Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: C/N ratio and light intensity

We investigated the effect of carbon/nitrogen (C/N) ratio on astaxanthin synthesis in Haematococcus pluvialis during photoautotrophic induction by continuous input of both CO₂–air mixture and intense light. When H. pluvialis was induced by constant irradiance induction at 200 μmol photon m⁻² s⁻¹, there was a positive correlation with astaxanthin content and C/N ratio, which was similar to the case for heterotrophic induction. Lower C/N ratios did not retard Haematococcus encystment, but did increase culture biomass, resulting in a decrease in astaxanthin production because of light limitation. However, induction using variable irradiance showed that reduction of astaxanthin production at low C/N ratios was successfully overcome by simply increasing the light intensity from 200 to 300 μmol photon m⁻² s⁻¹ to overcome the light limitation. This resulted in a greatly enhanced astaxanthin synthesis in proportion to cell density in cultures with low C/N ratios. Our results indicate that light intensity is more critical than C/N ratio in astaxanthin production by H. pluvialis during photoautotrophic induction.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Effects of temperature,photosynthetic photon flux density,photoperiod and O<Subscript>2</Subscript> and CO<Subscript>2</Subscript> concentrations on growth rates of the symbiotic dinoflagellate, <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m⁻² s⁻¹), three levels of CO₂ concentration (0.02–0.1%), and three levels of O₂ concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h⁻¹ at a temperature of 26°C and O₂ concentration of 5%, and the specific growth rate was saturated at a CO₂ concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m⁻² s⁻¹ and a photoperiod of 12 h day⁻¹ upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O₂ concentration.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Light-Dependency of Growth and Secondary Metabolite Production in the Captive Zooxanthellate Soft Coral Sinularia flexibilis

The branching zooxanthellate soft coral Sinularia flexibillis releases antimicrobial and toxic compounds with potential pharmaceutical importance. As photosynthesis by the symbiotic algae is vital to the host, the light-dependency of the coral, including its specific growth rate (μ day⁻¹) and the physiological response to a range of light intensities (10–1,000 μmol quanta m⁻² s⁻¹) was studied for 12 weeks. Although a range of irradiances from 100 to 400 μmol quanta m⁻² s⁻¹ was favorable for S. flexibilis, based on chlorophyll content, a light intensity around 100 μmol quanta m⁻² s⁻¹ was found to be optimal. The contents of both zooxanthellae and chlorophyll a were highest at 100 μmol quanta m⁻² s⁻¹. The specific budding rate showed almost the same pattern as the specific growth rate. The concentration of the terpene flexibilide, produced by this species, increased at high light intensities (200–600 μmol quanta m⁻² s⁻¹).     

Effects of Environmental Factors and Metal Ions on Growth of the Red Alga Gracilaria Chorda Holmes (Gracilariales, Rhodophyta)

Gracilaria is a potentially valuable source of marine biopolymers such as proteins and polysaccharides. In order to select suitable culture conditions, growth and tolerance of Gracilaria chorda Holmes from Shikoku Island in southwest Japan were investigated under variations of temperature (5–30 ^∘C), photon irradiance (20–120 μmol photons m⁻² s⁻¹), and photoperiod (12:12 h, 14:10 h light:dark regime) in a unialgal culture. Gracilaria chorda showed wide tolerances for all factors investigated, which is characteristic of eurythermal species. Maximum growth was observed at 18–24 ^∘C. The optimum photon irradiance for the algal growth was 60–120 μmol photons m⁻²s⁻¹. Instead of using ordinary sea salt (NaCl) to prepare artificial seawater, ultra pure salt was adopted. Gracilaria chorda grew faster in artificial seawater made with ultra-pure salt than that made with ordinary sea salt, probably because the former medium was clear, while the latter was milky. Effects of some metal ions on the growth were tested with artificial seawater. Iron ions affected algal growth, but cobalt ions did not. This study enables us to determine suitable culture conditions for G. chorda. A scaled-up 30 l culture of G. chorda under such conditions was successful.     

Cost reduction in the micropropagation of banana by using tubular skylights as source for natural lighting

Summary Daylight instead of artificial light was exploited for the in vitro culture of banana. Tubular skylights rediverted natural light into an interior enelosed room whereby each skylight, available for ca. US$600, could sufficiently illuminate an area of 3–5 m². The maintenance-free system allowed only a minimum of heat transfer and no cooling was necessary. The culture room required no electricity supply and under our conditions savings on costs for electricity of US$6 m⁻² wk⁻¹ were achieved, as compared to a standard growth room equipped with artificial lighting and controlled photoperiod and temperature regimes. Under natural light conditions, micropropagated plantlets were well developed at mean photosynthetic photon flux densities (PPFD) of 5–13 μmol m⁻² s⁻¹ and photoperiods of 9–14 h. Micropropagation rates were either the same or significantly higher than under artificial lighting. Single shoots on rooting medium showed some symptoms of etiolation, yet acclimatization rates averaged 95%. A step-like culture rack. rather than a vertical one, permitted uniform plant growth on all levels. This paper describes an established micropropagation system of low cost and simplicity.     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Effect of irradiation intensity on cell growth and kalata B1 accumulation in <Emphasis Type="Italic">Oldenlandia affinis</Emphasis> cultures

Light irradiation had remarkable effects on callus growth of Oldenlandia affinis with an optimum intensity of 35 μmol m⁻² s⁻¹. Biosynthesis of kalata B1, the main cyclic peptide in O. affinis, was induced and triggered with rising irradiation intensities. The highest concentration of kalata B1, 0.49 mg g⁻¹ DW characterised by the maximum productivity of 3.88 μg per litre and day was analysed at 120 μmol m⁻² s⁻¹, although callus growth was repressed. The light saturation point was established to be 35 μmol m⁻² s⁻¹, where kalata B1 productivity was in a similar order (3.41 μg per day) due to the higher growth index. O. affinis suspension cultures were shown to accumulate comparable specific kalata B1 concentrations in a delayed growth associated production pattern. These were dependent on irradiation intensity (0.16 mg g⁻¹ at 2 μmol m⁻² s⁻¹; 0.28 mg g⁻¹ at 35 μmol m⁻² s⁻¹). The batch cultivation process resulted in a maximum productivity of 27.30 μg per litre and day with culture doubling times of 1.16 d⁻¹. Submers operation represented a 8-fold product enhancement compared to callus cultivation.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Effects of photon flux density,CO2, aeration rate,and inoculum density on growth and extracellular polysaccharide production byPorphyridium cruentum

Growth and extracellular polysaccharide production byPorphyridium cruentum were measured as a function of several culture parameters. Photon flux density of 75 μmol m⁻² s⁻¹ and CO₂ concentration of 2.5% were found to be optimum for both growth and extracellular polysaccharide production. Interactive studies on these two parameters further confirmed that at these levels of photon flux density and CO₂, when applied together, both growth (5.9·10⁷ cells per mL) and extracellular polysaccharide production (1.9 g/L) were at the maximum. Maximum growth and extracellular polysaccharide production were observed at inoculum density of 10⁶ cells per mL and aeration rate of 500 mL air per min per liter.     

A two-stage solar photobioreactor for cultivation of microalgae based on solar concentrators

A novel two-stage experimental photobioreactor (PBR) with a total volume of 450 L and based uniquely on solar concentrators—linear Fresnel lenses—has been constructed and tested. Daily courses of irradiance, and also its distribution inside cultivation tubes, were studied in two unit types. The supra-high irradiance units in the ‘roof’ achieved a maximum summer value above 6 mmol photon m⁻² s⁻¹, while irradiance in the vertical-facade units was lower than ‘ambient’. In model cultivations, cultures of the cyanobacterium Arthrospira platensis were cultivated at much higher solar irradiances than those usually recorded outdoors in summer, indicating that this organism is resilient to high-irradiance (photoinhibition). Starting from a biomass density of 0.5 g L⁻¹ at optimum temperature, the cultures grew exponentially. A two-stage cultivation process of the green microalga Haematococcus pluvialis was investigated with respect to correlations between photochemical activities and astaxanthin production. The culture was first grown in low-irradiance units, and then exposed to supra-high irradiance when the rate of astaxanthin production was 30–50% higher than in the culture exposed to ‘ambient’ irradiance. Within 4 days, the astaxanthin content reached 3% of dry weight, whereas under ambient irradiance the astaxanthin content was 25% lower.     

Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis

This work describes the long-term acclimation of the halotolerant microalga Dunaliella viridis to different photon irradiance, ranging from darkness to 1500 μmol m⁻² s⁻¹. In order to assess the effects of long-term photoinhibition, changes in oxygen production rate, pigment composition, xanthophyll cycle and in vivo chlorophyll fluorescence using the saturating pulse method were measured. Growth rate was maximal at intermediate irradiance (250 and 700 μmol m⁻² s⁻¹). The increase in growth irradiance from 700 to 1500 μmol m⁻² s⁻¹ did not lead to further significant changes in pigment composition or EPS, indicating saturation in the pigment response to high light. Changes in Photosystem II optimum quantum yield (F_v/F_m) evidenced photoinhibition at 700 and especially at 1500 μmol m⁻² s⁻¹. The relation between photosynthetic electron flow rate and photosyntetic O₂ evolution was linear for cultures in darkness shifting to curvilinear as growth irradiance increased, suggesting the interference of the energy dissipation processes in oxygen evolution. Carbon assimilation efficiencies were studied in relation to changes in growth rate, internal carbon and nitrogen composition, and organic carbon released to the external medium. All illuminated cultures showed a high capability to maintain a C:N ratio between 6 and 7. The percentage of organic carbon released to the external medium increased to its maximum under high irradiance (1500 μmol m⁻² s⁻¹). These results suggest that the release of organic carbon could act as a secondary dissipation process when the xanthophyll cycle is saturated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Temperature and irradiance impacts on the growth,pigmentation and photosystem II quantum yields of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> (Chlorophyceae)

The microalga Haematococcus pluvialis Flotow has been the subject of a number of studies concerned with maximizing astaxanthin production for use in animal feeds and for human consumption. Several of these studies have specifically attempted to ascertain the optimal temperature and irradiance combination for growth of H. pluvialis, but there has been a great deal of disagreement between laboratories. “Ideal” levels of temperature and irradiance have been reported to range from 14 to 28°C and 30 to 200 μmol photons m⁻² s⁻¹. The objective of the present study was to simultaneously explore temperature and irradiance effects for a single strain of H. pluvialis (UTEX 2505) across an experimental region that encompassed the reported “optimal” combinations of these factors for multiple strains. To this end, a two-dimensional experimental design based on response surface methodology (RSM) was created. Maximum growth rates for UTEX 2505 were achieved at 27°C and 260 μmol photons m⁻² s⁻¹, while maximum quantum yield for stable charge separation at PSII (F_v/F_m) was achieved at 27°C and 80 μmol photons m⁻² s⁻¹. Maximum pigment concentrations correlated closely with maximum F_v/F_m. Numeric optimization of growth rate and F_v/F_m produced an optimal combination of 27°C and 250 μmol photons m⁻² s⁻¹. Polynomial models of the various response surfaces were validated with multiple points and were found to be very useful for predicting several H. pluvialis UTEX 2505 responses across the entire two-dimensional experimental design space.     

Effects of photon flux density and agricultural fertilizers on the development of <Emphasis Type="Italic">Sarcothalia crispata</Emphasis> tetraspores (Rhodophyta,Gigartinales) from the Strait of Magellan,Chile

Tetraspores of Sarcothalia crispata from San Juan Bay, Strait of Magellan, Chile, were cultivated under different combinations of photon flux densities and agricultural fertilizers in the laboratory. In the experiment, the S. crispata specimens were cultured in combinations of different photon flux densities (50, 100, 150 μmol photons m^-2 s^-1) and enriched seawater solutions (sodium nitrate + monocalcium phosphate, urea + monocalcium phosphate, ammonium nitrate + monocalcium phosphate), always adjusting the N and P concentrations to 10 and 3 mg L^-1, and in sea water as control. After 45 days, the tetrasporeling plants were found to be larger at photon flux densities of 50 and 100 μmol photons m^-2 s^-1 in the nutrient enrichment experiments; growth was greatest in the sea water enriched with ammonium nitrate and urea. An analysis of the combined effect of the photon flux density and nutrients revealed that the best combination for sporeling growth was the ammonium nitrate and urea solution at 50–100 μmol photons m^-2 s^-1.     

Changes in PAL,CHS, DAHP synthase (DS-Co and Ds-Mn) activity during anthocyanin synthesis in suspension culture of Fragaria ananassa

The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Mn, DS-Co), phenylalanine ammonia-lyase (PAL), and chalcone synthase (CHS) was monitored at various light intensities (dark, 8.88 μmol m⁻² s⁻¹, 88.8 μmol m⁻² s⁻¹) using a strawberry cell suspension culture. DS-Mn, PAL, and CHS were found to increase significantly (p>0.05) under light intensitie of 88.8 μmol m⁻² s⁻¹ compared to those of 8.88 μmol m⁻² s⁻¹ and dark. The activity of DS-Mn, PAL, and CHS were maximum at 88.8 μmol m⁻² s⁻¹. Anthocyanin content reached a maximum after 48–60 h of culturing at 88.8 μmol m⁻² s⁻¹. DS-Co showed greater activity than DS-Mn during cell culturing, but showed no correlation with anthocyanin production and light intensity. The CHS gene expression was continuous at a light intensity of 88.8 μmol m⁻² s⁻¹. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors that affect the re-attachment of Chondracanthus chamissoi (Rhodophyta, Gigartinales) thalli

Vegetative reproduction of Chondracanthus chamissoi by means of fragmentation and re-attachment of thalli is considered an effective strategy for maintaining natural populations of this species. Here, we evaluate the effects of (1) time of drifting thallus, (2) type of substratum, and (3) photon flux density, on the re-attachment capacity of thallus fragments of C. chamissoi. The results show that re-attachment decreases with the time after detachment, and was higher at the lower photon flux densities tested (10 and 40 μmol photons m⁻²s⁻¹), and on calcareous substratum. Secondary attachment discs are formed along the entire surface of the fragment.     

Seasonal variations of microsite light availability within aMiscanthus sinensis canopy

Photosynthetic photon flux density (PPFD) at 15 cm above the ground was measured at 20 microsites in gaps and grass patches within aMiscanthus sinensis Anderss community at 10 s intervals during 5 days every month from May to September 1989. Microsite light availability, which was characterized by daily total PPFD, sunfleck PPFD (PPFD above a threshold value of 50 or 400 μmol m⁻² s⁻¹) and the diffuse site factor, showed evident seasonal changes, with a marked reduction between June and July due to the rapid growth of the grass canopy. The monthly median value of daily total PPFD among the microsites decreased from 10.3 mol m⁻² day⁻¹ in May to 0.77 mol m⁻² day⁻¹ in September, with a reduction in the diffuse site factor from 31 to 4%. During the summer, the median value of the total time of sunflecks exceeding 50 μmol m⁻² s⁻¹ contributed 7–18% of measurement time, but the contribution of these sunflecks to daily total PPFD ranged from 29 to 59%. There was considerable microsite variation in light availability throughout the measurement period. Rank correlation analysis revealed that some microsites, such as those in gaps, consistently received more total PPFD, more sunfleck PPFD and had a higher diffuse site factor than those in grass patches. The diffuse site factor had a linearly positive relationship with daily total PPFD and total sunfleck PPFD for the 20 microsites during the measurement period, confirming that the diffuse site factor is a useful index for microsite light availability withinM. sinensis canopies.     

CO2 compensation concentration in bean Leaves: Effect of photon flux density and leaf age

Carbon dioxide compensation concentration,Г, net photosynthetic rate,Pn, and photorespiration rate,Rl, were measured in young, adult and old primary leaves ofPhaseolus vulgaris L. over a range of photon flux densities using a closed system with IRGA. Irrespective of leaf age,Г decreased rapidly with rising photon flux density up toca. 260 (μmol m⁻² s⁻¹. From this valueГ did not change with photon flux density under constant temperature, reaching on the average 178, 118 and 239 mg m⁻³ in young, adult and old leaves, respectively. Changes with age in curves relatingPn andRl to photon flux density were found.