Haematococcus Lacustris Research Articles

Impact of UV-B Radiation on 15N-Ammonium and 15N-Nitrate Uptake by Haematococcus lacustris (Volvocales). I. Different Response of Flagellates and Aplanospores

Summary The freshwater green alga Haematococcus lacustris accumulates 15N given as 15N-NH4+ to a higher extend than with 15N-NO3−. Special attention was focused to investigate the influence of UV-B radiation on the 15N-uptake. UV-B (1.25 W m−2) was applied additionally to the standard light conditions (8 W m−2) and nitrogen incorporation was determined for 5 hours during and after the irradiation period. The response of two different developmental stages of the cells on the UV-B-load (green flagellates and red coloured aplanospores), each in two different metabolic conditions (nitrogen-starved dark precultivated and nitrogen-supplied light precultivated cells, respectively) were examined. Results indicate (1) UV-B inhibits the incorporation of 15N-nitrogen in both cell types. (2) The degree of inhibition in aplanospores is generally lower than in the flagellates. This principle is valid also in activated aplanospores. (3) Activated cells of both types show a higher degree of inhibition in nitrate an ammonium uptake than cells precultivated under starvation. (4) Nitrogen incorporation is also inhibited when UV-B-radiation is applied before addition of the 15N-labelled compounds; Different uptake kinetics derived from these experiments are discussed with respect to the possible target of UV-B-influence.

CELL CYCLE AND ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

ABSTRACTSynchronous release of ellipsoidal biflagellated zoo‐spores from thick‐walled akinetes of Haematococcus lacustris (Gir.) Rostaf. (UTEX 16) was induced. After being released, the zoospores divided rapidly at a rate that depended on the initial concentration of urea in the culture medium. Cells fused after approximately five doublings, and the DNA content of most cells doubled within 50 h. Spherical nonmotile palmella cells and aplanospores appeared after 100 h of incubation in media containing high (1.7 g·L−1) and low (0.85 g·L−1) urea concentrations. Thereafter, the number of nonmotile cells increased with time, whereas motile cell numbers decreased with time. Nonmotile cells continued to grow and divide by forming 4–32 aplanospores, for up to 200 h of incubation in the high‐urea medium. The size of the nonmotile cells and the number of daughter cells formed within was inversely proportional to the growth rate of the cultures. Within the first 100 h of incubation, dry weight biomass of the zoo‐spores increased from about 0.3 to 0.8 g·L−1. In the following 180 h, dry weight biomass reached 1.7 g·L−1 in the low‐urea medium and 2.5 g·L−1 in the high‐urea medium. The astaxanthin content of zoospores decreased with time, whereas there was a net accumulation of astaxanthin in the nonmotile cells. The specific rate of accumulation of astaxanthin in motile and nonmotile cells, however, was practically identical.

FUNCTIONAL ASPECTS OF SECONDARY CAROTENOIDS IN HAEMATOCOCCUS LACUSTRIS (VOLVOCALES). III. ACTION AS A “SUNSHADE”1

ABSTRACTWe investigated the protection from photoinhibition by different developmental stages of Haematococcus lacustris [Girod] Rostafinski using chlorophyll fluorescence measurements of single cells and suspensions. An overall correlation between higher cellular content of secondary carotenoids and the capacity to withstand excessive irradiation was observed in flagellated cells and aplanospores of H. lacustris. Low‐light‐reversible spreading of extra‐chloroplastic secondary carotenoids occurred in the periphery of the cell during strong irradiation. This process resulted in increased shading of the cup‐shaped chloroplast as demonstrated by a decrease in chlorophyll fluorescence. Extrachloroplastic accumulation of secondary carotenoids in H. lacustris can be interpreted as a specific adaptation to habitats that exhibit strong insolation.

Functional aspects of secondary carotenoids in Haematococcus lacustris (Girod) Rostafinski (Volvocales). V. Influences on photomovement

ABSTRACTSecondary carotenoids are suspected to modulate photomovement in Haematococcus lacustris [Girod] Rostafinski (Volvocales). To investigate the influence of these extrachloroplastic ketocarotenoids on phototactic and photophobic responses in the flagellate stage of the green alga, flagellate suspensions differing in the content of secondary carotenoids were grown from green and red aplanospores. Photo‐orientation of these flagellates induced by unilateral irradiation was investigated using a computer‐aided system for microscopic image analysis. Results were hypothetically summarized as follows:(1) Diminution of precision of the positive phototaxis was found in red flagellates. This might be due to cellular shading of the blue‐light‐sensitive photoreceptor by secondary carotenoids.(2) Red flagellates exhibited an increase in the photophobic response. This finding is discussed in relation to an adaptive increase of the photoreceptor sensitivity, thought to be a result of the higher optical density of the corresponding cell suspension in the blue wavelength region.

Functional aspects of secondary carotenoids in Haematococcus lacustris (Girod) Rostafinski (Volvocales): I. The accumulation period as an active metabolic process.

Alterations of photosynthetic activity were investigated during accumulation of secondary carotenoids in Haematococcus lacustris (Girod) Rostafinski. Using several methods regarding (i) pigment pattern and pigment content (cytophotometry, computer-aided microscopic image analysis, in vivo absorption spectroscopy, high-performance liquid chromatrography) and (ii) photosynthesis (microfluorometry, modulated chlorophyll fluorescence and luminescence detection, O2 evolution and consumption measurement, 77 K fluorescence spectroscopy) we examined single cells and cell suspensions of the green alga. The results indicate decrease of both the photosystem II activity and the linear electron transport accompanying synthesis of secondary carotenoids. On the other hand, activation of those processes leading to an increased transthylakoid proton gradient could be established during early accumulation period. This is assumed to be an active adaptation of the photosynthetic apparatus to energetic requirements given by secondary carotenoid biosynthesis in H. lacustris.

Functional aspects of secondary carotenoids in Haematococcus lacustris [Girod] Rostafinski (Volvocales) IV. Protection from photodynamic damage

The function as an antioxidant seems to represent the central principle of chemopreventive activity of carotenoids against cancer initiation and promotion. The aim of this study was to clarify whether or not extrachloroplastic-accumulated secondary carotenoids (astaxanthin, canthaxanthin and echinenone) of Haematococcus lacustris [Girod] Rostafinski exhibit a similar antioxidative activity in protecting the cell of this green alga from photo-oxidative damage. In vivo experiments were performed, investigating the effect of UV radiation, artificial photosensitizers (rose bengal, toluidine blue) and copper-mediated lipid peroxidation on suspensions of flagellates which contained different amounts of secondary carotenoids. The results revealed a higher resistance of red flagellates to photo-oxidative stress. The findings are discussed with respect to the shading function of secondary carotenoids and known protective mechanisms involving quenching of reactive oxygen species and radical reactions in plant cells. A hypothesis for this functional aspect of secondary carotenoids in H. lacustris preventing injury by excessive insolation is suggested: ketocarotenoids, first accumulated in lipid vacuoles around the nucleus, might act as a physico chemical barrier, protecting particularly the genome from free radical-mediated damage.

The production of clonal and axenic cultures of microalgae using fluorescence-activated cell sorting

Unialgal cultures of the flagellate algae Cyanophora paradoxa, Haematococcus lacustris, Monomastix sp., Scherffelia dubia and Spermatozopsis similis which contained bacteria were sorted by flow cytometry to obtain axenic clonal cultures. The variables used for fluorescence-activated cell sorting (FACS) were chlorophyll autofluorescence, forward scatter and side scatter of the laser beam. To produce clonal cultures, a single cell was sorted into each culture flask. Depending on the species, about 20–30% of the sorted cultures grew successfully and at least 20% of these were axenic even if the numerical ratio betweeen bacteria and algae in the original cultures was as high as 300:1. FACS represents an effective and rapid method for the preparation of clonal and axenic cultures of microalgae.

ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

ABSTRACTIn N‐limited continuous chemostat cultures of the green alga Haematococcus lacustris (Gir.) Rostaf. (UTEX 16), the steady‐state astaxanthin content of the cells was determined by the specific growth rate of the cultures. The highest, pigment content was obtained at the lowest dilution rate. The specific rate of astaxanthin accumulation was, however, a function of the photon flux density measured at the illuminated culture surface. In nongrowing Haematococcus cultures, the specific rate of astaxanthin accumulation was determined by the growth rate of the culture during growth phase. The highest possible cellular astaxanthin content of all cultures was comparable and independent of the culture parameters.

Phototactic responses in Haematococcus lacustris and its modification by light intensity and the carotenoid biosynthesis inhibitor Norflurazon

At fluence rates below 45 W· m-2 cells of the flagellate stage of Haematococcus lacustris react only positively phototactically with a rather high degree of orientation (indicated by r values up to 0.66 with the Rayleigh test). The directedness of orientation decreases with decreasing irradiance. The degree of directedness of the phototactic response depends on the intensity of preirradiation: Low light intensity applied after strong light application results in a “dark reaction” (low r values), low light given after darkness stimulates a rather high degree of directedness of positive phototaxis. Weak blue light (λ=483 nm; 0.4 W · m-2) stimulates positive phototactic response, whereas comparable red light (λ=658 nm; 0.5 W · m-2) does not.

Note on the survival of algal resting cells during long‐term maintenance in darkness and minimum lake bottom temperature. Comparison of Anabaena (cyanobacteria) and Haematococcus (volvocales)

AbstractResting cell formation in algae and cyanobacteria is one of the strategies developed for withstanding adverse environmental conditions. This note informs about the different capacities of such resting cells to germinate after long‐term dark preservation at 4 °C in Anabaena variabilis (Cyanobacteria, Cyanophyta) and Haematococcus lacustris (Chlorophyta, Volvocales).

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.

Ultrastructural study of Haematococcus lacustris (Girod.) Rostafinski (Volvocales). II. Mitosis and cytokinesis.

In this article are presented the results of the ultrastructural study of the cell division in Haematococcus lacustris. The more significative data are as follows: the nuclear envelope is maintained intact practically throughout mitosis, as it only breaks down at the end of telophase when the daughter nuclei are separated. The disappearance of the nucleolus, which in the interphase nuclei display the two typical structural components (granulous and fibrillar), is sometimes delayed, since in late metaphase-early anaphase nucleolus clearly organised were observed. The spindle microtubules (NITS) seem to have an exclusively endonuclear origin, as neither through polar fenestrae nor nuclear pores they appear to be related with any extranuclear structure. In metaphase and anaphase we can see that the above endonuclear spindle is formed by “continuous” and chromosomic MTS. The former are persistent in the interzonal region up to telophase and the latter are deep in the chromatin, sometimes in apparently differentiated chromosomic regions (centromeres). In late anaphase-early telophase, the nucleus is seen more elongated, sometimes curved and repeatedly with dilated ends (dumbell shaped). The nuclear envelope of the telophasic nucleus breaks down in the middle-region and surrounds the two chromosomic groups (daughter nuclei). Between them, a growing cleavage furrow (invagination of the plasmalemma) ends by leading to the division of the protoplast (cytokinesis). This cleavage furrow is always associated to a microtubular system (phycoplast) similar to that which has been described in other algae.

Ultrastructural study of Haematococcus lacustris (Girod.) Rostafinski (Volvocales). I. Some aspects of carotenogenesis.

The process of accumulation of secondary carotenoids (astaxanthin) in Haematococcus lacustris was analysed in the optical and electron microscopes. The more relevant results of this cytological study are the following: a. In motile cells, the lipochromes are spherical inclusions, initially of small size, and located in the perinuclear cytoplasm apparently in preferential correlation with E. R. Although without a true limiting biomembrane, these sudanophilic and osmiophilic inclusions do not easily fuse when contacting. b. In the cysts, the pigment deposits, although being identical to the ones of the motile cells in what concerns their location and affinity for lipids “stains”, show a variety of shapes, including “long filaments”, rods and more or less complex reticulate forms. c. Cells of the older cultures (11-20 days) appear almost uniformly “stained” deep red. This process is reversible, i.e., these cultures again turn green when successively subcultured to fresh media. d. Apart from the stigma globules, the haematochrome is always localized outside the chloroplast, the photosynthetic apparatus of which remains practically intact through this process. Then, no sign of plastidal transformation, (chloroplast in chromoplast) comparable to the changes which take place in various organs of higher plants was observed.

Ketocarotenoid biosynthesis by Haematococcus lacustris

Haematococcus lacustris incubated on a nutrient-depleted medium utilised acetate-[2- 14C] from the medium and carbon fixed photosynthetically for the biosynthesis of ketocarotenoids. Conversion of β-carotene to astaxanthin occurs via the intermediates echinenone and canthaxanthin.

The antialgal activity of acti-dione.

Acti-dione inhibits the growth of Chlorophyceae, Xanthophyceae, and Bacillariophyceae at concentrations of 50 p.p.m. or less, but much higher concentrations have no inhibiting effect on the development of Myxophyceae. At concentrations of 50 to 100 p.p.m., it has been successfully employed in the isolation of unialgal cultures of two species of Myxophyceae. Haematococcus lacustris, a species of Chlorophyceae, has the lowest tolerance to Acti-dione of any microorganism yet found. Acti-dione interferes with cell division of this species at very low concentrations (0.016 to 0.032 p.p.m.), but much higher concentrations are required to inhibit motility. The effect of Acti-dione may be algistatic or algicidal depending upon the concentration used, the duration of the exposure, the species concerned, and the cell density.