were studied in two unit types. The supra-high irradiance units in the ‘roof’ achieved a maximum summer value above 6 mmol photon m−2 s−1, while irradiance in the vertical-facade units was lower than ‘ambient’. In model cultivations, cultures of the cyanobacterium Arthrospira platensis were cultivated at much higher solar irradiances than those usually recorded outdoors in summer, indicating that this organism is resilient to high-irradiance (photoinhibition). Starting from a biomass density of 0.5 g L−1 at optimum temperature, the cultures grew exponentially. A two-stage cultivation process of the green microalga Haematococcus pluvialis was investigated with respect to correlations between photochemical activities and astaxanthin production. The culture was first grown in low-irradiance units, and then exposed to supra-high irradiance when the rate of astaxanthin production was 30–50% higher than in the culture exposed to ‘ambient’ irradiance. Within 4 days, the astaxanthin content reached 3% of dry weight, whereas under ambient irradiance the astaxanthin content was 25% lower.

Isolation and characterization of a novel chytrid species (phylum Blastocladiomycota), parasitic on the green alga Haematococcus

A parasite was found in cultures of the green microalga Haematococcus pluvialis that grew epibiotically on algal cells and caused epidemics resulting in damage to the host cultures. The parasite was isolated into axenic culture on solid and liquid media. It was demonstrated to be the sole causative agent of the epidemics. According to its life cycle and phylogenetic analysis based on 18S ribosomal DNA sequences, the pathogen appears to represent a novel chytrid fungus closely related to the vascular plant pathogen Physoderma ( Blastocladiomycota), although it differs from all other known chytrids by its infective stage, a wall-less propagule endowed with amoeboid motion and lacking the group's typical flagellum.

Combined effects of light intensity and acetate concentration on the growth of unicellular microalga Haematococcus pluvialis

The green microalga Haematococcus pluvialis is known as one of the most useful photosynthetic microorganisms, due to its ability to produce a high value-added antioxidant (astaxanthin). However, this microalga is not easily commercially cultivated because its growth is slow under autotrophic conditions. In this study, the mixotrophic culture conditions were partially optimized using a sequential factorial design. The light intensity and acetate concentration were selected as major factors affecting the algal growth rate. The first factorial design (2 2) was performed with the light levels of 80–120 μE m −2 s −1 and 40–70 mM acetate. As a result, the optimum conditions were not present within the domain of the first experimental design. An ascent line obtained from the first design showed that the optimum conditions existed within a region of lower concentration of acetate, but similar intensity of light. On the basis of the ascent line, additional experiments were carried out using a central composite design, which enhanced the growth rate up to 0.243 g L −1 day −1 at a light intensity of 170 μE m −2 s −1 and acetate concentration of 30 mM.