Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.     

Effect of the Nutritional Status of Semi-continuous Microalgal Cultures on the Productivity and Biochemical Composition of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

The rotifer Brachionus plicatilis was cultured using the microalga Isochrysis aff. galbana clone T-ISO as feed. T-ISO was cultured semi-continuously with daily renewal rates of 10%, 20%, 30%, 40%, and 50% of the volume of cultures. The increase of renewal rate led to increasing nutrient and light availability in microalgal cultures, which caused differences in the biochemical composition of microalgal biomass. Growth rate, individual dry weight, organic content, and biomass productivity of rotifer cultures increased in response to higher growth rate in T-ISO cultures. Rotifer growth rate showed a strong negative correlation (R ² = 0.90) with the C/N ratio of microalgal biomass. Rotifer dry weight was also affected by nutrient availability of T-ISO cultures, increasing up to 50% from nutrient-limited to nutrient-sufficient conditions. Consequently, biomass productivity of rotifer cultures increased more than twofold with the increase of renewal rate of T-ISO cultures. Rotifer organic content underwent the same trend of total dry weight. Maximum content of polyunsaturated fatty acids was reached in rotifers fed T-ISO from the renewal rate of 40%, with percentages of docosahexaenoic acid (22:6ω-3, DHA) and eicosapentaenoic acid (20:5ω-3, EPA) of 11% and 5% of total fatty acids, respectively. Selecting the most appropriate conditions for microalgal culture can therefore enhance the nutritive quality of microalgal biomass, resulting in a better performance of filter feeders and their nutrient content, and may constitute a useful tool to improve the rearing of fish larvae and other aquaculture organisms that require live feed in some or all the stages of their life cycle.     

Renewal rate and nutrient concentration as tools to modify productivity and biochemical composition of cyclostat cultures of the marine microalga Dunaliella tertiolecta

A factorial experimental design with two nutrient concentrations (2 and 4 mmol Nl^–1 in the form of NaNO₃) and five rates of daily renewal of the cultures (10%, 20%, 30%, 40% and 50%) was carried out in cyclostat, light/dark-synchronized cultures of the marine microalga Dunaliella tertiolecta Butcher. Steady-state cellular density was a linear function inversely proportional to renewal rate. Maximal cellular productivity, 3 × 10⁹ cells1^–1 day^–1, equivalent to 0.24 g1^–1 day^–1 dry weight and 0.17 g1^–1 day^–1 organic weight, was found with renewal rates of 20%–30% and 4mmol N1^–1, but maximal protein productivity, 0.066 g1^–1 day^–1, was obtained with a renewal rate of 40% for both nutrient concentrations. The protein content ranged between 30% and 70% of the organic fraction depending on the culture conditions. Carbohydrates were the only fraction accumulating in response to nutrient stress, ranging from 57% to 10% of the organic fraction, meanwhile the lipid content was increased by increase of nutrient availability. Under non-nitrogen-limited conditions the C:N ratio stabilized around 5.2–5.3 and the protein content of the organic fraction around 70%, but the cell nitrogen quota decreased under these conditions with increasing renewal rates, owing to the lower organic content of cells obtained with high growth rates. The high capacity for changing the biochemical composition, demonstrated for D. tertiolecta in the cyclostat system, has interesting implications for the management of continuous cultures of microalgae and its applications in biotechnological processes.     

Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal

The appearance of differentiated cells in embryonal carcinoma (EC) cultures can be inhibited by culturing the cells on fibroblast feeder layers. To determine whether or not feeder layers act by increasing the probability of stem cell renewal, growth and differentiation were monitored in cultures of F9 (subclone OTF9 -63) EC cells exposed to retinoic acid (RA) in either the presence or absence of feeder layers. By measuring the fraction of laminin-positive TROMA 1-positive or alkaline phosphatase-negative cells, it was determined that the frequency of differentiated cells in RA-treated F9 cultures was reduced by 70-80% when cells were cultured on fibroblast feeder layers instead of gelatin-coated dishes. Experiments in which EC cells were cultured in close proximity to a feeder layer demonstrated that cell-cell contact was required for maximal inhibition of differentiation. The probability of stem cell renewal was determined by measuring the number of colony-forming cells in RA-treated cultures as a function of time. Analysis of the data demonstrated that the probabilities of stem cell renewal were 0.5 and 0.25 during the first and second 48 h periods, respectively, following addition of RA for cells cultured without feeder layers. Cultures maintained on feeder layers exhibited a stem cell renewal probability of 0.72. Thus, feeder layers reduce the frequency of differentiated cells in RA-treated cultures by increasing the probability of stem cell renewal. Determining the mechanism by which feeder layers counteract the effect of a chemically defined differentiation inducer should help to uncover the processes that regulate the probability of stem cell renewal.     

Cadmium removal by living cells of the marine microalga Tetraselmis suecica

Cadmium removal by living cells of the marine microalga Tetraselmis suecica was tested in cultures exposed to different cadmium concentrations (0.6, 3, 6, 15, 30 and 45 mg/l). The EC50 for growth was 7.9 mg Cd/l after six days of exposure. The cadmium removed was proportional to the concentration of this metal in the medium and it was dependent on the time of exposure; cultures with higher cadmium concentration removed a higher amount of this metal. In cultures exposed to 0.6 mg/l, T suecica cells removed 98.1% of added cadmium with 0.392 x 10(-6) microg Cd/cell, whereas in cultures with 45 mg/l only 7.7% was removed with 16.052 x 10(-6) microg Cd/cell. The highest amount of cadmium removed per liter of culture was observed in cultures exposed to 6 mg/l, with 3.577 mg/l of cadmium. After six days of incubation, the higher proportion of cadmium was bioaccumulated intracellularly in all cultures except in 45 mg/l cultures, the percentage of intracellular cadmium being always more than 50%. The highest percentage of bioadsorbed cadmium (60.1%) was found in cells of cultures with the highest cadmium concentration (45 mg/l). Furthermore, a relation between intracellular cadmium and the concentration of sulfhydryl groups was observed.     

Pyridine nucleotides in normal and nicotinamide depleted adrenal tumor cell cultures

Mouse adrenal tumor cell line Y-129 was grown in cell culture in medium without nicotinamide. Inhibition of growth occurred after the second subculture in the vitamin-deficient medium. Pyridine nucleotides were measured in control and nicotinamide-starved cultures. DPN⁺ decreased to less than 10% of the normal level after 7 days of vitamin starvation. TPNH dropped to 25% of its normal level in the same period. Plating efficiency decreased from 20% in controls to 10% in 7-day nicotinamide-starved cultures. Steroid production was reduced by approximately 50% in the starved cultures. Sensitivity of the cultures to the lethal effects of the nicotinamide antagonist, 3-acetylpyridine, was inversely proportional to the pyridine nucleotide content of the cells. Reconstitution of nicotinamide in depleted cultures caused a rapid renewal of the pyridine nucleotide levels and complete protection against the effects of 3-acetylpyridine. Recovery and subsequent growth of the cultures did not restore the decreased capacity to produce steroids. Nicotinic acid was not utilized by the cells for the synthesis of pyridine nucleotides in depleted cultures and azaserine did not inhibit the utilization of nicotinamide indicating that DPN⁺ was synthesized directly via nicotinamide ribonucleotide.     

Factors controlling eicosapentaenoic acid production in semicontinuous cultures of marine microalgae

Three marine microalgal species with a high content of eicosapentaenoic acid (EPA), Phaeodactylum tricornutum, Isochrysis galbana and Porphyridium cruentum, were cultured semicontinuously in order to study the effect of renewal rate on EPA productivity. The percentage of EPA in total fatty acids increased with increasing renewal rates in nitrogen limited cultures, but while for Phaeodactylum tricornutum and Isochrysis galbana a plateau around 20–25% of total fatty acids was reached with renewal rates that were not nitrogen-limiting, in Porphyridium cruentum EPA percentage increased continuously with increasing renewal rate even for those cultures that were nitrogen sufficient. Maximal EPA productivities of4.6 mg L^-1 day^-1 for Isochrysis galbana and 5.2 mg L^-1 day^-1 for Phaeodactylum tricornutum were achieved with renewal rates of 20% and 30% respectively. On the other hand for Porphyridium cruentum maximal EPA productivity, 5.3 mg L^-1 day^-1, was obtained with the maximal renewal rate tested. Results indicate that different culture strategies should be adopted for the production of a particular polyunsaturated fatty acid depending on the microalgal species being used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Density dependent regulation of growth in suspension cultures of L-929 cells

Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 × 10⁶ cells/ml through daily centrifugation and resuspension in fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are in a G₀ or early G₁ phase, resuming growth readily in response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring in the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated in the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.     

Exopolysaccharide, pigment and protein production by the marine microalga Chroomonas sp. in semicontinuous cultures

The marine microalga Chroomonas sp. isolated from Venezuela was grown in semicontinuous culture in order to study the effect of renewal rate and nutrient concentration on alloxanthin, chlorophyll a, carotenoid, carbohydrate, exopolysaccharide, protein and cell productivity. Maximal cell productivity of 8.43 ± 1.8 and 8.81 ± 2.3 × 10⁹ cell l^–1 day^–1 were achieved with renewal rates of 30 and 40%. Maximal protein and chlorophyll productivity of 64.64 ± 2.3 and 2.72 ± 0.3 mg l^–1 day^–1 were obtained with renewal rate of 20 and 30%. Biochemical composition of Chroomonas sp. was influenced by renewal rate. Nutrient concentration seems not to affect cell, protein, chlorophyll and carotenoid productivity. However, carbohydrate and exopolysaccharide productivity of 7.56 ± 0.4 and 9.57 ± 1.2 mg l^–1 day^–1 were increased at 12 mM NaNO₃(P < 0.05). Also, alloxanthin and chlorophyll a production analysed by HPLC, were higher between 8 and 12 mM NaNO₃ at a renewal rate of 30%. Results demonstrated that a renewal rate of 30% and nutrient concentration at 8 mM NaNO₃ optimize the cell, protein, carbohydrate, chlorophyll a, and exopolysaccharide productivity in semicontinuous cultures of Chroomonas. This microalga, as biological source of commercially valuable compounds, shows high capacity for changing its productivity and biochemical composition in semicontinuous system on the basis of nutrient concentration and the renewal rate.     

Baculovirus expression system scaleup by perfusion of high-density Sf-9 cell cultures

A perfusion system based on a 4-L stirred tank bioreactor and a custom-designed tangential (cross-flow) filter was assembled to realize a scaleup of the Baculovirus Expression Vector System (BEVS). When perfused with 1 to 1.5 vol/day, Spodoptera frugiperda (Sf-9) insect cell cultures grew from 4 x 10(6) to 15 x 10(6) cells/mL over 3 to 4 days. The possibility of maintaining high specific production of recombinant VP6 protein (from bovine rotavirus) after baculovirus infection of the high-density cultures was then assessed. The process consisted of a growth phase in TNMFH + 10% FBS, followed by infection with Bac-BRV6L recombinant baculovirus and a shift to a low-serum (0 to 1%) medium for perfusion during the production phase. Multiple runs were executed, each including a battery of shaker flask controls at various cell densities and serum concentrations. On average, specific rVP6 production in the bioreactor amounted to 76% of that found in 20-mL shaker cultures simulatingthe bioreactor's high cell density, low serum concentration, and medium renewal rate. Mechanical stress generated by cell/medium separation in theperfusion process reduced cell growth rate but had minimal effect on rVP6production. Our results also indicated that serum concentration during the infection phase affected the rVP6 specific production in a cell density-dependent fashion. Although the feasibility of the cell density scale up was demonstrated, optimization is still needed to achieve a truly cost-effective process.     

Growth and Metabolism of L Cells in a Chemically Defined Medium in a Controlled Environment Culture System: I. Effects of O2 Tension on L-Cell Cultures

Six water-jacketed 500-ml Bellco spinner flasks were equipped to monitor and control environmental variables to study their effects on the growth and metabolism of mammalian cells. Studies with automated control of pO(2) levels of l-cell cultures, grown at pH 6.9 +/- 0.1, showed that dissolved O(2) tensions of ca. 9% were optimal for cell growth. At pO(2) values of 5 and 20%, maximum cell yields as well as growth rates were reduced by approximately 20%. Peak yields of L-cell cultures exceeded 5 x 10(6) cells/ml when grown for 4 days without medium renewal from inocula of ca. 10(6) cells/ml in a defined medium sparged with 5% CO(2) and maintained at 9% dissolved O(2) tension. The redox potentials of L-cell cultures reflected the pO(2) levels in the medium and ranged from -45 to +160 mv (versus calomel reference) for O(2) values ranging from 2 to 20% dissolved oxygen tension. Increased utilization of glucose per cell occurred in the presence of increased pO(2), whereas minimal accumulation of ammonia occurred with a pO(2) value maintained at 9%.     

Soluble fractions of Solanum tuberosum enchnce call and pigment production of semi-continous cultures of the microlga Phaeodactylum tricornutum

J. FÁBREGAS, E. MORALES, N. POLANCO, M. PATINO AND A. OTERO. 1996. Soluble fractions of Solanum tuberosum extracted with different methods were tested on semicontinuous mixotrophic cultures of the marine microalga Phaeodactylum tricornutum maintained with renewal rate at 15%. The highest stabilization cell density, 78.2 x 10⁶ cells ml⁻¹, was obtained with an autoclaved non-fermented soluble fraction obtained with distilled water. Highest productivities and carotenoids, 0.9 mg 1⁻¹ d⁻¹, and chlorophyll, 2.9 mg 1⁻¹, d⁻¹, were obtained with a non-autoclaved nonfermented fraction extracted in sea water. The bacterial population associated to the microalgal cultures changed depending on the nutrient availability of each of the potato-soluble fractions.     

Structure and dynamics of the community associated to cultivated Gracilariopsis tenuifrons (Gracilariacea) in Chacopata, Sucre, Venezuela. I: Faunistic inventory

The associated fauna of Gracilariopsis tenuifrons (Gracilariaceae, Rhodophyta) cultures was collected between October 1994 and December 1996 in Chacopata (Sucre State, Venezuela) and preserved in 10% formaldehyde. The species list includes 17 Crustacea, 14 Mollusca and six Polychaeta, the remaining taxa added ten species (total 47 species in eight Phyla). Grazing by mesoherbivores affected the algae and the mollusk Aplysia protea damaged new cultures significantly. The abundance of tubes built by the amphipod Euricthonius brasiliensis impairs algal aspect and facilitates colonization by other organisms.     

In vitro culture of cells derived from larvae of the staghorn coral <Emphasis Type="Italic">Acropora</Emphasis> <Emphasis Type="Italic">millepora</Emphasis>

Previous attempts to culture cells from corals or other cnidarians have been unsuccessful. These efforts have, however, generally made use of adult tissue as starting material. Early developmental stages are potentially more appropriate for the initiation of cell cultures, as the expectation is that a greater proportion of the cell population is undifferentiated and may have the intrinsic ability of unlimited cell renewal. To explore this idea, cell cultures were initiated from five key stages of coral development, and the presence of coral cells monitored by polymerase chain reaction (PCR) using coral-specific primers. After 4 weeks, semi-quantitative PCR implied that coral cells were better represented in cultures initiated from planulae than in those derived from earlier developmental stages. Coral cells were detected in cultures initiated from planulae for up to 10 weeks, but after this time, extensive contamination by the protist Thraustochytrium sp. was observed.     

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.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Influence of Cadmium on Growth and Nitrogen Metabolism of Anabaena cylindrica Lemm

A study was made of the effects of cadmium on the Cyanobacterium(blue-green alga) Anabaena cylindrica Lemm. as part of the paddy-fieldecosystem. A simple culture vessel has been designed, which allows periodicalmeasurement of growth (optical density) and nitrogenase activity(C₂H₂-C₂H₄ method). The influence of medium renewal was checked:the renewal of the medium maintained a higher growth rate andhigher nitrogen fixation ability. The cadmium effects were studied using six concentration levelsranging from 0 (control) to 2 parts 10^–6 with renewedmedia (10% every day). No significant differences could be seen up to 1 part 10^–6for nitrogenase activity and relative percentage of heterocysts(decreasing as a function of time from ±4% to ±1.5%). Inhibition of growth (OD and dry weight) was weak at 1 part10^–6 but important at 2 parts 10^–6; at this concentrationcadmium induced morphological and physiological effects: chlorosis,cellular malformations and destruction, and increase in heterocystfrequency (up to 7.72% ±0.19). The cadmium concentration factors were much lower than thosereported for other plants like Chlorella and water pests     

Cell cycle and cell protein content of Chinese hamster cells grown in culture with daily renewal of medium

Microspectrophotometric and autoradiographic procedures were used to determine cell protein content and cell cycle parameters of Chinese hamster V79 cells growing as monolayer cultures with daily renewal of medium. The frequency of G 1 cells decreased and then increased as the cultures grew. Cell size at mitosis and at the G 1/S boundary changed only slightly during growth. The changes were very similar to those reported earlier for cultures of the same cell line grown without renewal of medium, but changes in the frequency of G 1 were somewhat slower and those in cell size were much less marked when the medium was renewed daily. The results of the two studies taken together show that cell cycle parameters and cell size change even during periods of exponential growth, and that daily renewal of medium slows but does not prevent the changes. Both studies show that the onset of DNA synthesis is more sensitive to conditions in older cultures than is the onset of mitosis. Our results indicate that radiation from incorporated isotopes might seriously affect the results of the labeled mitoses method of cell cycle analysis, especially when cell crowding is involved.     

Un nouveau procede de culture continue a dialyse pour le phytoplancton

Summary Equipment and methods for continuous dialysis culture are described which make feasible the continous aseptic production of dense cultures of phytoplankton (unicellular marine algae). New features include the use of independently-replaceable fibre dialysis units and renewal of the nutrient medium (sea water) by controlled hydraulic diffusion. Cell densities of up to 4 × 10⁷ are attained.