Optimizing the population density inIsochrysis galbana grown outdoors in a glass column photobioreactor

Particularly high population densities are readily sustainable in newly designed glass column reactors. The optimal density ofIsochrysis galbana in these columns in summer was 4.6 g L^–1 dry algal mass at which value the highest sustainable productivity obtained was a record of 1.6 g L^–1 d^–1. The population density exerted a direct effect on productivity: The higher the light intensity, the more pronounced was the dependence of the output rate on the population density, variations of 10%± from the optimal density resulting in a significant decline in productivity. The population density had also a very significant effect on the course of photoadaptation which took place during the first days after transferring the cultures from the laboratory to the outdoors. The output rate was lower by 5 to 35% on the first day of such transfer as compared to the light-adapted control. The higher the cell density, the faster was the process of photoadaptation as indicated by the rise of the productivity and O₂ tension to the control level. The potential for excess light damages was most prominent in the column reactors used, in which the light path was much reduced compared with that in open raceways. Significant photoinhibition took place at below optimal population density (2.8–3.8 g L^–1), and when cell density was further reduced (1.9 to 1.1 g L^–1), exposure to full sunlight caused photooxidative death within a few hours. The pattern of O₂ concentration in the culture that emerged along the day served as a useful indicator of photolimitation.Author for correspondence     

Annual productivity of Spirulina (Arthrospira) and nutrient removal in a pig wastewater recycling process under tropical conditions

An evaluation was made of the annual productivity of Spirulina (Arthrospira) and its ability to remove nutrients in outdoor raceways treating anaerobic effluents from pig wastewater under tropical conditions. The study was based at a pilot plant at La Mancha beach, State of Veracruz, Mexico. Batch or semi-continuous cultures were established at different seasons during four consecutive years. The protein content of the harvested biomass and the N and P removal from the ponds were also evaluated. Anaerobic effluents from digested pig waste were added in a proportion of 2% (v/v) to untreated sea-water diluted 1:4 with fresh water supplemented with 2 g L^–1 sodium bicarbonate, at days 0, 3 and 5. A straight filament strain of Spirulina adapted to grow in this complex medium was utilized. A pH value 9.5 ± 0.2 was maintained. The productivity of batch cultures during summer 1998 was significantly more with a pond depth of 0.10 m than with a depth 0.065 m. The average productivity of semi-continuous cultures during summer 1999 was 14.4 g m^–2 d^–1 with a pond depth of 0.15 m and 15.1 g m^–2 d^–1 with a depth of 0.20 m. The average annual productivity for semi-continuous cultures operating with depths of 0.10 m for winter and 0.15 and 0.25 m for the rest of the year, was 11.8 g m^–2 d^–1. This is the highest value reported for a Spirulina cultivation system utilising sea-water. The average protein content of the semi-continuous cultures was 48.9% ash-free dry weight. NH₄-N removal was in the range 84–96% and P removal in the range of 72–87%, depending on the depth of the culture and the season.     

Outdoor cultivation ofArthrospira platensis during autumn and winter in temperate climates

Arthrospira (Spirulina) platensis M2 was grown outdoors in 50-mm diameter tubular reactors under the climatic conditions of central Italy (Florence) from September to December 1995 and in March 1996. Except for September, the cultures temperature was regulated. Mean productivities of 0.83, 0.44 and 0.61 g dry wt L^–1 d^–1 were achieved in autumn (September–October), winter (November–December) and March, respectively. In autumn and winter, the photosynthetic efficiency of the cultures and the degree of correlation between productivity and solar irradiance were significantly greater than in summer. The effect of cell density and aeration rate on productivity was evaluated in September. The productivity of cultures operated at high supra-optimal population density was about 30% less at high aeration rate (1.0 LL^–1 min^–1), and 50% less at standard aeration rate (0.17 LL^–1 min^–1), than that of control cultures kept at optimal population density and standard aeration rate. The reduction of productivity in high-density cultures was due to lower daylight output rates and higher night biomass losses (the latter were particularly relevant under standard aeration conditions). The main factor limiting productivity in closed reactors during autumn was the night temperature. Heating the cultures during daylight hours on sunny days did not cause any significant increase of the yields, since under sunlight the unheated cultures also reached the optimal temperature for growth early in the morning. On cloudy days, the day-time temperature of the unheated cultures remained well below the optimum, however this had only a limited effect on productivity since algal growth was mainly light-limited.     

Harvesting of Aphanizomenon flos-aquae Ralfs ex Born. & Flah. var. flos-aquae (Cyanobacteria) from Klamath Lake for human dietary use

In western cultures, certain cyanobacteria have beenan accepted source of microalgal biomass for food forabout 30 years, in particular Spirulina(Arthrospira) platensis and S. maxima. Beginning in the early 1980s, another species, Aphanizomenon flos-aquae was adopted for similaruses. This is harvested from Upper Klamath Lake, thelargest freshwater lake system in Oregon. In 1998 theannual commercial production of Aphanizomenonflos-aquae was about 1 × 10⁶ kg. Since thisspecies is not cultured like Spirulina inoutdoor ponds or raceways, it requires very differentprocedures for harvesting and processing. These arereviewed here and include extensive off-lake screensor on-lake barges, which dewater and concentrate thecells. Other procedures, such as those for removal ofdetritus and mineral materials, and those formonitoring and reducing the amounts of certaincontaminant cyanobacteria, which can producecyanotoxins, have also become important in qualitycontrol and marketing.     

Recombination protein A gene,recA, fromSpirulina platensis IAM-M135

We report the cloning and sequencing of therecA gene fromSpirulina platensis. A genomic library ofSpirulina was constructed in pUC19 and screened by PCR using oligonucleotides corresponding to the conserved amino acid sequences ofAnabaena variabilis andSynechococcus RecA proteins. TheSpirulina recA gene consists of an open reading frame (ORF) of 1095 nucleotides encoding a protein (365 residues) which shares an identity of 79%, 70% and 57% with the RecA proteins ofAnabaena variabilis, Synechococcus andEscherichia coli respectively. TherecA gene is located close to one end of the clonedBglII fragment and has only 53 bp of 5 nucleotides. The isolation of this gene has implications for the development of gene transfer system(s) forSpirulina.     

Drought, differential mortality and the coexistence of a native and an introduced fish species in a south east Australian intermittent stream

Synopsis The longitudinal distribution ofSalmo trutta andGalaxias olidus, a small salmoniform fish, was mapped over four summers (1985–1988) in the upper reaches of the Lerderderg River, an intermittent stream in central Victoria. Over the four successive summers of the study, the distribution ofS. trutta expanded upstream. Coincident with the expansion ofS. trutta was a contraction in the distribution ofG. olidus upstream. In the summer of 1988, an extended dry spell resulted in high levels ofS. trutta mortality at the upstream limits of their distribution. This suggested that the upstream distribution ofS. trutta could ultimately be constrained by an inability to tolerate prolonged periods of low stream flow and high temperatures that tended to occur in the headwaters of the stream. A study of summer mortality was conducted over three successive summers (1989–1991) at three reaches along the river. The two downstream reaches contained onlyS. trutta, the upstream reach contained bothS. trutta andG. olidus. Salmo trutta mortality was consistently higher at the most upstream reach compared with lower reaches. High levels ofS. trutta mortality at the two downstream reaches were only observed in 1991, a particularly dry year. In contrast,G. olidus survived in scattered small pools throughout the upper reaches of the stream. The results of the study suggest a shifting pattern ofS. trutta andG. olidus distribution determined by the duration of low stream flow periods and summer high temperatures.     

Kraft black liquor for improving the productivity of Spirulina biomass

Summary Mass cultivation of Spirulina for commercial application suffers from poor productivity when measured against laboratory results or theoretical projections. Wider applications of algal products require that this gap be reduced. Addition of eucalyptus kraft black liquor at a maximum of 0.1% to Spirulina cultures enhanced biomass productivity by at least 40%. The factors enhancing Spirulina biomass productivity were insoluble at low pH, of low molecular mass and stable to high temperature. Single addition of kraft black liquor in outdoor continuous cultures afforded sustained enhancement in biomass productivity for at least eight weeks.     

Increasing productivity of Spirulina platensis in photobioreactors using artificial neural network modeling

Although production of microalgae in open ponds is conventionally practiced due to its economy, exposure of the algae to uncontrollable elements impedes achievement of quality and it is desirable to develop closed reactor cultivation methods for the production of high value products. Nevertheless, there are several constraints which affect growth of in closed reactors, some of which this study aims to address for the production of Spirulina. Periodic introduction of fresh medium resulted in increased trichome numbers and improved algal growth compared to growth in medium that was older than 4 weeks in 20 L polycarbonate bottles. Mixing of the cultures by bubbling air and use of draft tube reduced the damage to the growing cells and permitted increased growth. However, there was better growth in inclined cylindrical reactors mixed with bubbling air. The oxygen production rates were very similar irrespective differences in the maintained cultures densities. The uniformity in oxygen production rate suggested a tendency towards homeostasis in Spirulina cultures. The frequency of biomass harvest on the productivity of Spirulina showed that maintenance of moderate culture density between 0.16 and 0.32 g/L resulted in about 14% more productivity than maintaining the cell density between 0.16 and 0.53 g/L or 48% more than by daily harvest above 0.16 g/L. An artificial neural network based predictive model was developed, and the variables useful for predicting biomass output were identified. The model could predict the growth of Spirulina up to 3 days in advance with a coefficient of determination >0.94.     

A new tubular reactor for mass production of microalgae outdoors

A novel reactor for outdoor production of microalgae is described. Air-lift is used for circulation of the culture in transparent tubes lying on the ground and interconnected by a manifold. Dissolved O₂ is removed through a gas-separator placed 2.0 m above the tubes and water-spray is used for cooling. The manifold permits short-run durations between leaving the gas separator and re-entering it, preventing thereby damaging accumulation of dissolved oxygen. Day temperature control in summer is attained using water-spray. In winter, temperature in the tubes rises rapidly in the morning, as compared to an open raceway even if placed in a greenhouse. The number of hours along which optimal temperature prevails in the culture throughout the year increased significantly. Very high daily productivity computed on a volumetric basis (e.g. 550 mg dry wt l^–1 culture) was obtained and preliminary observations indicate that a significantly higher output, e.g. 1500 mg dry wt l^–1 d^–1 is attainable. Much more research is required to assess the year-round, sustained productivity attainable in this reactor.     

Role of light and photosynthesis on the acclimation process of the cyanobacteriumSpirulina platensis to salinity stress

The response ofSpirulina platensis cells to salinity stress was studied. Once adapted to the higher osmoticum, photosynthetic parameters such as the maximum rate of photosynthesis under saturating irradiance (P_max) and the initial slope of the P-I curve () are reduced by 15% and 25% in 0.5 M NaCl grown cells, respectively. Salt-adapted cells have a modified biochemical composition; reduced protein and chlorophyll content, and an increased level of carbohydrates. The reduction in the photosynthetic capacity of the salt-adaptedSpirulina cells reflects a lower ability to utilize light energy and results in an increase in the susceptibility of the stressed cells to photoinhibition. This conclusion is supported by the finding that cultures exposed to salt stress show not only a decrease in growth rate (), but lose the ability to respond to increased irradiance with an increase in growth. The use of variable fluorescence as a fast and reliable measurement to follow the changes in PSII of salt-stressesSpirulina cells enables following the early events of salinity shock. It indicates that as soon as the cells are exposed to salt, a protection mechanism is induced. This mechanism does not require any protein synthesis and may take place even in the dark, though at somewhat reduced effectiveness. The significance of the result in providing a better understanding of the interaction between two environmental stresses — light and salinity — and their application in the outdoor mass cultivation ofSpirulina are discussed.Author for correspondence     

Productivity and photosynthetic efficiency ofSpirulina platensis as affected by light intensity,algal density and rate of mixing in a flat plate photobioreactor

The effect of the rate of mixing on productivity of algal mass in relation to photon flux density and algal concentration was quantitatively evaluated in cultures ofSpirulina platensis grown in a newly designed flat-plate photobioreactor. Special emphasis was placed on elucidating the principles underlying efficient utilization of high photon flux density for maximal productivity of algal-mass. Whereas the rate of mixing exerted little influence on productivity and photosynthetic efficiency in cultures of relatively low algal density, its effect became ever more significant as algal concentration was increased. Maximal mixing-enhanced cell concentrations and productivity of biomass were obtained at the highest light intensity used. At each level of incident light intensity, maximum productivity and photosynthetic efficiency could be achieved only when algal concentration and mixing rates were optimized. The higher the intensity of the light source, the higher became the optimal culture density, highest algal concentrations and productivity of biomass being obtained at the highest light intensity used. The rate of mixing required careful optimization: when too low, maximal productivity resulting from the most efficient utilization of light could not be obtained. Too high a rate of mixing resulted in cell damage and reduced output rate.Author for correspondence     

Gelidium pristoides in South Africa

Gelidium pristoides has been harvested commercially from the eastern Cape, South Africa, since 1951, with 40–80 t y^–1 (dry wt) collected in recent years. This species has been intensively studied since 1983, and we briefly review knowledge of its biology in relation to harvesting. We describe a new study of intertidal epiphytic animals, showing that none is specific to G. pristoides, and that only 2.8% of these animals (numbers) inhabit this agarophyte, while the rest are found in other intertidal algal communities: harvesting is considered to have negligible effects on epifauna. Over the past 3 y, we have monitored, at two sites, the effects of the harvesting of G. pristoides on other benthic algae and animals. In only two of the seven main components analysed, did we find any difference between harvested and control plots. At one site only, the number of limpets and percentage cover of Gelidium was higher in harvested plots. These results show that harvesting has no significant biological effect. Regulations governing seaweed exploitation in South Africa were amended in 1988, to encourage local processing of products, and these changes are discussed in relation to the local Gelidium industry. Despite experimental results predicting a higher yield per unit effort if harvesting is limited to summer, harvesting continues throughout the year for practical reasons.     

TUBE SIZE COMPARISON AND AUTOMATED GROWTH MEASUREMENT IN A TUBULAR REACTOR USING SPIRULINA PLANTENSIS

A tubular photobioreactor for outdoor cultivation of Spirulina platensis was successfully operated for the last two years. The reactor was made of transparent 2.4‐cm diameter tubes with a total length of approximately 101‐m and a volume of 124‐liter. Flow was induced using an airlift pumping system. To optimize the system further, a larger tube diameter was also tested. Preliminary results have suggested that a larger tube diameter might provide increased output with reduced surface area requirements. Results of experiments comparing the productivity of the same culture volume in tubes of 2.4 cm and 5.0 cm will be presented which show a small decrease in productivity by volume, but a large increase in areal productivity. This suggests that the larger tube diameter would be an appropriate choice for larger scale systems. Additionally, data will be presented demonstrating the effectiveness of an on‐line surface scatter turbidimeter for accurate measurement of Spirulina density when correlated to manual dry weight measurements.     

In situ monitoring of chlorophyll fluorescence to assess the synergistic effect of low temperature and high irradiance stresses inSpirulina cultures grown outdoors in photobioreactors

A chlorophyll fluorescence technique was applied to anin situ study on the effects of low temperature and high light stresses onSpirulina cultures grown outdoors in controlled tubular photobioreactors at high (1.1 g L^–1) and low (0.44 g L^–1) biomass concentrations. Diurnal changes in PSII photochemistry (F _v/F _m) after 15 min of darkness, or in the light (dF/F _m), and non-photochemical (qN) quenching were measured using a portable, pulse-amplitude-modulated fluorometer. The depression of theF _v/F _m ratio ofSpirulina cultures grown outdoors at 25°C (i.e. 10°C below optimum for growth) and 0.44 g L^–1, reached 30% at the middle of the day. At the same time of the day thedF/F _m ratio showed a reduction of up to 52%. The depression of bothF _v/F _m anddF/F _m was lower in the cultures grown at 1.1 g L^–1. Photoinhibition reduced the daily productivity of the culture grown at 0.44 g L^–1 and 25°C by 33% with respect to that grown at 35°C. Changes in the growth yields of the cultures grown under different temperatures and growth rates correlate well with analogous changes in photon yield (dF/F _m). Simple measurements of photochemical yield (F _v/F _m) can be used to test the physiological status ofSpirulina cultures. The results indicate that the saturating pulse fluorescence technique, when usedin situ, is a powerful tool for assessment of the photosynthetic characteristics of outdoor cultures ofSpirulina.     

Growth and amino acid contents of<Emphasis Type="Italic">Spirulina platensis</Emphasis> with different nitrogen sources

The growth and amino acid contents of the cyanobacterium,Spirulina platensis strain NIES 46, were investigated using ammonium, nitrate, nitrite, or urea as the sole nitrogen source in a batch culture. Chlorophylla concentration was highest at 2,096 μg/L in the nitrate group after 10 days of cultivation, while the dry weight ofS. platensis was highest at 4.5 g/L in the ammonium group after 30 days of cultivation. The total amino acid content was highest at 174 mg/g dry weight ofS. platensis in the urea group at the end of the cultivation period, yet the amino acid patterns forS. platensis were similar for all the experimental groups. Therefore, it seemed that the growth and amino acid composition ofS. platensis varied depending on the type of nitrogen sources, while the amino acid patterns were not changed. Also, the most efficient harvesting time forS. platensis seemed to be approximately 10 days after cultivation.     

Analysis of photosynthetic productivity of microalgal mass cultures

The calculated value of microalgal massproductivity is an important parameter incommercial mass production and derivativecompound production. Mathematical analysiswas conducted in order to predict the rateof microalgal mass production, which wascalculated from the factor of oxygenevolution rate and the respiration rate percell. Calculated productivities of twomutants with small light-harvestingpigment, a phycocyanin deficient mutant ofSynechocystis PCC 6714 (strain PD-1)and a mutant with small light-harvestingpigment of Chlamydomonasperigranulata (strain LHC-1), wereevaluated compared with the wild-types ofthese mutants, respectively. The resultsshow that calculated productivity isimproved by reducing the content oflight-harvesting pigment, which issupported by the actual values ofphotosynthetic productivity. Productiveimprovement by reducing the content oflight-harvesting pigment is not limited toa special strain but applies to a widevariety of photosynthetic organisms.     

Matter-economical roles of the evergreen foliage ofAucuba japonica,an understory shrub in the warm-temperate region of Japan

Leaf demography and productivity ofAucuba japonica, an understory shrub in the warm-temperate region, were examined and dry matter economy was analyzed to evaluate the roles of the evergreen foliage. Turnover of leaves occurred during a short period in spring. The mean leaf life span was about 2.6 years. Annual NAR (net assimilation rate) of each sample shoot was calculated from the biomass and the total dead mass estimated from scars of leaves and floral parts. The average NAR was 1.34±0.22 g·g⁻¹·yr⁻¹. The ratio of dry matter produced by leaves during their whole life span to the initial investment was 3.45±0.37. The annual NAR calculated for individual plants was negatively related to the life span of their leaves. The seasonal change in SLW (specific leaf weight) showed that the reserve material in leaves was accumulated from autumn to early spring and was consumed for the growth of new organs in the following season. The dry matter withdrawn in spring from the overwintering foliage amounted to 40% of dry mass of the new organs developed.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors

CO₂ at different concentrations were added to cultures of the eukaryotic microalgae, Chlorella kessleri, C. vulgaris and Scenedesmus obliquus, and the prokaryotic cyanobacterium, Spirulina sp., growing in flasks and in a photobioreactor. In each case, the best kinetics and carbon fixation rate were with a vertical tubular photobioreactor. Overall, Spirulina sp. had the highest rates. Spirulina sp., Sc. obliquus and C. vulgaris could grow with up to 18% CO₂.     

Outdoor cultivation of lutein-rich cells of <Emphasis Type="Italic">Muriellopsis</Emphasis> sp. in open ponds

The growth performance of the chlorophycean microalga Muriellopsis sp. outdoors in open tanks agitated with a paddlewheel and its ability to accumulate carotenoids have been evaluated throughout the year. The cells grown in the open system had free lutein as the main carotenoid, with violaxanthin, β-carotene, and neoxanthin also present. Lutein content of the dry biomass ranged from 0.4 to 0.6%, depending on the growth and environmental conditions. In addition, the biomass of Muriellopsis sp. had a high content in both protein and lipids with about half of the fatty acids being of the polyunsaturated type, with α-linolenic acid accounting for almost 30% of the total fatty acids. The effect of determinant parameters on the performance of the cultures in open tanks was evaluated. Operating conditions that allow the maintenance of productive cultures were established under semicontinuous regime for 9 months throughout the year. Biomass and lutein yields in the open system were not far from those in closed tubular photobioreactors, and reached productivity values of 20 g dry biomass, containing around 100 mg lutein m⁻² day⁻¹ in summer. The outdoor culture of Muriellopsis sp. in open ponds thus represents a real alternative to established systems for the production of lutein.