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PubMed2003

Nitric oxide alleviates oxidative damage in the green alga Chlorella pyrenoidosa caused by UV-B radiation.

The effect of ultraviolet-B radiation (UV-B; 280-320 nm) on induction of nitric oxide was estimated in the suspensions of green alga Chlorella pyrenoidosa with or without the NO scavenger N-acetyl-L-cysteine, and reductants such as 1,4-dithiothreitol, glutathione (reduced form), and ascorbic acid. Exogenously added sodium nitroprusside (NO donor), glutathione, 1,4-dithiothreitol, and ascorbic acid were able to prevent chlorophyll loss mediated by UV-B. Addition of NO to algal suspensions irradiated by UV-B increased the activity of catalase and superoxide dismutase but lowered the activity of phenylalanine ammonia-lyase. UV-B thus appears to be a strong inducer of NO production, exogenously added NO and reductants protecting the green alga against UV-B-induced oxidative damage.

Acetylcysteine↗
PubMed1999

Expression of human growth hormone by the eukaryotic alga, Chlorella.

A method to use Chlorella to express a recombinant heterologous protein that can be recovered from the extracellular medium has been developed. Plasmids are constructed with an extracellular secretion signal sequence inserted between a promoter region and a gene for human growth hormone (hGH). The plasmids also contain a Kanr region which confers resistance to the antibiotic G418. Protoplasts are prepared by enzymatic treatment, and the plasmid is introduced by incubation of the protoplasts with polyethylene glycol and dimethyl sulfoxide. Cells are then grown in the presence of G418, and the medium is collected from 6 days after transfection. hGH is measured by immunoassay, and values for expressed hGH of about 200-600 ng/ml are obtained.

Amino Acid Sequence↗
PubMed2002

PAM fluorometry in the determination of the sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper.

The pulse-amplitude-modulation (PAM) fluorometric method was used in the past as a sensitive and rapid method for assessing toxic effect of pollutants in plants. Here, we used the advantages of this method to evaluate the difference in sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper toxicity. We found by using the PAM-fluorescence parameters (phiM, phi'M, QP, and QN), that C. reinhardtii is the most sensitive species, followed by S. capricornutum and C. vulgaris. Indeed, the copper 5-, 48-, and 96-h EC50 for phiM, phi'M, and QP vary between 11 and 34 microg/L for C. reinhardtii, while for S. capricornutum this value is between 25 and 50 microg/L. However, the same parameters obtained from C. vulgaris did not show susceptibility to copper, even at very high concentrations (100 microg/L). In this study, the estimated 5-h EC50 for C. reinhardtii and S. capricornutum are lower than the 24-h EC50 reported earlier for these species with other bioassays, indicating that the use of PAM fluorometry in copper bioassay provides a more sensitive method than the other biotests in aquatic toxicology.

Biological Assay↗
PubMed2000

Reclamation of wastewater for polyculture of freshwater fish: bioassays using Chlorella and Gambusia.

The feasibility of using the secondary effluents from two sewage treatment plants in Hong Kong (Yuen Long and Shek Wu Hui) for fish culture was assessed. Total ammonia contents in the two sewage effluents surpassed the level of 2 mg L(-1) recommended for wastewater fish culture. The two sewage effluents both needed essential elements for supporting algal Chlorella vulgaris growth, whereas only sewage effluent from Yuen Long had contaminants at toxic levels. Total ammonia in water explained more than 80% variations of toxicity of water samples to Gambusia patruelis, mosquito fish, according to regression analysis. Removal of ammonia from the sewage effluent is necessary before being used for fish culture. In addition, the rather high levels of heavy metals (Cu, Zn, Cd) in the effluent should be closely monitored.

Ammonia↗
PubMed2003

Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta).

When grown photoautotrophically, Chlorella zofingiensis strain CCAP 211/14 accumulates a significant amount of valuable carotenoids, namely astaxanthin and lutein, of increasing demand for use as feed additives in fish and poultry farming, as colorants in food, and in health care products. Under standard batch-culture conditions, this microalgal strain exhibits high values of both growth rate (about 0.04 h(-1)) and standing cell population (over 10(11) cells l(-1), or 7 g dry weight l(-1)). Lutein, in a free (unesterified) form, was the prevalent carotenoid during early stages of cultivation (over 0.3 pg cell(-1), equal to 4 mg g(-1) dry weight, or 20 mg l(-1) culture), whereas esterified astaxanthin accumulated progressively, to reach a maximum (over 0.1 pg cell(-1), equal to 1.5 mg g(-1) dry weight, or 15 mg l(-1) culture) in the late stationary phase. A differential response of lutein and astaxanthin accumulation was also recorded with regard to the action of some environmental and nutritional factors. C. zofingiensis CCAP 211/14 represents a unique model system for analyzing the differential regulation of the levels of primary (lutein) and secondary (astaxanthin) carotenoids. Relevant also from the biotechnological viewpoint, this photosynthetic organism, with outstanding attributes for fast photosynthetic growth and carotenoid accumulation, might prove most valuable for its application to the mass production of either or both lutein and astaxanthin.

Chlorella↗
PubMed2005

Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta).

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a beta-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting beta-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4'-beta-ionone ring oxygenase.

Blotting, Southern↗
PubMed2001

Concurrent sorption of Ni2+ and Cu2+ by Chlorella vulgaris from a binary metal solution.

Kinetics and capacity of Ni2+ and Cu2+ sorption by Chlorella vulgaris were studied using single and binary metal solutions at various concentrations of these metal ions. The second-order rate law best described the kinetics of metal sorption from both single and binary metal systems. C. vulgaris preferentially sorbed Cu2+ over Ni2+ in the binary system. In comparison to the single metal system, the amounts of Ni2+ and Cu2+ sorbed at equilibrium (qe) were respectively 73% and 25%, and the initial rate of sorption (h) was ca. 50% in the case of the binary metal system. The test metals inhibited sorption of each other, thereby indicating competition between Ni2+ and Cu2+ for sorption onto non-specific binding sites. The present study showed that C. vulgaris has specific as well as non-specific sites for the binding of Ni2+ and Cu2+. Participation of these sites for sorption depended on the ratio of Ni2+ and CU2+ in solution. The maximum metal sorption capacity of C. vulgaris was 6.75 mmol g(-1) from the binary metal solution at the tested biomass concentration (100 mg dry weight l(-1)). Total metal sorption was enhanced with increasing total concentration of both the metals up to 1.6 mM, beyond which a decrease occurred. Two-dimensional contour plots were successfully used for the first time for the evaluation of metal sorption potential.

Adsorption↗
PubMed2001

Attenuation of monochromatic and polychromatic lights in Chlorella vulgaris suspensions.

A quantitative description of light attenuation in microalgal suspensions is a prerequisite for kinetic modeling of microalgal photosynthesis and/or growth activity depending upon the light distribution inside photobioreactors. In this study, the light attenuation coefficients in Chlorella vulgaris suspensions were theoretically calculated from light absorption spectra and spectral irradiances of various light sources. By using this method, errors occurring in the direct measurement of the attenuation coefficients can be avoided. The obtained light attenuation coefficients were used for evaluating light attenuation models such as the Beer-Lambert, Cornet, and hyperbolic models. Furthermore, advantages and disadvantages of these models are discussed with respect to prediction of performance, mechanistic background, and usefulness for further application to calculation of the light distribution inside photobioreactors.

Bioreactors↗
PubMed1996

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil.

An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD50 value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4-9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent.

Animals↗
PubMed1998

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation.

A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25+, CD54+, CD69+ or CD71+ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression.

Animals↗
PubMed2003

Temperature-induced greening of Chlorella vulgaris. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching.

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.

Chlorella↗
PubMed2003

Effects of brassinazole, an inhibitor of brassinosteroid biosynthesis, on light- and dark-grown Chlorella vulgaris.

Treatment of cultured Chlorella vulgaris Beijerinck cells with 0.1-10 microM brassinazole (Brz2001), an inhibitor of brassinosteroid (BR) biosynthesis, inhibits their growth during the first 48 h of cultivation in the light. This inhibition is prevented by the co-application of BR. This result suggests that the presence of endogenous BRs during the initial steps of the C. vulgaris cell cycle is indispensable for their normal growth in the light. In darkness, a treatment with 10 nM brassinolide (BL) promotes growth through the first 24 h of culture, but during the following 24 h the cells undergo complete stagnation. Treatment of dark-grown cells with either Brz2001 alone, or a mixture of 10 nM BL and 0.1/10 microM Brz2001, also stimulates their growth. The effects of treatment with 10 nM BL mixed with 0.1-10 microM of a mevalonate-pathway inhibitor (mevinolin), or a non-mevalonate-pathway inhibitor (clomazone), were also investigated. Mevinolin at these concentrations did not inhibit growth of C. vulgaris; however, clomazone did. Addition of BL overcame the inhibition. These results suggest that the mevalonate pathway does not function in C. vulgaris, and that the non-mevalonate pathway for isopentenyl diphosphate biosynthesis is responsible for the synthesis of one of the primary precursors in BR biosynthesis.

Brassinosteroids↗
PubMed2006

Granule-bound starch synthase cDNA in Chlorella kessleri 11 h: cloning and regulation of expression by CO(2) concentration.

The cDNA for the granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) of Chlorella kessleri 11 h was isolated and characterized. CkGBSS encodes a 609-amino acid polypeptide (65,627 Da) that includes an N-terminal hydrophobic signal peptide of 55 amino acids. The deduced amino acid sequence of the mature CkGBSS polypeptide shares a greater identity (65%) to that of the GBSS protein of Chlamydomonas reinhardtii, than to those of vascular plant species, but does not have the extra-long C-terminal sequence found in C. reinhardtii. When CO(2 )concentration was decreased from 3 to 0.04% (air level) in light, the levels of CkGBSS mRNA, CkGBSS protein, and GBSS activity increased. Under this condition, pyrenoid and pyrenoid starch developed, and the relative amount of amylose in starch increased. These observations suggest that low CO(2) level up-regulates GBSS biosynthesis at the transcriptional level.

Algal Proteins↗
PubMed2000

The role of growth rate, redox-state of the plastoquinone pool and the trans-thylakoid deltapH in photoacclimation of Chlorella vulgaris to growth irradiance and temperature.

The long-term photoacclimation of Chlorella vulgaris Beijer (UTEX 265) to growth irradiance and growth temperature under ambient CO2 conditions was examined. While cultures grew at a faster rate at 27 than at 5 degrees C, growth rates appeared to be independent of irradiance. Decreases in light-harvesting polypeptide accumulation, increases in xanthophyll pool size and changes in the epoxidation state of the xanthophyll cycle pigments were correlated linearly with increases in the relative reduction state of QA, the primary quinone receptor of photosystem II, when estimated as 1-qP under steady-state growth conditions. However, we show that there is also a specific temperature-dependent component, in addition to the redox-state of the QA, involved in regulating the content and composition of light-harvesting complex II of C. vulgaris. In contrast, modulation of the epoxidation state of the xanthophyll pool in response to increased 1-qP in cells grown at 5 degrees C was indistinguishable from that of cells grown at 27 degrees C, indicating that light and temperature interact in a similar way to regulate xanthophyll cycle activity in C. vulgaris. Because C. vulgaris exhibited a low-light phenotype in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but a high-light phenotype upon addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone, we conclude that the plastoquinone pool acts as a sensor regulating the accumulation of light-harvesting polypeptides in C. vulgaris. However, concomitant measurements of non-photochemical fluorescence quenching (qN) and the epoxidation state of the xanthophyll pool appear to indicate that, in addition to the redox-state of the plastoquinone pool, the trans-thylakoid deltapH may also contribute to sensing changes in irradiance and temperature that would lead to over-excitation of the photosynthetic apparatus. We suggest that sink capacity as reflected in photosynthate utilization and cell growth ultimately regulate photoacclimation in C. vulgaris.

Adaptation, Physiological↗
PubMed2006

Symbiotic alga Chlorella vulgaris of the ciliate Paramecium bursaria shows temporary resistance to host lysosomal enzymes during the early infection process.

Paramecium bursaria free of symbiotic Chlorella species can be experimentally reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. Isolated symbiotic algae were cloned, mixed with the algae-free P. bursaria at 25 +/- 1 degrees C for 1.5 min, washed and chased, with or without fixation 3 h after mixing. Though genetically identical, a few of the algae were not digested but coexisted with the digested ones in the same vacuole after lysosomal fusion. Light microscopy showed that algal fate did not depend on cell cycle stage or location in the vacuole. Electron microscopy showed that the nondigested algae were not protected by a perialgal vacuole membrane in the digestive vacuole. Moreover, this phenomenon was also observed in the presence of cycloheximide and puromycin, which are known to inhibit algal and host protein synthesis, respectively. These observations suggest that a few algae can acquire temporary resistance to host lysosomal enzymes in order to establish endosymbiosis without algal protein synthesis.

Animals↗
PubMed2004

Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlorella vulgaris (Chlorophyta, Trebouxiophyceae).

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17-21 nm) emerged as a small, but complete, autospore.

Cell Cycle↗
PubMed2004

Lycopene accumulation and cyclic carotenoid deficiency in heterotrophic Chlorella treated with nicotine.

We studied the effects of nicotine on Chlorella regularis Y-21 grown under heterotrophic conditions. Nicotine repressed growth, doubled cell size, and changed the culture coloration from dark-green to orange. These effects are likely due to the change of the chloroplast into a red irregular vesicle. This morphological change was associated with alteration of the carotenoid composition. Lycopene accounted for more than 80% of total carotenoids in nicotine-treated cells. The red irregular vesicles had a high electron density; we supposed them to be immature chloroplasts accumulating lycopene.

Carotenoids↗
PubMed2005

Cell volume distribution dynamics of Chlorella vulgaris Beij. in batch cultures under continuous light.

Cell volume distribution in Chlorella vulgaris cultures coming out of senescence was measured by flow cytometry every 6 h for 114 h in a full-factorial experiment with initial nitrate (420-4200 microg NO3-N l(-1)), phosphate (9-186 microg PO4-P l(-1)), and continuous light (50-330 microE m(-2) s(-1)) as treatments. The maxima in median and median absolute deviation (MAD) of cell volume were achieved within 6 h of each other and their timing was not affected by any treatment. Population specific growth rate during the first 66 h calculated from volume distribution changes was significantly affected by light treatment only (p=0.002).

Bioreactors↗