Marine Microalga Isochrysis Galbana Research Articles

Immobilized Isochrysis galbana (Haptophyta) for long-term storage and applications for feed and water quality control in clam (Meretrix lusoria) cultures

The marine microalga Isochrysisgalbana was cultivated and entrapped inalginate beads for long-term storage. Theentrapped cells were alive and maintainedtheir physiological activities after oneyear of storage in absolute darkness at4 °C without a liquid medium. Thenumber of cells in the beads increased morethan 32 times when they were subsequentlyre-cultured in an aqueous medium for fiveweeks, showing that they had remained aliveduring storage. TEM observations showedthat the entrapped cells reduced their cellcovering and pyrenoid size compared withthe normal free-living cells afterlong-term storage. The algal beads werealso applied to feed and water qualitycontrol in clam cultures' leading to amarked decrease in ammonium concentrations.Algal cells escaped from the beads provideda food source for the clams. This mightreduce the cost of clam culture compared totraditional culture methods. Therefore,immobilized I. galbana can be usedfor long-term preservation of algal stockin the laboratory and applied practicallyto clam cultures.

A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures

Isochrysis galbana, one of the most widely usedmarine microalgae in the rearing of finfish and shellfish larvae, is masscultured frequently in outdoor tanks. Under prolonged and repeated culture,severe contamination occurs. Axenic isolation of I.galbanafrom such cultures was best achieved by using a ternary procedure involvingpercoll-gradient centrifugation, treatment with antibiotics, and growth on agarmedium. Protozoa and other algae were removed most effectively by isolation ofI. galbana at the 30–40% density layer on apercoll-gradient. Removal of bacteria was accomplished using a mixture of 5antibiotics (250 μg mL−1 ampicillin, 50μg mL−1 gentamycin, 100 μgmL−1 kanamycin, 500 μgmL−1 neomycin, 50 μgmL−1 streptomycin). Axenic colonies were isolated fromasolid medium prepared from 1% purified agar. The ternary procedure isconsideredapplicable to the isolation of other axenic single-celled microalgae fromheavily contaminated cultures.

Biomass production and biochemical variability of the marine microalga Isochrysis galbana in relation to culture medium

We have studied the autotrophic growth of the marine microalga, Isochrysis galbana Parke, in a batch photobioreactor, comparing five different culture media and analysing the influence of each on growth kinetics as well as on the fatty-acid composition and protein content of the biomass. All the experiments were performed at 15°C, with the culture medium at pH 8.0, a specific rate of air supply of 1 v v −1 min −1 and a continuous illumination of 40–43 W m −2. The results show no parallel between good nutritional characteristics and high values of the kinetic parameters. Nevertheless, a compromise between the nutritional factors and growth kinetics could be provided by Ukeles medium, which provided a biomass with a good composition in polyunsaturated fatty acids (quotient n3/ n6=3.2), an adequate protein content (25.3%) and relatively high values, although not the highest registered, for maximum specific growth rate ( μ m=0.018 h −1) and biomass productivity (1.9×10 −3 kg m −3 h −1).

Photolimitation and photoinhibition as factors determining optimal dilution rate to produce eicosapentaenoic acid from cultures of the microalga Isochrysis galbana

Eicosapentaenoic acid (EPA) productivity from continuous cultures of the marine microalga Isochrysis galbana was studied, taking into account the irradiance on the reactor surface, that is, the photolimitation/photoinhibition regime to which the cells are exposed. Experiments were conducted under a wide variety of operating conditions. The dilution rate ranged from 0.005 h−1 to 0.040 h−1 at five external irradiances (820, 1620, 2050, 2450 and 3270 μmol photons m−2 s−1) covering photolimited to photoinhibited growth. Under these conditions, the specific growth rate (μ) was found to be the main factor influencing EPA content (ranging from 2.35% to 5.23% dryweight) and productivity (up to 0.88 mg l−1 h−1). The fatty acid content was not significantly affected by the external irradiance, but was influenced by the state of growth of the microalga, depending on whether the light regime was photolimiting or photoinhibiting. It might be suggested that light should no longer be considered an isolated factor affecting EPA synthesis, but an indirect influence through the photolimitation/photoinhibition regime and growth rate. At a given dilution rate, EPA content and biomass concentration are lower under photoinhibiting external irradiances than those corresponding to photolimiting conditions, and consequently EPA productivity decays. Since the effect of photoinhibition is less marked at high biomass concentration, a strategy to optimize EPA productivity from microalgal cultures could consist of reducing the dilution rate when the external irradiance increases above the phoinhibition threshold.

Effects of nitrogen source and growth phase on proximate biochemical composition, lipid classes and fatty acid profile of the marine microalga Isochrysis galbana

The marine microalga Isochrysis galbana was cultured in nitrate, nitrite or urea media to examine changes in the gross biochemical composition, with special emphasis on the growth phase associated changes in the compositions of the lipid classes and fatty acids. The gross biochemical composition was affected more by the growth phase than by the nitrogen source. Protein was higher during exponential growth (about 37–45% AFDW) but the pattern changed as the cultures aged, and lipids were the main algal constituents on all nitrogen sources in the late stationary phase. In all cultures, the relative abundance of neutral lipid increased in the late stationary phase concomitant with a proportional reduction in phospholipids, whereas galactolipids only slightly changed during the growth of the cultures. Total fatty acid content was affected by nitrogen source and growth phase. Maximal PUFA values were obtained at the early stationary phase and decreased throughout the stationary phase. The highest fatty acid contents in the early stationary phase were produced in urea cultures; these cultures also had higher PUFA content, with EPA and DHA contents of 27.66% and 14.13% of total fatty acids, respectively.

Growth activation of the microalga Isochrysis galbana by the aqueous extract of the seaweed Monostroma nitidum

Cell growth of the marine microalga Isochrysis galbana was regulated by the addition of seaweed extracts in the culture medium. Methanol-soluble extracts from 27 species of seaweed showed growth activation only from Enteromorpha linza, and growth inhibition from Ishige foliacea and Sargassum sagamianum. Water-soluble extracts from Grateloupia turuturu and Monostroma nitidum showed growth activation, while none of the seaweed showed growth inhibition. From results of growth activation of extracts on I. galbana, the water extract of M. nitidum was the most effective up to two-fold increase in cell density with the addition of 1 mg mL-1 of extract to the medium. The cell growth rate was increased from 0.52 to 0.65 d-1. Cell size, gross biochemical compositions, fatty acid compositions, and digestion efficiency by shellfish differed marginally between cultures of I. galbana grown with and without the M. nitidum aqueous extract. This extract has also enhanced the growth of other feed microalgae tested, including Dunaliella salina.

Manipulation of the biochemical composition of the eicosapentaenoic acid‐rich microalga Isochrysis galbana in semicontinuous cultures

The marine microalga Isochrysis galbana was cultured semicontinuously with two nutrient concentrations [2 and 4 mmol of nitrogen (N)/litre] and five rates of daily renewal of culture medium (from 10 to 50%) in order to optimize the productivity of the valuable polyunsaturated fatty acids eicosapentaenoic acid (EPA, C 20:5n-3 ) and docosahexaenoic acid (DHA, C 22:6,n-3 ). The N conversion rate and chlorophyll content indicated that a nutrient concentration ol 2 mmol of N.l -1 was limiting, except for a renewal rate of 50%, while cultures maintained with 4 mmol of N.I -1 were nutrient-limited only up to a renewal rate of 20%. Carbohydrates, but not lipids, were stored by I. galbana under nutrient deficiency, but the composition of the fatty acid fraction was strongly affected by culture conditions. The degree of unsaturation of fatty acids increased with increasing renewal rate, mainly at the expense of C 16:0 and C 16:1 fatty acids, which decreased under the same conditions. EPA increased from 9% of total fatty acids with 2 mmol of N.I -1 and a renewal rate of 10% to values around 25% of total fatty acids achieved in nutrient saturated cultures for both nutrient concentrations. On the other hand, the percentage of DHA was not strongly affected by either renewal rate or nutrient concentration, which was reflected in very little variation in the productivity, which remained in the range 0.9-1.3 mg.day -1 .I -1 . Owing to the decrease of cell density with increasing renewal rates, maximal EPA productivity, 4.6 mg.day -1 .I -1 , was achieved with 4 mmol of N.I -1 and intermediate renewal rates.

Evaluation of photosynthetic efficiency in microalgal cultures using averaged irradiance

The growth yield of the PUFA-producing marine microalga Isochrysis galbana grown in light-limited continuous operation was measured under a wide variety of conditions of incident irradiance ( I 0) and dilution rates ( D). The experiments were conducted under laboratory conditions at 20°C with continuous light. D ranged from 0.0024–0.0410 h −1 at three values of I 0 (820; 1,620; and 3,270 μE m −2 s −1) close to those found in outdoor cultures. A maximum efficiency of Ψ max = 0.616 g E −1 was obtained at I 0 = 820 μE m −2 s −1 and D = 0.030 h −1, and the maximum capacity of the biomass to metabolize light was found to be 13.1 μE g −1 s −1. Above this value, a significant drop in system efficiency was observed. A new approach based on averaged irradiance is used to assess the photon flux absorbed by the biomass. The biomass productivity to be expected in an outdoor tubular photobioreactor growing I. galbana was predicted from efficiency data obtained in laboratory cultures and was found to be in agreement with the actual value.

Growth yield determination in a chemostat culture of the marine microalgaIsochrysis galbana

The growth yield of the PUFA-producing marine microalgaIsochrysis galbana ALII-4 grown in a light limited chemostat, was measured under a wide variety of conditions of incident irradiance (I O ) and dilution rates (D). The experiments were conducted under laboratory conditions at 20 °C under continuous light. D ranged from 0.0024 to 0.0410 h−1 at three intensities of Io (820, 1620 and 3270 µmol photon m−2 s−1) close to those found in outdoor cultures. A maximum efficiency Ψ max = 0.616 g mol photon−1 was obtained at I O = 820 µmol photon m−2 s−1 and D = 0.030 h−1 and the maximum capacity of the biomass to metabolize the light harvested was found to be 13.1 µmol photon g−1 s−1. Above this value, a significant drop in the system efficiency was observed. A new approach based in the averaged irradiance is used to assess the photon flux absorbed by the biomass.

A study on simultaneous photolimitation and photoinhibition in dense microalgal cultures taking into account incident and averaged irradiances

From chemostat cultures of the marine microalga Isochrysis galbana (CCAP 927/15) simultaneous photolimitation and photoinhibition was observed. The extent of each phenomenon depends on the light gradient inside the culture and therefore on the incident irradiance. Variations in biomass concentration and average irradiance inside the culture with dilution rate at three incident irradiances, I o , were studied (from I o = 820 to 3270 μE m −2 s −1). At I o above 1630 μE m −2 s −1 a photoinhibition effect was observed, although the specific growth rate remained a hyperbolic function of average irradiance regardless of incident irradiance. To calculate average irradiance, a three-dimensional irradiance distribution model for cylindrical geometry is proposed, improving the estimation of the irradiance field inside culture with regard to other methods used up to now since the variations in illumination along the vertical axis are considered. Lastly, a new approach to model simultaneous photolimitation and photoinhibition is proposed by considering that specific growth rate is related to average irradiance and that parameters representing the cell adaptability to light are a function of the maximum irradiation at which cells are exposed, that is, the incident irradiance.

Concentration and purification of stearidonic, eicosapentaenoic, and docosahexaenoic acids from cod liver oil and the marine microalgaIsochrysis galbana

n‐3 Polyunsaturated fatty acids (n‐3 PUFA) from the marine microalgaIsochrysis galbana were concentrated and purified by a two‐step process—formation of urea inclusion compounds followed by preparative high‐performance liquid chromatography. These methods had been developed previously with fatty acids from cod liver oil. By the urea inclusion compounds method, a mixture that contained 94% (w/w) stearidonic (SA), eicosapentaenoic (EPA), plus docosahexaenoic (DHA) acids (4:1 urea/fatty acid ratio and 4°C crystallization final temperature) was obtained from cod liver oil fatty acids. Further purification of SA, EPA, and DHA was achieved with reverse‐phase C18 columns. These isolations were scaled up to a semi‐preparative column. A PUFA concentrate was isolated fromI. galbana with methanol/water (80:20, w/w) or ethanol/water (70:30, w/w). With methanol/water, a 96% EPA fraction with 100% yield was obtained, as well as a 94% pure DHA fraction with a 94% yield. With ethanol/water as the mobile phase, EPA and DHA fractions obtained were 92% pure with yields of 84 and 88%, respectively.

Variation of fatty acid profile with solar cycle in outdoor chemostat culture ofIsochrysis galbana ALII-4

Outdoor chemostat cultures of the marine microalgaIsochrysis galbana at constant dilution rate (0.034 h−1 ) have been carried out under different weather conditions. Steady-state biomass concentrations were 1.61±0.03 kg m−3 in May and 0.95±0.04 kg m−3 in July, resulting in biomass output rates of 0.54 kg m−3 d−1 and 0.32 kg m−3 d−1 in May and July, respectively. Two patterns of daily variation with the solar cycle were observed in the fatty acid content. Saturated and mono-unsaturated fatty acids (16:0 and 16:1n7) show significant variation with the solar cycle, associated with short-term changes in environmental factors. Palmitic and palmitoleic acids are generated during daylight and consumed during the dark period. However, polyunsaturated fatty acids do not show a significant response to the solar cycle and their changes are associated with long-term variation in environmental factors. The maximum EPA productivity was obtained in May, 14.1 g m−3 d−1, which is close to that found in the literature for indoor continuous cultures. Nonetheless, the outdoor EPA content (up to 2.61 % d.wt) was lower than the indoor EPA content from a previous study (5% d.wt).

N-3 Polyunsaturated fatty acid productivity of the marine microalgaIsochrysis galbana. Growth conditions and phenotypic selection

Two set of isolates were obtained in an isolation/selection programme to select eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) rich strains ofIsochrysis galbana. EPA content was improved up to an average of 5.3% (d.wt) for the second set of isolates. On the other hand, with the aeration rate, pH and irradiance kept at low levels, the growth rate was slow and EPA synthesis was enhanced, but productivity increased when growth rates were maximum. A model relating steady-state dilution rates in chemostat cultures ofI. galbana to internal average irradiance is proposed. The greatest productivities were obtained between 0.0295 h−1 and 0.0355 h−1 with 300 mg m−3 h−1 for EPA and 130 mg m−3 h−1 for DHA.

EPA from Isochrysis galbana. Growth conditions and productivity

The marine microalga Isochrysis galbana Parke is presented as an alternative source of eicosapentaenoic acid (EPA). The effects of growth conditions on polyunsaturated fatty acid productivity with special attention to EPA and docosahexaenoic (DHA) acids were studied. The variables tested were: aeration flow rate, nitrate concentration, temperature, light intensity, micronutrient and vitamin concentration, and pH. Growth conditions producing the greatest EPA yields of over 200 μg/litre/h, are indicated as a starting point for a large scale mass culture system which would develop I. galbana's potential.

Outdoor cultivation of the marine microalga Isochrysis galbana in open reactors

Experiments concerning the optimization of growth conditions in the laboratory and mass production of the marine microalga Isochrysis galbana in open reactors are described. In the laboratory, a maximum specific growth rate (μmax) of 0.073 h −1 was obtained, which is equivalent to a doubling time of 9.5 h. In a small reactor (2.5-m 2 pond) the production rate of biomass was 29.5±2 g m −2 day −1 and that of lipids was 6.5±0.5 g m −2 day −1. In a larger reactor (100-m 2 pond) in which algae were grown first by batch and then by semi-continuous mode, the average output rates of biomass and of lipids were 23.5±1.5 g m −2 day −1 and 5.6±0.5 g m −2 day −1, respectively. A complete removal of the biomass was achieved in the 100-m 2 pond by means of flocculation with FeCl 3 (180 mg l −1) and dissolved air flotation without a prior recycling of part of the effluent. Monoalgal cultures were maintained in the different outdoor experiments throughout the entire 4-month production period.

Biomass production and biochemical composition in mass cultures of the marine microalga Isochrysis galbana Parke at varying nutrient concentrations

Mass cultures of Isochrysis galbana were carried out with four nutrient concentrations ranging from 2 to 16 m M of NaNO 3 and salinity 35‰. An air flow of 15 l/min maintained a CO 2 transference rate sufficient to keep the pH below 8.4. Using these conditions, equations were calculated by a multiple non-linear least squares regression of order four, enabling predictions to be made of growth kinetics and chemical composition. Maximum cellular density of 65.5 × 10 6 cells/ml was obtained with 4 m M NaNO 3. Cellular volume was constant in the different nutrient concentrations. Protein content reached a maximum value of 374 μg/ml at 4 m M of NaNO 3, and this concentration also presented the maximum efficiency of transformation from nitrate to protein, i.e. 114%. As a result, lowest costs for harvesting are obtained at a nutrient concentration of 4 m M NaNO 3. Efficiencies decreased to 15% as nutrient concentration increased. Maximum values of chlorophyll a (21.9 μg/ml) and carbohydrates (213 μg/ml) were also obtained with 4 m M NaNO 3. In the logarithmic phase, the contents of protein, chlorophyll a, carbohydrates, RNA and DNA per cell were constant. Chlorophyll a reached values between 0.15 and 0.33 pg/cell in the stationary phase. Carbohydrate levels reached the maximum value of 3.16 pg/cell with 4 m M NaNO 3 in the stationary phase. The levels of RNA/cell and DNA/cell were constant in all the nutrient concentrations tested and in both growth phases, and ranged from 1.15 to 1.71 pg/cell for RNA and from 0.006 to 0.014 pg/cell for DNA. Growth in mass cultures is closely coupled to changes in nutrient concentrations and variations occur in protein, chlorophyll a and carbohydrate contents, showing differences of 177%, 220% and 136%, respectively, in the stationary phase. This biochemical variability, mainly in protein content, must have a marked effect on the nutritive value of this microalga as a feed in mariculture.

Biomass production and biochemical composition in mass cultures of the marine microalga Isochrysis galbana Parke at varying nutrient concentrations : Faregas, Jaime, Concepcion Herrero, Buenaventura Cabezas and Julio Abalde, 1986. Aquacultute, 53(2):101–113

Biomass production and biochemical composition in mass cultures of the marine microalga Isochrysis galbana Parke at varying nutrient concentrations : Faregas, Jaime, Concepcion Herrero, Buenaventura Cabezas and Julio Abalde, 1986. Aquacultute, 53(2):101–113

Growth, chlorophyll a and protein of the marine microalga Isochrysis galbana in batch cultures with different salinities and high nutrient concentrations

Cultures of the marine microalga Isochrysis galbana were grown under 56 different nutrient concentration-salinity conditions, ranging from 1 to 64 m M NaNO 3 and from 0 to 35‰ salinity. Salinity and nutrient concentration were found to be closely related to I. galbana growth and to the biochemical composition. Optimal growth conditions were between 15 and 35‰ salinity and nutrient concentrations of 2, 4 and 8 m M NaNO 3, resulting in one doubling/day and a maximum cellular density of 20 × 10 6 cells/ml. Variations in salinity and in nutrient concentration had a greater effect on the final biomass than on the growth velocity. Maximum values of chlorophyll a ml were found with 2, 4 and 8 m M NaNO 3 and between 15 and 35‰ salinity. Chlorophyll a cell values were more homogeneously distributed between 15 and 35‰ salinity and 1 to 8 m M NaNO 3, although maximum concentrations (37 pg chlorophyll a cell ) were reached at 10–15‰ with all the nutrient concentrations. Protein per ml of culture and protein per cell were closely related to salinity and nutrient concentration. Maximum values of 387 μg/ml and 18.6 pg/cell were obtained at 15–35‰ salinity and 4–8 m M NaNO 3. The nitrate-protein transformation rate was related to nutrient concentration. Maximum rate was 84% at 15‰ salinity and 1 m M NaNO 3. Nutrient concentrations higher than 16 m M NaNO 3 produced a strong decrease in the efficiency at all salinities.