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.     

A Modular Flat Panel Photobioreactor (MFPP) for indoor mass cultivation of Nannochloropsis sp. under artificial illumination

Nannochloropsis sp. was grown in a Modular FlatPanel Photobioreactor (MFPP) consisting of sixalveolar panels each with 20.5 L culture volume and3.4 m² illuminated surface area. The panelsformed a closely-packed unit with illuminationprovided by banks of fluorescent tubes placed betweenthe panels. The whole unit was contained in athermoregulated cabinet. Continuous illumination ofone side of the panels with 115 molphoton m^-2 s^-1 attained a mean volumetricproductivity of 0.61 g (d. wt) L^-1 24 h^-1,increasing to 0.97 g (d. wt) L^-1 24 h^-1 whenthe same irradiance was provided on both sides of thepanels. With 230 mol photon m^-2 s^-1 onone side of the panel, a mean productivity of 0.85 g(d. wt) L^-1 24 h^-1 was achieved, whichreached 1.45 g (d. wt) L^-1 24 h^-1 when bothsides were illuminated. Increasing the amount of lightprovided to the culture (either by increasingirradiance or the illuminated surface area) decreasedpigment and enhanced the total fatty acid content, butdid not change significantly the content ofeicosapentaenoic acid. A MFPP of the presentdimensions could produce sufficient microalgae tosupport a hatchery producing 6 million sea breamfingerlings annually.     

Light/dark cyclic movement of algal culture (<Emphasis Type="Italic">Synechocystis aquatilis</Emphasis>) in outdoor inclined tubular photobioreactor equipped with static mixers for efficient production of biomass

Synechocystis aquatilis SI-2 was grown outdoors in a 12.5cm diam. tubular photobioreactor equipped with static mixers. The static mixers ensured that cells were efficiently circulated between the upper (illuminated) and lower (dark) sections of the tubes. The biomass productivity varied from 22 to 45g m^–2d^–1, with an average of 35g m^–2d^–1, etc which corresponded to average CO₂ fixation rate of about 57 g CO₂ m^–2 d^–1. The static mixers not only helped in improving the biomass productivities but also have a high potential to lower the photoinhibitory effect of light during the outdoor cultures of algae. Revisions requested 27 July 2004; Revisions received 12 November 2004     

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).     

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.     

Production potential of eicosapentaenoic acid by the diatom Nitzschia laevis

To investigate the production potential of eicosapentaenoic acid (EPA) by the diatom Nitzschia laevis, the growth characteristics and fatty acid composition of the cells were studied under photoautotrophic, mixotrophic and heterotrophic conditions of growth. The specific growth rate and maximum biomass concentration were respectively 0.466 d^–1 and 2.27 g l^–1 for mixotrophic culture, 0.344 d^–1 and 2.04 g l^–1 for heterotrophic culture, and 0.167 d^–1 and 0.5 g l^–1 for photoautotrophic culture, respectively. As for EPA production, the yield and productivity were respectively 52.32 mg l^–1 and 10.46 mg l^–1 d for mixotrophic culture, 35.08 mg l^–1 and 6.37 mg l^–1 d for heterotrophic culture, and 6.78 mg l^–1 and 3.39 mg l^–1 d for photoautotrophic culture, respectively. Results suggest that mixotrophic culture is the most suitable growth mode for the production of EPA by the diatom Nitzschia laevis. The results are useful for the development of a cost-effective fermentation process for EPA production by Nitzschia laevis.     

Cultivation of Gracilaria verrucosa (Gracilariales,Rhodophyta) Strain G-16 for agar

The agarophyte, Gracilaria verrucosa Strain G-16, has been grown in sustained cultivation over a period of five years. During this period, a number of experiments were conducted to examine the productivity, agar yield and agar gel strength of this strain. Productivities range from 3–31 g dry wt m^–2 d^–1 and are generally highest in the summer when annual water temperatures and daily irradiances are highest. In the summer months agar yield from Strain G-16 appears to be lowest whereas the gel strength of the agars was highest (> 750 g cm^–2).     

Overall assessment of Monodus subterraneus cultivation and EPA production in outdoor helical and bubble column reactors

The outdoor production of Monodus subterraneus wasstudied in bubble column and helical reactors, mainly analysing the influenceofdilution rate, air flow rate and solar irradiance on growth rate andbiochemicalcomposition. Photoinhibition and photo-oxidation phenomena were also analysed.The cultures were stressed at high solar irradiance and dissolved oxygenconcentrations. A clear relationship between stress of the cultures and thefluorescence from PSII measurements was observed, the Fv/Fm ratio being lowerinthe helical reactor than in the bubble column. Growth rate and biomassproductivity were both a function of the average irradiance and the Fv/Fmratio;maximum values of 0.040 h^–1 and 0.54 gL^–1 d^–1 were measured. The influenceofphotoinhibition and average irradiance was modelled, the model also fitting theexperimental data reported by another author. The chlorophyll contenthyperbolically decreased, whereas the carotenoid content decreased linearlywiththe average irradiance. The higher the dilution rate the higher the protein andcarbohydrate content of the biomass, and the lower the lipid content. Theeicosapentaenoic acid (EPA) content ranged from 2.3 to 3.2% d.wt, the higherthe dilution rate, the lower EPA content, although the higher the EPAproportion. Maximum EPA productivity was only 9 mg L^–1d^–1, due to the stress to which the cultures wereexposed.     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

The cell composition of Nannochloropsis sp. changes under different irradiances in semicontinuous culture

Nannochloropsis sp. was grown semicontinuously with a rate of daily renewal of the culture media of 40% of the volume of the culture under different irradiances (40, 60, 80, 220 and 480 mol quanta m^–2 s^–1). Under the conditions tested, light saturation was achieved at 220 mol quanta m^–2 s^–1 with no significant increase in steady-state cell density or of dry weight productivity with higher irradiance, reaching values of 115 × 10⁶ cells ml^–1 and 375 mg l^–1 day^–1 respectively. C/N ratios clearly indicated the point of light saturation, decreasing with increasing irradiance for light-limited conditions and increasing for light-saturated conditions. Under light-limited conditions, an increase in the irradiance produced an increase in the protein percentage of the organic fraction to the detriment of lipids and carbohydrates, while small changes were recorded under light-saturated conditions. The degree of unsaturation of fatty acids was lower with increasing irradiance, with a three-fold decrease of the percentage of total n–3 fatty acids, from 29 to 8% of total fatty acids, caused mainly by a decrease of eicosapentaenoic acid (EPA) (20:5n–3). The microalga reached its maximal value of dry weight productivity (375 mg l^–1 day^–1), EPA productivity (3.2 mg l^–1 day^–1) and maximal protein content (36% of the organic content) at the point at which light saturation was achieved. Results demonstrate the efficiency of the use of the irradiance for the modification of the biochemical composition of Nannochloropsis sp.     

Intensive rotifer cultures using chemostats

Continuous production of the rotifer Brachionus plicatilis rotundiformis (S-type) in an intensive chemostat culture system has been investigated. The production dynamics of rotifers in relation to different flow rates and feed regimes show that the growth rate and production depends on the type of algal feed and flow rate utilized in the culture system. It was possible to achieve a mean production of up to 318.84 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 6 1 h^–1 in 100 1 chemostats and up to 261.21 × 10⁶ rotifers m^–3 d^–1 at a flow rate of 40 1 h –1 while using 1 m³ capacity rotifer chemostats as production units. The 3 fatty acid composition of rotifers while using Chlorella and Nannochloropsis in the culture system has been described. The results of this investigation show that the rotifer productivity in the continuous culture system is considerably higher than in any of the conventional culture systems described to date for aquacultural purposes.This research was financed by the Kuwait Foundation for the Advancement of Sciences (KFAS), Kuwait, under a contract research project code 86-04-02.     

Optimization of eicosapentaenoic acid production byPhaeodactylum tricornutumin the flat panel airlift (FPA) reactor

Biomass and eicosapentaenoic acid (EPA) productivities were investigated in a flat panel airlift loop reactor ideally mixed by static mixers. Growth with ammonium, urea and nitrate as nitrogen source were performed at different aeration rates. Cultures grew on ammonium but the decay of pH strongly inhibited biomass increase. On urea biomass productivity reached 2.35 g L^–1d^–1at an aeration rate of 0.66 vvm (24 h light per day, 1000 mol photon m^–2s^–1). Aeration rates between 0.33 vvm and 0.66 vvm and maximal productivities on urea were linearly dependent. Productivity on nitrate never exceeded 1.37 g L^–1d^–1. In the range of maximum productivity photosynthesis efficiency of 10.6% was reached at low irradiance (250 mol photon m^–2s^–1). Photosynthesis efficiency decreased to 4.8% at 1000 mol photon m^–2s^–1. At these high irradiances the flat panel airlift reactor showed a 35% higher volume productivity than the bubble column. At continuous culture conditions the influence of CO₂concentration in the supply air was tested. Highest productivities were reached at 1.25% (v/v) CO₂where the continuous culture yielded 1.04 g L^–1d^–1(16 h light per day, 1000 mol photon m^–2s^–1). The average EPA content amounted to 5.0% of cell dry weight, that resulted in EPA productivities of 52 mg L^–1d^–1(continuous culture, 16 h light per day) or 118 mg L^–1d^–1(batch culture, 24 h light per day).     

A closed solar photobioreactor for cultivation of microalgae under supra-high irradiance: basic design and performance

An account is given of the setting up and use of a novel type of closed tubular photobioreactor at the Academic and University Centre in Nove Hrady, Czech Republic. This "penthouse-roof" photobioreactor was based on solar concentrators (linear Fresnel lenses) mounted in a climate-controlled greenhouse on top of the laboratory complex combining features of indoor and outdoor cultivation units. The dual-purpose system was designed for algal biomass production in temperate climate zone under well-controlled cultivation conditions and with surplus solar energy being used for heating service water. The system was used to study the strategy of microalgal acclimation to supra-high solar irradiance, with values as much as 3.5 times the ambient value, making the approach unique. The cultivation system proved to be fully functional with sufficient mixing and cooling, efficient oxygen stripping and light tracking. Experimental results (measurement of the maximum photochemical yield of PSII and non-photochemical quenching) showed that the cyanobacterium Spirulina (= Arthrospira) platensis cultivated under sufficient turbulence and biomass density was able to acclimate to irradiance values as high as 7 mmol photon m^–2 s^–1. The optimal biomass concentration of Spirulina cultures in September ranged between 1.2 to 2.2 g L^–1, which resulted in a net productivity of about 0.5 g L^–1 d^–1 corresponding to a biomass yield of 32.5 g m^–2 d^–1 (based on the minimum illuminated surface area of the photobioreactor).     

Growth physiology of a marine nitrogen-fixing cyanobacterium (Nodularia harveyana) in outdoor culture

The performance ofNodularia harveyana, a N₂-fixing cyanobacterium isolated from seawater, has been studied outdoors in two different culture systems: open pond (OP) and tubular photobioreactor (TPR). The productivity in both devices was influenced by areal density. The maximum yield obtained was 12.0 g (d.wt) m^–2 day^–1 in OP and 14.0 g (d.wt) m^–2 day^–1 in TPR in August, corresponding to the highest solar radiation received. In a month-long experiment with the cyanobacterium cultivated in TPR at high circulation speed, a net increase in productivity was obtained over that at low circulation speed. The influence of temperature on the productivity of the cultures grown in open ponds and tubular photobioreactors has been investigated. The higher productivity obtained in TPR compared to OP was attributed to its better controlled temperature conditions. In outdoor culture the maximum nitrogenase activity did not coincide with the maximum light intensity, but occurred in early afternoon. The amount of carbohydrate accumulated during the day probably influenced the rate of dark nitrogenase activity and its duration in the night.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

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.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

A study of polynucleotide phosphorylase production by <Emphasis Type="Italic">Escherichia coli</Emphasis> in a hollow fibre reactor

A 30-l hollow fibre reactor with continuous fermentation for cell recycling of Escherichia coli AS 1.183 was used to remove the inhibitory effects on cell growth and extend the fast growth phase to increase the yield of polynucleotide phosphorylase (PNPase) in E. coli cells. When the dilution rate was 1.5 h⁻¹, the cell concentration of E. coli reached 235 g/l (wet wt, 70% moisture content), with PNPase activity above 90 u/g (wet wt). With the dilution rate is 1.0 h⁻¹, the fermentor volumetric productivity of PNPase in a hollow fiber reactor can reach 974 (u/h * l) compared to 20 (u/h * l) in a conventional batch culture.     

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.     

Batch xylitol production from wheat straw hemicellulosic hydrolysate using Candida guilliermondii in a stirred tank reactor

Batch production of xylitol from the hydrolysate of wheat straw hemicellulose using Candida guilliermondii was carried out in a stirred tank reactor (agitation speed of 300 rpm, aeration rate of 0.6 vvm and initial cell concentration of 0.5 g l^–1). After 54 h, xylitol production from 30.5 g xylose l^–1 reached 27.5 g l^–1, resulting in a xylose-to-xylitol bioconversion yield of 0.9 g g^–1 and a productivity of 0.5 g l^–1 h^–1.