Genetic polymorphism in eight Chilean strains of the carotenogenic microalga Dunaliella salina Teodoresco (Chlorophyta)

Eight Chilean strains of Dunaliella salina obtained within a restricted geographic range, but exhibiting a high variability in their morphology, rate of growth and carotenogenic capacity, were analyzed by Random Amplified Polymorphic DNA (RAPD-PCR) Twenty of the 50 random primers (D, P, OPA and OPD series) that were tested amplified reproducible bands and were useful for comparative analysis of the strains. Of 107 polymorphic genetic markers, 49 were strain-specific. A great genetic variability was found among the strains in spite of their geographic proximity. In addition, phenetic analysis of the data showed close agreement between the morphophysiological attributes and the genetic diversity of the strains.     

PCR-identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics

The saline pond microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's (basal) medium under laboratory conditions was confirmed by amplifying the chromosomal DNA of the microalga by PCR with specific primers MA1 and MA2. Seaweed extracts obtained from Sargassum wightii and Ulva lactuca were amended separately at 1.0%, 1.5%, 2.0% and 2.5% levels to the basal medium in order to assess their potential on the growth and concentration of pigments, viz. Chl a, Chl b and beta-carotene of the alga. beta-Carotene was isolated and visible absorption spectrum was taken at 443 and 475 nm confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene isomers. Maximum yield, highest division rate (mu) and highest pigment concentrations were observed in the cells grown in 1.5% S. wightii and 2.0% U. lactuca amended medium and these cells were subjected to DAPI staining. The results of epifluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear area of the microalgae.     

Effect of stressful conditions on the carotenogenic activity of a Colombian strain of Dunaliella salina

The objective was evaluate the carotenogenic activity of Dunaliella salina isolated from the artificial salt flats of municipality of Manaure (Department of La Guajira, Colombia). Two experimental testings were designed, in triplicate, to induce the reversibility of the cell tonality depending on the culture conditions. In the first test (A), to induce the reversibility from green to red tonality in D. salina cells, these were cultured in J/1 medium at a concentration of 4.0 M NaCl, 390 µmol m^?2 s^?1, 0.50 mM KNO₃. In the second test (B), to induce the reversibility from red to green cell tonality, the cultures were maintained in J/1 medium 1 M NaCl, 190 µmol m^?2 s^?1, 5.0 mM KNO₃ and pH 8.2. The population growth was evaluated by cell count and the pigment content was performed by spectrophotometric techniques. It was found that in both tests the culture conditions influenced the population growth and the pigments production of D. salina. There was a significant difference between the mean values of total carotenoids in the test A with 9.67 ± 0.19 μg/ml and second test with 1.54 ± 0.08 μg/ml at a significance level of p < 0.05. It was demonstrated that the culture conditions of test A induce the production of lipophilic antioxidants, among these carotenoids. The knowledge of the stressful conditions for the production of carotenoids from D. salina isolated from artificial saline of Manaure opens a field in implementation of this biotic resource for biotechnological purposes, production of new antibiotics, nutraceuticals and/or biofuels production.     

Bioenergetic and metabolic processes for the survival of sulfur-deprived Dunaliella salina (Chlorophyta)

The work addressed bioenergetic, metabolic and physiological responses ofthe green alga Dunaliella salina to sulfur (S)-deprivation. Photo-autotrophically grown cells were suspended in a medium in which thesulfates were replaced by chloride salts. Growth characteristics, pigmentcontent, rates of photosynthesis and respiration, as well as endogenoussubstrate (starch and protein) accumulation were monitored as a functionof time under S-deprivation. Lack of S from the growth medium had adifferential effect on photosynthesis and respiration. The rate oflight-saturated photosynthesis declined semi-exponentially with time,whereas the activity of respiration remained fairly constant over a periodof up to 100 h in S-deprived medium. Cell division and `packed cell volumeincrease' declined in tandem with the decline in the rate of photosynthesis. There was gradual loss of chlorophyll from the cells and a concomitant lossof photochemically competent system-II reaction centers, whereas theconcentration of system-I remained largely unaffected under S-deprivation. Cells altered the partition of photosynthate between starch and protein sothat control steady-state starch/protein ratios in the light (0.1: 1, w: w)gradually increased up to about 1: 1 as a function of S-deprivation. SealedDunaliella salina cultures, in which the capacity of photosynthesisdeclined to levels lower than that of respiration, consumed dissolvedoxygen and became anaerobic in the light. These cultures, however, did notactivate the reversible `hydrogenase pathway' and did not produceH₂ gas. Instead, under extended S-deprivation, cells maintained alow-level cycling of O₂ and CO₂ between photosynthesis andrespiration that resulted in no net exchange of gases. Such low-level cyclingof photosynthesis and respiration was sufficient to ensure the generation ofATP needed for survival of the organism under protracted S-deprivationconditions.     

Protective effect of Dunaliella salina (Volvocales, Chlorophyta) against experimentally induced fibrosarcoma on wistar rats

The beta-carotene-yielding microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's medium was harvested and lyophilized. Fibrosarcoma was induced in rats by 20-methylcholanthrene. 0.5 g and 1.0 g of lyophilized D. salina powder was administered to the rats orally through carboxy methyl cellulose. Cisplatin was administered along with vitamin E to compare the protective effect of D. salina against fibrosarcoma. Administration of D. salina decreased the levels of cholesterol and lactate dehydrogenase as well as the activities of catalase, superoxide dismutase, serum aspartate aminotransaminase, serum alanine aminotransferase, when compared to control. A significant reduction in the levels of hepatic and renal RNA and DNA was observed in the sarcoma rats when treated with D. salina powder. Histopathological studies of tumor tissues showed regenerative and regressive changes. beta-carotene globules isolated from the powder of Dunaliella salina confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene.     

Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta)

A stable transformation system for theexpression of foreign genes in theunicellular marine green alga Dunaliella salina was established. Amongfive antibiotics, 60 g mL^-1chloramphenicol completely inhibitedgrowth. Of five promoters tested, theubiquitin-$Ohgr; promoter yielded thehighest -glucuronidase (GUS) activity.The hepatitis B surface antigen (HBsAg)gene was introduced into the cells by usingelectroporation. PCR and Southern blotanalysis amd it was shown that the gene wasintegrated into the genome. The stableexpression of HBsAg protein was confirmedby enzyme-linked immunosorbent assay(ELISA) and Western blot analysis. Theintroduced DNA and HBsAg expression weremaintained stable for at least 60generations in medium devoid ofchloramphenicol. This is an important steptoward the production of useful foreignproteins in the alga.     

The effect of temperature on the post-embryonic growth of Neomysis intermedia Czernlawsky (Crustacea, Mysidacea) under laboratory conditions

The effect of temperature on post-embryonic growth of Neomysisintermedia was investigated under unlimited food conditionsin the laboratory. The effect of temperature on the size ofnewly released animals was negligibly small, but body size wasinversely related to temperature in adults. This was mainlycaused by the difference in the number of molts before maturation.The specific growth rate of N. intermedia increased exponentiallywith a temperature coefficient, Q₁₀ of 4.6 from 0.018 d^–13C to 0.21 d^–1 at 20C in juveniles, and with a temperaturecoefficient of 2.7 from 0.006 d^–1 at 3C to 0.05 d^–1at 25C in adults. The rate in juveniles levelled off above20C, and dropped at 29C. Brood size and brood interval decreasedwith temperature increase, while the daily specific reproductionrate increased. The specific growth rate of gravid females,including production of egg matter, increased exponentiallywith a temperature coefficient of 3.3 from 0.015 d^–1 at10C to 0.093 d^–1 at 25C. The present laboratory experiments confirmed the temperaturecontrol on the growth of N. intermedia suggested in a hyper-eutrophiclake.     

Characterization of a novel Dunaliella salina (Chlorophyta) strain and the assessment of its cultivation parameters

Physiological characteristics of a novel strain of the halophilic alga Dunaliella salina IPPAS D-232 isolated from the hypersaline lake Razval (Sol-Iletsk, Orenburg region) were studied. It was shown that the extract from the cells of this strain manifested antioxidant properties and also antibacterial activity relative to opportunistic bacteria. The influence of medium stirring and salinity on the level of D. salina antioxidant activity was assessed: it was established that medium salinization (93 g/L) and continuous stirring favored the highest accumulation of D. salina IPPAS D-232 biomass. It was found that the extremely halophilic archaea Halorubrum tebenquichense and moderately halophilic bacterium Marinococcus halophilus stimulated D. salina development and increased its antioxidant activity. The results obtained expand our knowledge of specific ecological features of the studied microalgal species; they could form the basis for the development of new approaches to the mass cultivation of D. salina.     

A mutant of the green alga Dunaliella salina constitutively accumulates zeaxanthin under all growth conditions

A novel mutant (zea1) of the halotolerant unicellular green alga Dunaliella salina is impaired in the zeaxanthin epoxidation reaction, thereby lacking a number of the beta-branch xanthophylls. HPLC analysis revealed that the zea1 mutant lacks neoxanthin (N), violaxanthin (V) and antheraxanthin (A) but constitutively accumulates zeaxanthin (Z). Under low-light physiological growth conditions, the zea1 (6 mg Z per g dry weight or 8 x 10(-16) mol Z/cell) had a substantially higher Z content than the wild type (0.2 mg Z per g dry weight or 0.5 x 10(-16) mol Z/cell). Lack of N, V, and A did not affect photosynthesis or growth of the zea1 strain. Biochemical analyses suggested that Z constitutively and quantitatively substitutes for N, V, and A in the zea1 strain. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin, a high-value bioproduct.     

Effect of salinity on the quantity and quality of carotenoids accumulated by Dunaliella salina (strain CONC-007) and Dunaliella bardawil (strain ATCC 30861) Chlorophyta

Dunaliella salina and D. bardawil are well-known microalgae accumulating high levels of beta-carotene under growth-limiting conditions. In both taxa, this pigment is primarily composed of the isomers 9-cis and all-trans. The 9-cis beta-carotene occurs only in natural sources and is the most attractive from a commercial point of view. The conditions that enhance the preferred accumulation of 9-cis beta-carotene in D. salina are controversial and they have not been well established yet. This study examined the effect of salinity on the quantity and quality of total carotenoids and beta-carotene isomers accumulated by D. salina (strain CONC-007) and D. bardawil (strain ATCC 30861) grown in two media with different nutritional compositions (PES and ART) and at salt concentrations of 1M, 2M and 3M NaCl. Total carotenoids were determined by spectrophotometry and beta-carotene isomers, by HPLC. The highest carotenoid contents per cell were obtained at 2M NaCl in both taxa. In both media, an increase of the 9-cis/all-trans beta-carotene ratio was observed in D. bardawil when the salt concentration increased, with a maximum value of 2.6 (in ART medium at 3M NaCl). In D. salina this ratio did not exhibit the same pattern, and the salt concentrations for maximal ratios were different in both media. The highest ratio obtained for this strain was 4.3 (in ART medium at 2M NaCl).     

Effect of nitrate concentration on growth and pigment synthesis of Dunaliella salina cultivated under low illumination and preadapted to different salinities

A wild strain of Dunaliella salina was isolated from a solar evaporation salt-pond in Araya (Estado Sucre, Venezuela) and grown in batch culture using relatively low illumination (80 μmol photon m-2 s-1). After the alga had been adapted to various salinities (9, 14, 21% w/v NaCl), the influence of nitrate concentration (882, 435, 212 μmol L-1 N) on growth rate and chlorophyll a and total carotenoid concentrations was measured. Low nitrate concentration negatively affected growth, but enhanced carotenoid accumulation. A slight increase in carotenogenesis was also observed in alga grown at the highest salinity. There were no significant additive or synergistic effects of salinity and nutrient concentrations on the concentrations of chlorophyll a or total carotenoid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of temperature and irradiance on the growth and reproduction of the green macroalga, Chaetomorpha valida (Cladophoraceae, Chlorophyta)

“Green tides” are vast accumulations of unattached green macroalgae associated with eutrophicated marine environments. It had major ecological and economic impacts globally, thus understanding of their detailed conditions was required to inform management decisions. Chaetomorpha valida (Cladophoraceae, Chlorophyta), as a fouling green alga, was found in aquaculture ponds along the coast of Dalian and Rongcheng cities, People’s Republic of China in the past few years. Seasonal abundances of this macroalga in China have presented a significant nuisance and caused great loss to the local aquaculture. Using unialgal cultures, the effect of temperature and irradiance on growth and reproduction were investigated. Healthy growth and reproduction occurred in the range of 17–29°C while the upper lethal limit was at 33°C. A suitable temperature range over 21–29°C and a relatively high irradiance of 108?μmol photons?m^?2?s^?1 were more favorable for growth and reproduction. According to our observations, the diversity of reproductive approaches and wide adaption to temperature can be considered to be key factors that facilitate its excessive growth and colonization in spring and summer. Our results provide a basis for establishing a forecast system and taking remediation measures against seasonal green tides of this macroalga.     

High temperature Chlorellaceae (Chlorophyta) strains from the Syrian-African Rift Valley: the effect of salinity and temperature on growth,morphology and sporulation mode

Two algal cultures, TvB and SH, were isolated from extreme habitats along the Syrian-African rift Valley (Israel). These cultures were initially identified as Chlorella spp. according to their morphology and lack of bristles, but following molecular phylogenetic analyses, re-identified as Micractinium spp. closely related to Chlorella. The strains were subjected to a bi-factorial study in the search for algae that grow well at elevated temperatures and salinities for future biotechnological uses. Cell density (CD) and optical density (OD) were measured for each strain at three temperatures: 35, 40 and 45ºC, and five salinities of seawater (SW): 34.8 ppt (100% SW), 26.5 ppt (75% SW), 18.3 ppt (50% SW) 10 ppt (25% SW) and 1.8 ppt (0% SW). Both strains grew best at 35–40ºC and at 0–50% SW. Increased salinity enhanced temperature tolerance to 45ºC, particularly for strain TvB. At 45ºC, following a short initial growth spurt, cultures underwent a lag period of c. 7 days, followed by a significant growth phase. During the lag period, algae underwent a substantial increase in average cell diameter (ACD). These enlarged or gigantic cells with diameters of up to ~20 μm, produced and eventually released multiple autospores. By day 13, original size distribution was almost restored. The observed morphological alterations appear to enable these strains to survive and grow autotrophically at supra-optimal temperatures (SOT). These natural adjustments may be exploited for reducing costs associated with both cooling and desalination in future cultivation.     

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.     

Evaluation of total reducing capacity in three Dunaliella salina (Dunal) Teodoresco isolates

To evaluate reducing capacity of the halophilic green alga Dunaliella salina, total reducing substances together with total carotenoids were extracted from three D. salina isolates at both the logarithmic and the stationary growth phases. Extractions were performed using hexane (H), ethyl acetate (EA), methanol (M), and water (W). Total reducing capacities of the extracts were determined by ferric ion reducing antioxidant power (FRAP), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), and Folin-Cioculteu (F-C) assay methods. As the best solvent, ethyl acetate was more efficient in extracting total carotenoids and total reducing substances, as is evident by the higher total reducing capacities measured by the FRAP, the DPPH, and the F-C assays. Isolate difference in reducing capacity was noticeable in the isolate MSI-2 with significantly higher extractable total reducing substances and total carotenoids. In the stationary growth phase, total reducing capacity was higher compared with the logarithmic growth phase; in particular, with water as the solvent, fivefold increase in total reducing capacity was observed. As a result, solvent extracting efficiency changed from EA?>?H, M, W at the logarithmic phase to EA?>?W?>?H, M at the stationary phase with the F-C assay, and from EA?>?H?>?M?>?W to EA?>?W?>?M?>?H (P?<?0.05) when the FRAP and the DPPH assays were used. Patterns of changes in total reducing capacity were similar in the three assays with highest correlations of 0.940 and 0.916 at P?<?0.05 between the F-C and the FRAP assays at the logarithmic and at the stationary growth phases, respectively. Weakest correlation (R ²?=?0.518, P?<?0.05) was observed between total carotenoids and the DPPH assay at the stationary growth phase. It is concluded that D. salina not only is a good source of β-carotene, but also, its reducing substances may contribute to the antioxidant capacity of this microalga.     

The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta)

It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.     

The effect of acute high light and low temperature stresses on the ascorbate–glutathione cycle and superoxide dismutase activity in two Dunaliella salina strains

Dunaliella species accumulate carotenoids and their role in protection against photooxidative stress has been investigated extensively. By contrast, the role of other antioxidants in this alga, has received less attention. Therefore, the components of the ascorbate–glutathione cycle, along with superoxide dismutase (E.C. 1.15.1.1) and peroxidase (E.C. 1.11.1.11) activity were compared in two strains of Dunaliella salina. Strain IR‐1 had two‐fold higher chlorophyll and β‐carotene concentration than Gh‐U. IR‐1 had around four‐fold higher superoxide dismutase, ascorbate peroxidase and pyrogallol peroxidase activities than Gh‐U on a protein basis. Ascorbate and glutathione concentrations and redox state did not differ between strains and there was little difference in the activity of ascorbate–glutathione cycle enzymes (monodehydroascorbate reductase [E.C. 1.6.5.4], dehydroascorbate reductase [E.C. 1.8.5.1] and glutathione reductase [E.C. 1.8.1.7]). The response of these antioxidants to high light and low temperature was assessed by transferring cells from normal growth conditions (28°C, photon flux density of 100 μmol m^?2 s^?1)to 28°C/1200 μmol m^?2 s^?1; 13°C/100 μmol m^?2 s^?1; 13°C/1200 μmol m^?2 s^?1 and 28°C/100 μmol m^?2 s^?1 for 24 h. Low temperature and combined high light‐low temperature decreased chlorophyll and β‐carotene in both strains indicating that these treatments cause photooxidative stress. High light, low temperature and combined high light‐low temperature treatments increased the total ascorbate pool by 10–50% and the total glutathione pool by 20–100% with no consistent effect on their redox state. Activities of ascorbate–glutathione cycle enzymes were not greatly affected but all the treatments increased superoxide dismutase activity. It is concluded that D. salina can partially adjust to photooxidative conditions by increasing superoxide dismutase activity, ascorbate and glutathione.