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.     

Combined carbon and nitrogen removal from acetonitrile using algal–bacterial bioreactors

When compared with Chlorella vulgaris, Scenedesmus obliquus and Selenastrum capricornutum, C. sorokiniana presented the highest tolerance to acetonitrile and the highest O₂ production capacity. It also supported the fastest acetonitrile biodegradation when mixed with a suitable acetonitrile-degrading bacterial consortium. Consequently, this microalga was tested in symbiosis with the bacterial culture for the continuous biodegradation of acetonitrile at 2 g l^–1 in a stirred tank photobioreactor and in a column photobioreactor under continuous illumination (250 E m^–2 s^–1). Acetonitrile removal rates of up to 2.3 g l^–1 day^–1 and 1.9 g l^–1 day^–1 were achieved in the column photobioreactor and the stirred-tank photobioreactor, respectively, when operated at the shortest retention times tested (0.4 days, 0.6 days, respectively). In addition, when the stirred-tank photobioreactor was operated with a retention time of 3.5 days, the microbial culture was capable of assimilating up to 71% and nitrifying up to 12% of the NH₄⁺ theoretically released through the biodegradation of acetonitrile, thus reducing the need for subsequent nitrogen removal. This study suggests that complete removal of N-organics can be combined with a significant removal of nitrogen by using algal–bacterial systems and that further residual biomass digestion could pay-back part of the operation costs of the treatment plant.     

Feeding rate inhibition in crowded Daphnia pulex

Feeding rates of Daphnia pulex fed a range of levels of the alga Chlamydomonas reinhardi of 15 °C are strongly density-dependent. At lower densities, Daphnia (30 1^–1) fed at higher rates than crowded (270 1^–1) Daphnia which manifest a relatively depressed saturation feeding response. At 30 individuals/liter, Daphnia consumed 8.5 – 15.7 × 10⁴ cells d^–1h^–1 (on a volume basis, 12.1 – 22.2 × 10⁶ m³), at 270 L^–1 3.7 – 3.9 × 10⁴ (5.2 – 5.5 = 10⁶ m³ cells d^–1h^–1 when feeding on algae at 80 000 cells ml^–1 (11.3 × 10⁶ m³ ml^–1). The feeding rate data best fit an Ivlev feeding function. An autoallelopath might be causing the repression. Water preconditioned with crowded Daphnia completely repressed feeding in uncrowded Daphnia after six hours.     

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d⁻¹) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell⁻¹) compared with cells grown by other culture methods (0.27–0.49 pg cell⁻¹). The highest cell density and cellular toxin content were 17190 cells mL⁻¹ and 1.26 pg cell⁻¹ respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.     

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).     

Biology of Moina mongolica (Moinidae,Cladocera) and perspective as live food for marine fish larvae: review

Moina mongolica, 1.0-1.4 mm long and 0.8 mm wide, is an Old World euryhaline species. This paper reviewed the recent advances on its autecology, reproductive biology, feeding ecology and perspective as live food for marine fish larviculture. Salinity tolerance of this species ranges from 0.4–1.4 to 65.2–75.4. Within 2–50 salinity, Moina mongolica can complete its life cycle through parthenogenesis. The optimum temperature is between 25 °C and 28 °C, while it tolerates high temperature between 34.4 °C and 36.0 °C and lower temperature between 3.2 °C and 5.4 °C. The non-toxic level of unionised ammonia (24 h LC₅₀) for M. mongolica is <2.6 mg NH₃–N l^–1. Juvenile individuals filter 2.37 ml d^–1 and feed 9.45×10⁶ algal cells d^–1, while mature individuals filter 9.45 ml d^–1 and consume 4.94×10⁶ algal cells d^–1. At 28 °C, M. mongolica reaches sex maturity in 4 d and gives birth once a day afterward; females carry 7.3 eggs brood^–1 and spawn 2.8 times during their lifetime. A variety of food can be used for M. mongolica culture including unicellular algae, yeast and manure, but the best feeding regime is the combination of Nannochloropsis oculata and horse manure. Moina mongolica reproduces parthenogenetically during most lifetime, but resting eggs can be induced at temperature (16 °C) combined with food density at 2000–5000 N. oculata ml^–1. The tolerance to low dissolved oxygen (0.14–0.93 mg l^–1) and high ammonia makes it suitable for mass production. Biochemical analyses showed that the content of eicospantanoic acid (20:53) in M. mongolica accounts for 12.7% of total fatty acids, which is higher than other live food such as Artemia nauplii and rotifers. This cladoceran has the characteristics of wide salinity adaptation, rapid reproduction and ease of mass culture. The review highlights its potential as live food for marine fish larvae.     

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     

A novel photobioreactor with transparent rectangular chambers for cultivation of microalgae

An open tank photobioreactor containing transparent rectangular chambers (TRCs) was developed to improve the photosynthetic efficiency of microalgal cultivation. The TRCs, made of transparent acrylic, conducted light deep into the photobioreactor, especially at high cell concentrations. The average irradiance, I_av, was calculated by Lambert–Beer's law, and was used to determine the light conditions in the cultivation system. The photobioreactor provided large areas of illumination that improved the effective utilization of light energy for microalgae growth and created a good artificial environment for a high rate of cell growth, even at low I_av. The biomass concentration of Chlorella sp. reached 3.745 g L⁻¹ on the 13th day, with biomass productivity of 0.340 g L⁻¹ d⁻¹. The total biomass obtained was 56% more than that of similar culture systems without TRCs.     

Thermophilic H2 production from a cellulose-containing wastewater

Cellulose in wastewater was converted into H₂ by a mixed culture in batch experiments at 55°C with various wastewaters pH (5.5–8.5) and cellulose concentrations (10–40 g l^–1). At the optimal pH of 6.5, the maximum H₂ yield was 102 ml g^–1 cellulose and the maximum production rate was 287 ml d^–1 for each gram of volatile suspended solids (VSS). Analysis of 16S rDNA sequences showed that the cellulose-degrading mixed culture was composed of microbes closely affiliated to genus Thermoanaerobacterium.     

Outdoor pond cultivation of the subtropical marine red algaGracilaria tenuistipitata in brackish water in Sweden. Growth,nutrient uptake,co-cultivation with rainbow trout and epiphyte control

Pond cultivation of the subtropical, euryhaline macroscopic red algaGracilaria tenuisipitata var.liui Zhanget Xia was carried out in brackish seawater (6–7) in the Gryt archipelago on the east coast of Sweden, using four outdoor tanks of 30–40 m³. Growth rate and nutrient uptake in batch culture were measured with the aim of estimating the water purification capacity ofG. tenuisipitata in outdoor conditions. Its ability to withstand epiphytic infections was also studied. An average growth rate of 4 biomass increase per day was recorded during two seasons with a maximum growth rate of 9 d^–1. The initial biomass was usually 1 kgFW m^–3 (FW, fresh weight). The nutrient uptake capacity was on average ca. 1 g N_i kgFW^–1 d^–1 and 0.08 g P_i kgFW^–1 d^–1 and the uptake rates for NH₄ ⁺-N were higher than those for NO₃ ^–-N. Both the growth rate and the nutrient uptake rate were highest at the highest water temperature. Co-cultivation with rainbow trout (Oncorhynchus mykiss) was tested: with trout fodder as the only nutrient inputG. tenuistipitata could grow and maintain low levels of N_i and P_i with optimum efficiency at a trout: alga ratio of 1:1 (w:w). Epiphytic growth of filamentous green and brown algae was limited, probably as a result of the high pH values caused by inorganic carbon uptake byG. tenuistipitata. The growth ofEnteromorpha intestinalis, the only significant epiphyte, was completely inhibited and the majority of plants died by a few days treatment with 100 µg 1^–1 Cu²⁺, a concentration that did not severely affectG. tenuistipitata. We conclude thatG. tenuistipitata can be cultivated in outdoor ponds in southern Sweden during 5–6 months of the year using aerated or unaerated batch cultures and that wastewater from trout cultivation may be used as a nutrient source, resulting in purification with respect to N and P.     

Ethanol acetylation by mycelium-bound carboxylesterase of Aspergillus oryzae: estimation of thermodynamic parameters and integral productivity

The results collected at different temperatures for ethanol acetylation by cell-bound carboxylesterase from lyophilized cells of Aspergillus oryzae have been used to investigate the kinetics and thermodynamics of this esterification in n-heptane. The occurrence of reversible unfolding followed by irreversible denaturation of the enzyme has been proposed to explain the increase in the starting rate of ethyl acetate formation with temperature observed up to 55 °C and the consequent fall beyond this threshold. The Arrhenius model has been used to estimate the apparent activation enthalpies of both the acetylation reaction (H = 29–33 kJ mol^–1) and reversible enzyme unfolding (H _u = 56–63 kJ mol^–1). The results of residual activity tests performed with cells previously exposed at different temperatures for variable times enabled us also to estimate the first-order rate constant of irreversible denaturation (2.40 × 10^–3 h^–1 < k _d < 8.11 × 10^–3 h^–1) as well as the related thermodynamic parameters (H _d = 22 kJ mol^–1; S _d = –0.29 kJ mol^–1 K^–1). This last phenomenon proved particularly slow for the system under consideration, probably because the biocatalyst link to the mycelium was able to improve its thermostability. In view of future continuous application, the effects of operating time, starting substrate concentration and temperature on the theoretical integral productivity of a fixed-bed column filled with this biocatalyst have been investigated.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate     

Effect of dilution rate on eicosapentaenoic acid productivity ofPhaeodactylum tricornutum utex 640 in outdoor chemostat culture

Summary Eicosapentaenoic acid (EPA) volumetric productivity from an outdoor chemostat culture ofPhaeodactylum tricornutum UTEX 640 in a 50-l tubular photobioreactor varies with dilution rate, reaching a maximum of 47.8 mg l^–1 d^–1 at D=0.36 d^–1. Continuous culture at high dilution rates' is proposed as the most adequate operating mode to maximize polyunsaturated fatty acid production.     

Propylphenols are metabolites in the anaerobic biodegradation of propylbenzene under iron-reducing conditions

The metabolism of monoaromatic hydrocarbons by an iron-reducing bacterial enrichment culture originating from diesel-contaminated groundwater was examined using d₇-propylbenzene as a model hydrocarbon. Sequence analysis of the 16S rDNA gene showed that the dominant part (10 of 10 clones) of the enrichment culture consisted of a bacterium closely related to clones found in benzene-contaminated groundwater and to the iron-reducing -proteobacterium, Rhodoferax ferrireducens (similarity values were 99.5% and 98.3%, respectively). In degradation studies conducted over 18 weeks, d₇-propylphenols were detected by gas chromatography–mass spectrometry (GC/MS) as intra-cellular metabolites concomitant with cell growth in the cultures. The amount of propylphenols increased during the exponential growth phase, and by the end of this phase 4 × 10^–14 moles of ferric iron were reduced and 3 × 10^–15 moles propylphenol produced for every cell formed. During the stationary growth phase the cell density was approximately 10⁷ ml^–1, with significantly correlated amounts of propylphenols. Succinate derivates of propylbenzene or phenylpropanol previously shown to be the initial metabolites in the anaerobic degradation of alkylbenzenes could not be identified. This study is the first to report that oxidation of propylbenzene to propylphenols can initiate anaerobic propylbenzene degradation and that iron-reducing bacteria are responsible for this process. In addition, the study shows the importance of taking account of the metabolites adhering to solid phases when determining the extent of biodegradation, so as not to underestimate the extent of the process.     

A simple and low-cost airlift photobioreactor for microalgal mass culture

A simple, low-cost, and efficient airlift photobioreactor for microalgal mass culture was designed and developed. The reactor was made of Plexiglas, and composed of three major parts: outer tube, draft tube and air duct. The fluid-dynamic characteristics of the airlift reactor were studied. The system proved to be well suited to the mass cultivation of a marine microalga, Chlorella sp. In batch culture, the biomass volumetric output rate of 0.21 g l^–1 d^–1 was obtained at the superficial gas velocity of 4 mm s^–1 in the draft tube.     

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

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

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence,spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-GalNAc and for jack fruit agglutinin (JFA) is Me-Gal. Examination of the percentage enhancement and association constants (1.51×10⁶, 6.56×10⁶ and 4.17×10⁵ M^–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×10⁵, 1.3×10⁴, and 11.7×10⁵ M^–1 sec^–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10^–2, 83.3 sec^–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.Abbreviations SBA Soybean agglutinin - WBA I Winged bean agglutinin (Basic) - JFA Jack fruit agglutinin - PNA Peanut agglutinin - Con A Concanavalin A - Dansyl (Dns) 5-dimethylaminonaphthalene-I-sulphonyl - 2GaINDns N-dansylgalactosamine - dGal 2-deoxygalactose - l-Ara l-arabinose - d-Fuc d-fucose - l-Rha l-rhamnose - N-acetyllactosamine Galß4GlcNAc - melibiose Gal6Glc     

Productivity analysis of outdoor chemostat culture in tubular air-lift photobioreactors

Net productivity and biomass night losses in outdoor chemostat cultures ofPhaeodactylum tricornutum were analyzed in two tubular airlift photobioreactors at different dilution rates, photobioreactor surface/volume ratios and incident solar irradiance. In addition, an approximate model for the estimation of light profile and average irradiance inside outdoor tubular photobioreactors was proposed. In both photobioreactors, biomass productivity increased with dilution rate and daily incident solar radiation except at the highest incident solar irradiances and dilution rates, when photoinhibition effect was observed in the middle of the day. Variation of estimated average irradiance vs mean incident irradiance showed two effects: first, the outdoor cultures are adapted to average irradiance, and second, simultaneous photolimitation and photoinhibition took place at all assayed culture conditions, the extent of this phenomena being a function of the (incident)¹ irradiance and light regime inside the culture. Productivity ranged between 0.50 and 2.04 g L^–1 d^–1 in the tubular photobioreactor with the lower surface/volume ratio (S/V = 77.5 m^–1) and between 1.08 and 2.76 g L^–1 d^–1 in the other (S/V = 122.0 m^–1). The optimum dilution rate was 0.040 h^–1 in both reactors. Night-time biomass losses were a function of the average irradiance inside the culture, being lower in TPB0.03 than TPB0.06, due to a better light regime in the first. In both photobioreactors, biomass night losses strongly decreased when the photoinhibition effect was pronounced. However, net biomass productivity also decreased due to lower biomass generation during the day. Thus, optimum culture conditions were obtained when photolimitation and photoinhibition were balanced.     

Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis

Taxus chinensis suspension cells were cultured in the modified Gamborg's B5 medium. Addition of 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ resulted in the greatest paclitaxel production, at 25 mg l^–1 in the cultures, being almost 40 times higher than that of the control culture, 10 times higher than that of the culture exposed to Ag⁺, 6 times higher than that of the culture elicited by chitosan and almost double that of the culture elicited by methyl jasmonate.     

Mixotrophic growth ofChlorella sorokiniana in outdoor enclosed photobioreactor

Chlorella sorokiniana was cultured in heterotrophic or mixotrophic mode in outdoor enclosed tubular photobioreactor. The culture temperature was maintained at 32–35 °C. At night, theChlorella culture grew heterotrophically, and 0.1 M glucose was completely consumed. The biomass growth yield of glucose was 0.35 ± 0.001 g-biomass g-glucose^–1. During the day, the algal culture grew mixotrophically and the biomass growth yield was 0.49 g-biomass g-glucose^–1 in low density culture (initial biomass concentration, X_o = 2 g l^–1), 0.56 g-biomass g-glucose^–1 in medium density culture (X_o = 4 g l^–1) and 0.46 g-biomass g-glucose^–1 in high density culture (X_o = 7 g l^–1). The daily area productivity of the culture, with X_o = 4 g l^–1 corresponded to 127 g-biomass m^–2 d^–1 during the day and 79 g-biomass m^–2 d^–1 during the night. In all the cultures, the dissolved O₂ concentration increased in the morning, reached the maximum value at noon, and then decreased in the afternoon. The dissolved CO₂ concentration remained at 3 mBar in the morning and increased in the afternoon. Glycolate was not found to accumulate in culture medium.