Increase of the yields of eicosapentaenoic and docosahexaenoic acids by the microalga Pavlova lutheri following random mutagenesis

The high commercial values of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids have driven a strain-improvement program, aimed at increasing the content of those fatty acids in the microalga Pavlova lutheri (SMBA 60) as parent strain. After a round of mutation using UV-light as mutagenic agent, an isolate strain (tentatively called II#2) was obtained, the EPA and DHA contents of which (in % dry biomass) were 32.8% and 32.9% higher than those of the control, native strain. The final EPA yields, when the cultures were maintained under appropriate conditions, were 17.4 and 23.1 mg. g(-1) dry biomass, for the wild-type and the II#2 strain, respectively, whereas the final DHA yields were 8.0 and 10.6 mg. g(-1) dry biomass, respectively. These results suggest that random mutagenesis can successfully be applied to increase the yield of n-3 fatty acids by microalgae.     

Identification of a very long chain polyunsaturated fatty acid Delta4-desaturase from the microalga Pavlova lutheri

Pavlova lutheri, a marine microalga, is rich in the very long chain polyunsaturated fatty acids (VLCPUFAs) eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Using an expressed sequence tag approach, we isolated a cDNA designated Pldes1, and encoding an amino acid sequence showing high similarity with polyunsaturated fatty acid front-end desaturases. Heterologous expression in yeast demonstrated that PlDES1 desaturated 22:5n-3 and 22:4n-6 into 22:6n-3 and 22:5n-6 respectively, and was equally active on both substrates. Thus, PlDES1 is a novel VLCPUFA Delta4-desaturase. Pldes1 expression is four-fold higher during the mid-exponential phase of growth compared to late exponential and stationary phases.     

Polyunsaturated Fatty Acids and Betaine Lipids from Pavlova lutheri

The polar lipids and fatty acids of the microalgae Pavlova lutheriwere analyzed. The principal polar lipid components were monogalactosyldiacylglycerol(MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol(SQDG), 1,2-diacylglyceryl-O-2'-hydroxymethyl-(N,N,N-trimethyl)-rß-alanine(DGTA) and 1,2-diacylglyceryl-3-O-carboxyhydroxymethylcholine(DGCC). Each polar lipid had a different set of combinationsof fatty acids, the most characteristic feature being the localizationof polyunsaturated fatty acids in the betaine lipids. The percentagesof polyunsaturated fatty acids in DGTA and DGCC were 70% and50%, respectively, and these fatty acids were localized at theC-2 position in the betaine lipids. An analysis of the incorporationof ¹⁴C-labelled compounds into the algal cells indicated theinvolvement of DGCC in acyl exchange. (Received October 17, 1994; Accepted October 2, 1995)     

Kinetic modeling of the autotrophic growth of Pavlova lutheri: study of the combined influence of light and temperature

The optimization and control of biochemical processes require the previous establishment of mathematical models that can describe the effect of process variables on their actual kinetics. Environmental temperature is a modulating factor to which the algal cells respond continuously by adjusting their rates of cellular reactions, their nutritional requirements, and, consequently, their biomass composition. Light intensity is an exhaustible resource, indispensable to autotrophic organisms. The effects of light intensity and temperature on growth of the microalga Pavlova lutheri, which have hardly been considered to date in a simultaneous fashion, were experimentally assessed using a factorial experimental design; in this way, the effects of each variable independently and their interactions could be quantified, using maximum biomass (X(max)) or maximum specific growth rate (mu(max)) as objective functions. The preliminary results produced indicated that light intensity plays a more important role on mu(max) than temperature; in the case of X(max), both temperature and, to a lesser extent, light intensity do apparently play a role. The highest values of X(max) were associated with low temperatures and high light intensities; a similar behavior could be observed for mu(max) concerning light intensity, although the dependency on temperature did not seem to be as important. A more complex mechanistic model was then postulated, incorporating light and temperature as input variables, which was successfully fitted to the experimental data generated during batch cultivation of P. lutheri.     

Effects of photoperiod,salinity and pH on cell growth and lipid content of Pavlova lutheri

The aim of the present work was to study the effects of photoperiod, salinity and pH on growth and lipid content of Pavlova lutheri microalgae for biodiesel production in small-scale and large-scale open-pond tanks. In a 250-mL flask, the cultures grew well under 24 h illumination with maximum specific growth rate, μ _max , of 0.12 day^?1 and lipid content of 35 % as compared to 0.1 day^?1 and 15 % lipid content in the dark. The salinity was optimum for the cell growth at 30–35 ppt, but the lipid content of 34–36 % was higher at 35–40 ppt. Algal growth and lipid accumulation was optimum at pH 8–9. Large-scale cultivation in 5-L and 30-L tanks achieved μ _max of 0.13–0.14 day^?1 as compared to 0.12 day^?1 in small-scale and 300L cultures.     

Productivity and biochemical composition of cyclostat cultures of the marine microalga Tetraselmis suecica

Cyclostat, light/dark-synchronized cultures of the marine microalga Tetraselmis suecica were carried out with six nutrient concentrations: 0.5, 1, 2, 4, 8, and 16 mmol N/l. A renewal of 50% culture volume, equivalent to a dilution rate of 1 day^–1 was applied. Maximal steady-state cell density (2.5 × 10⁶ cells/ml) and dry-weight productivity (0.2 g l^–1 day^–1) were achieved with 4 mmol N/l. Maximal protein (94.3 pg/cell) and lipid contents (25.4 pg/cell) were achieved with8 mmol N/l. The high variability reported in chlorophyll cellular content invalidates any estimation of biomass or cell number based on chlorophyll concentration or fluorescence, especially when nutrient concentration is being varied. The total fatty acid cellular content increased with increasing nutrient concentration although the amount of eicosapentaenoic acid decreased. Results indicated that a different desaturation pathway may be present in this species. The change of the nutrient concentration in the cyclostat system is a powerful tool to manipulate the biochemical composition of microalgae regarding protein, lipid, carbohydrates and fatty acid content.     

Influence of irradiance on the growth and biochemical composition of Nitzschia aff. pellucida

Journal of Applied Phycology - In this paper the influence of irradiance and the operation modes on biomass production and biochemical composition, especially the fatty acid profile, on the diatom...     

Effect of organic carbon sources and environmental factors on cell growth and lipid content of Pavlova lutheri

The present study aimed to investigate the effects of organic carbon sources, cultivation methods, and environmental factors on growth and lipid content of Pavlova lutheri for biodiesel production. In the 250-mL flask bioreactors, P. lutheri was cultivated in the modified artificial seawater (ASW) medium containing glucose, glycerol, sodium acetate, or sucrose as an organic carbon substrate. The effects of different growth conditions (phototrophic, mixotrophic, and heterotrophic) and environmental factors such as photoperiod, light intensity, and salinity were evaluated. Growth of P. lutheri was inhibited under heterotrophy but was enhanced in mixotrophy as compared to phototrophy. Biomass and lipid content of P. lutheri were significantly (p < 0.05) affected by changing photoperiod, light intensity, and salinity. Higher biomass concentration and lipid content were observed at a light intensity of 100 ± 2 μmol photons m−2 s−1, 18 h photoperiod, and 30% salinity, in a modified ASW medium supplemented with 10 mmol sucrose. An increase in biomass concentration from 320 ± 25.53 to 1106 ± 18.52 mg L−1 and high lipid content of 31.11 ± 1.65% (w/w) were observed with the optimized culture conditions, demonstrating a significant (p < 0.05) enhancement in biomass and lipid content due to the improved culture conditions. The present study emphasizes the possible use of sucrose for biomass and lipid production with P. lutheri under the optimized culture conditions. Using low-cost and relatively easy accessible feedstock such as sucrose would be a valuable alternative for growing microalgae with enhanced lipid content.     

Isolation of two cDNAs encoding E2 ubiquitin-conjugating enzymes from Pavlova lutheri (Haptophyceae)

Two novel cDNAs, Plubc1 and Plubc2, encoding ubiquitin-conjugatingenzyme E2, were isolated from a Pavlova lutheri cDNA library. They areeach encoded by single copy genes in thealgae genome. Sequence comparison withplant, yeast and algal E2 sequences showedthat PlUBC1 and PlUBC2 are members of newE2 subfamilies. Time-course expressionanalysis of the two cDNAs revealed thatPlubc1 is transitionallyover-expressed at the end of theexponential phase of growth of the culture,while Plubc2 is constitutivelyexpressed at the same level throughout thecell growth. The phylogenetic study and thedifferent expression patterns suggest thatthese two enzymes could exhibit differentphysiological functions in P. lutheri.The partial sequence of the 18S rRNA geneand the full-length cDNA sequence of Plubc1 and Plubc2 reported in thispaper will appear in the Genbank databaseunder the accession numbers AY135218,AY135219 and AY135220 respectively.     

Effect of nitrogen limitation on growth,biochemical composition,and cell ultrastructure of the microalga Picocystis salinarum

Journal of Applied Phycology - In recent years biodiesel production has attracted worldwide attention due to the awareness of fossil fuel depletion and microalgae biomass is considered a promising...     

Effects of phosphorus-induced changes on the growth,nitrogen uptake,and biochemical composition of Pavlova pinguis and Hemiselmis cf. andersenii

Journal of Applied Phycology - The understanding of the phosphorus-induced changes in the biochemical composition of microalgae is of great importance for achieving efficiency in high-value lipid...     

Glycolate Metabolism in Low and High CO(2)-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by O-Labeling

Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring ¹⁸O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO₂-grown cells than in air-grown cells when both were assayed under the same O₂ and CO₂ concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O₂ levels and inhibited by high CO₂ levels. Glycolate synthesis in 1.5% CO₂-grown Chlorella, when exposed to a 0.035% CO₂ atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O₂ concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O₂ concentration was lowered to 2% or the CO₂ concentration was raised to 1.5%. Glycolate excretion was also sensitive to O₂ and CO₂ concentrations in 1.5% CO₂-grown cells and the glycolate that was excreted was ¹⁸O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory ¹⁸O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.     

Stimulation or inhibition of growth of the unicellular alga Pavlova lutheri by bacteria isolated from larval turbot culture systems

During growth of larval turbot in aquaculture the first food supplied is usually the rotifer, Brachionus plicatilis and algae are commonly included in the system as food for the rotifers, thereby maintaining their nutrient quality. As bacteria are known to influence markedly the survival of larval turbot, the effect of bacteria, isolated from larval turbot, on growth of Pavlova lutheri was measured over a 3-d period. Of 41 bacteria tested, 23 inhibited growth to various degrees, eight had no effect and 10 were weak growth stimulants. Four bacteria, identified as a Flavobacterium, Vibrio fluvialis, Vibrio natrigens and a Vibrio sp., were strongly inhibitory and the Flavobacterium inhibited growth of Pavlova lutheri from an inoculum of 10³ colony-forming units per ml. Inhibition was due to a heat-labile factor released by the Flavobacterium into the culture medium. The Flavobacterium also produced bacteriocin(s) which inhibited the growth of a range of vibrios. Bacteria antagonistic towards algae would be undesirable in larval rearing and if bacteria are to be selected which are beneficial (probiotics) in larval rearing systems their possible interaction with algae must be considered.     

Combined effects of irradiance level and carbon source on fatty acid and lipid class composition in the microalga Pavlova lutheri commonly used in mariculture

Pavlova lutheri, a marine Pavlovophyceae, has been well documented as it is commonly used as a food source in mariculture. In this study, we investigated the combined effects of carbon sources and irradiance levels on the growth, lipid classes and fatty acid profiles of this microalga. The microalga was cultured at 15 °C with a 14 h photoperiod in artificial seawater containing bicarbonate or acetate as carbon source.The growth and lipid composition of P. lutheri were more sensitive to variations in light intensity than in carbon source. However, P. lutheri seems to be able to use acetate to growth cell and lipid metabolism. With the both carbon source, the lowest cellular lipid contents were obtained under low light intensity. The proportions of PUFAs, especially EPA, were significantly higher under low light, and saturating fatty acids and DHA levels were significantly higher under high light. In P. lutheri, galactolipids, a major component of chloroplast lipid membranes, made up approximately 54-66% of total lipids. The highest PUFA levels, such as those of EPA, were predominantly found in the galactolipid fraction when the cells were grown at low light, regardless of the carbon source. The consequent accumulation of n-3 fatty acids in the galactolipids could facilitate thylakoid membrane fluidity, and therefore the velocity of electron flow involved in photosynthesis during light acclimatization. These results could be used to optimize the culture conditions and the nutritional value of this microalga, which is used to feed marine invertebrates and fish larvae in mariculture hatcheries, and to produce n-3 fatty acids for human health care and nutrition.     

Coupled effects of irradiance and iron on the growth of a harmful algal bloom-causing microalga Scrippsiella trochoidea

Effects of irradiance and iron on the growth of a typical harmful algal blooms (HABs) causative dinoflagellate, Scrippsiella trochoidea, were investigated under various irradiances (high light: 70 μmol m^?2 s^?1 and low light: 4 μmol m^?2 s^?1) and iron concentrations (low iron: 0.063 mg L^?1, medium iron: 0.63 mg L^?1 and high iron: 6.3 mg L^?1), and evaluated by the parameters of algal cell density, specific growth rate, optical density and chlorophyll a content. The results indicated that there was significant difference in the cell density of dinoflagellate S. trochoidea between high light and low light intensity treatments across the entire experiments, 7-fold higher at high irradiance as compared with low irradiance, which was further enhanced by the iron concentration. It was found that the maximum cell density of 25 × 10⁴ cell mL^?1 occurred under the combination of high light intensity and high iron concentration, followed by 23 × 10⁴ cell mL^?1 in the combination of high light and medium iron, and 20 × 10⁴ cell mL^?1 in the combination of high light and low iron. There was no significant effect of iron concentration on the cell density under low light intensity. The cell density maintained about 3 × 10⁴ cell mL^?1 across all combinations of iron concentrations and low light in the end of experiments. Such interactive effects of light intensity and iron level dependent were also observed for the specific growth rate, OD₆₈₀ and chlorophyll a content of S. trochoidea. The maximum values of specific growth rate, OD₆₈₀ and chlorophyll a content peaked at the condition of high irradiance and high iron, which were 0.22 d^?1, 0.282 and 0.673 mg L^?1, respectively. In general, their values increased significantly with the increasing of iron concentration at high irradiance, whereas no significant difference was observed among three iron concentrations at low irradiance, all remaining approximately 0.06 d^?1, 0.03 and 0.050 mg L^?1, respectively. Those results suggest that there may be a strong interactive effect between irradiance and iron on microalgal growth and their physiological characteristics. The combination of high light and high iron concentration may accelerate algal cell growth and pigment biosynthesis, thus leading to massive occurrence of HABs.     

Effect of high irradiance and high temperature on chloroplast composition and structure of Dioscorea zingiberensis

High irradiance (HI) and high temperature (HT) increased in chloroplasts the content of monogalactosyldiacylglycerol (MGDG) and decreased the contents of digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylinositol (PI). HI and HT accelerated the transformation of DGDG to MGDG. The contents of unsaturated fatty acids in chloroplasts increased, while those of saturated fatty acids decreased. The contents of total carotenoids, neoxanthin, violaxanthin, lutein, and -carotene increased first, then decreased. The content of chlorophyll decreased. HI caused the unfolding of thylakoids that was not resumed after a 72-h recovery.This revised version was published online in March 2005 with corrections to the page numbers.     

The effect of nitrogen starvation on the ultrastructure and pigment composition of chloroplasts in the acidothermophilic microalga Galdieria sulphuraria

We studied the effect of nitrogen starvation on growth indices, vitality, ultrastructure, and the photosynthetic apparatus of unique acidothermophilic microalga Galdieria sulphuraria (Galdieri) Merola. Long-term nitrogen starvation ceased G. sulphuraria growth and cell division. During the first days of starvation, phycobiliproteins degraded first, then the content of chlorophyll and carotenoids decreased to trace amounts, chloroplast reduced, cell wall became thinner, and storage compounds accumulated. However, the cells were alive. A comparison with the effects of nitrogen starvation on other photosynthesizing organisms showed that suppression of cell division, reduction of the photosynthetic apparatus to some minimum, and accumulation of storage compounds are a universal response to this stress.     

The effect of temperature on the lipid composition of Botryococcus

The lipid composition of the green alga Botryococcus was studied at three different cultivation temperatures: suboptimal, optimal, and supraoptimal. Cultivation at the supraoptimal temperature was found to considerably inhibit the synthesis of nearly all intracellular lipids, except for triacylglycerides, and to influence their fatty acid composition. In particular, the content of trienoic fatty acids was significantly lower at the supraoptimal than at the optimal cultivation temperature. At the same time, the fatty acid composition of the extracellular lipids of the alga virtually did not depend on cultivation temperature.     

温度和光照强度对鼠尾藻生长和生化组成的影响

在实验室条件下,研究了温度(10、15、20、25 ℃)和光照强度(20、60、100、140、180 μE·m^-2·s^-1)对鼠尾藻生长和生化组成的影响.结果表明,温度、光照强度及两者的交互作用对鼠尾藻生长均具有极显著影响.鼠尾藻在15 ℃和20 ℃下生长速率较高,随着温度的升高,达到最大生长速率所需要的光照强度有上升趋势.在10 ℃和15 ℃下,较高的光照强度对鼠尾藻生长产生了一定抑制作用,而在20 ℃和25 ℃下,其生长速率总体随光照强度的增加而增加.温度和光照强度对鼠尾藻叶绿素a、墨角藻黄素的含量影响极显著,其中光照强度的影响大于温度.总体上,叶绿素a和墨角藻黄素的含量均随着光照强度的升高而显著下降,随着温度的升高而升高.鼠尾藻碳水化合物含量随着光照强度的升高而显著升高,而温度对碳水化合物含量影响不显著.鼠尾藻蛋白质含量随着光照强度的增加而显著下降,在10 ℃和15 ℃下含量较高,随着温度的继续升高,蛋白质含量呈下降趋势.光照强度和温度的变化可改变鼠尾藻的藻体成分含量,这种改变可能是鼠尾藻为了适应环境因子改变而做出的积极的生理调节,对其生长和生存具有重要的生态意义.     

The effect of temperature,irradiance and animal size on incorporation rates of Simocephalus vetulus

Carbon incorporation rates of Simocephalus vetulus were measured to study the effects of the physical state of the animals, size of the animal, varying temperature and light conditions. Physical state of the animal showed little effect on incorporation rates after the first hour. Incorporation rates increased in proportion to the third power of animal size. Experimental animals collected at temperatures of 12, 20 or 25°C fed maximally at 10, 15 and 25°C respectively, when subjected to a feeding temperature range of 5 to 30°C. We have interpreted this as an indication that S. vetulus is able to acclimate and incorporate maximally at various temperatures after prolonged exposure to that temperature. When fed over an irradiation range of 0 to 4.68 × 10^–3 cal cm^–2 s^–1 incorporation rates were indirectly proportional to irradiance. This suggests a response to decreased irradiance in the weedy, littoral habitat of these animals.