Culture ofGelidium sesquipedale (Clem.) Born.et Thur. in a chemostat system. Biomass production and metabolic responses affected by N flow

Gelidium sesquipedale is the most important raw material used for extraction of agar in Spain. Based on chemostats, a system of culture for macroalgae with a continuous flow of culture medium has been developed. A stressed morphotype from the South of Spain was cultured, and the effects of different rates of NO ₃ ^– flow on growth and internal constituents were investigated in the laboratory. Cultivation was successful after optimizing factors affecting growth, such as irradiance level, renewal rate and water movement. Mass production was dependent on N supply. With a flow of 35 mol NO₃ ^– g^–1 DW d^–1, optimal values of growth (2.1% d^–1) and biomass yield were obtained. In these conditions, biomass yield resembled the values observed in natural populations (about 500 g DW m^–2 y^–1). When the flow of N was reduced to 15 mol NO ₃ ^– g^–1 DW d^–1, growth rate and biomass yield were reduced three-fold, and were null when N was supplied as 7 mol NO ₃ ^– g^–1 DW d^–1. C:N ratio was an index of the physiological status of the tissue, remaining low when N was sufficient and raised to critical values when N supply was limited. Phycobiliproteins, kept at a constant irradiance level, were affected by N supply, acting as an internal nitrogen reserve, unlike chlorophylla. An effective phycobiliprotein synthesis took place when the flow of N was sufficient. Agar yield, on dry weight basis, was similar as a function of N flow, whereas agar yield of the culture was higher when N was sufficient as a result of growth not being limited by N.This system of culture, commonly used in microalgal studies, may have an important use in macroalgae as a system to obtain biomass of high quality as well as a good tool for physiological studies in conditions of continuous and controlled flow of nutrients.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

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     

Dependence of temperature on loss rates of rotifers,lipids, and ω3 fatty acids in starved Brachionus plicatilis cultures

Rotifer cultures of Brachionus plicatilis (SINTEF-strain, length 250 m) rich in 3 fatty acids were starved for > 5 days at variable temperature (0–18 °C). The net specific loss rate of rotifer numbers were 0.04 day^–1 (range 0–0.08 day^–1) at 5–18 °C, but reached values up to 0.25 day^–1 at 0–3 °C. The loss rate was independent on culture density (range 40–1000 ind ml^–1), but was to some extent dependent on the initial physiological state of the rotifers (i.e., egg ratio).The loss rate of lipids was 0.02–0.05 day^–1 below 10 °C, where the potential growth rate of the rotifer is low (0–0.09 day^–1). The loss rate of lipids increased rapidly for higher temperatures where the rotifer can maintain positive growth, and reached 0.19 day^–1 at 18 °C. The Q₁₀ for the lipid loss rate versus temperature was higher than the Q₁₀ for respiration found in other strains. This may suggest that other processes than respiration were involved in lipid catabolism. The content of 3 fatty acids became reduced somewhat faster than the lipids (i.e. in particular 22:6 3), but the fatty acid per cent distribution remained remarkably unaffected by the temperature during starvation.The results showed that rotifer cultures could be starved for up to 4 days at 5–8 °C without essential quantitative losses of lipids, 3 fatty acids, and rotifers. The rotifers exhausted their endogenous lipids through reproduction (anabolism) and respiration (including enhanced locomotion) at higher temperatures. At lower temperatures, the mortality rate became very high.     

Re-appearance of rotifers in hypersaline Mono Lake, California, during a period of rising lake levels and decreasing salinity

The surface elevation of Mono Lake, California, rose 2 m and mixed-layer salinities declined about 5 g kg^–1 during the 3 years (1995–1997) following the decision to restrict water diversions out of the Mono Basin. Abundant (18000 m^–2) Hexarthra jenkinae de Beauchamp were noted in pelagic samples in October 1997 after three decades of absence or very low abundance. Abundance subsequently increased to 100000 m^–2 in December 1997 before declining to low numbers through 1998 and 1999. The re-appearance of Branchionus plicatilis Müller in pelagic samples occurred in September 1998. B. plicatilis areal abundance increased to 15000 m^–2 in October–December of both 1998 and 1999 but was low throughout the rest of the year. Both rotifers were noted in nearshore ponds, but were only abundant in those with salinities below 53 g kg^–1. During 1998–1999 when the salinities of the upper water column were 73–75 g kg^–1, less saline shoreline habitats may have been seeding the offshore rotifer populations.     

Production and nutritional quality of the rotifer Brachionus plicatilis fed marine Chlorella sp. at different cell densities

The effect of different cell densities of marine Chlorella sp. on the growth rate, doubling time and production of the rotifer Brachionus plicatilis was investigated. A significant increase in rotifer production was achieved at a density of 50 × 10⁶ Chlorella cells ml^–1. The nutritional quality of rotifers grown at different concentrations of Chlorella is discussed.     

Characterization of the bacterial flora of mass cultivated Brachionus plicatilis

Bacterial density and composition in association of mass cultivated rotifers (Brachionus plicatilis, SINTEF-strain) was investigated, during experimental conditions identical to the procedures used for preparing rotifers as live food for marine cold water fish larvae. These procedures include cultivation, enrichment with squid meal and acclimation to low temperature by storage of the rotifer culture at 6 °C. Large variations were observed in the number of rotifer associated (1.8–7.6 · 10³ colony forming units per rotifer^–1) and free-living (0.6–25 10⁷ cells·ml^–1) bacteria. An increase of 50–150% in the bacterial number was normally observed after feeding the rotifer with squid meal, but after three days of acclimation at 6 °C, the bacterial numbers decreased to the initial level.After enrichment of the cultures with squid meal, the similarity in the composition of the bacterial flora between the rotifers and water was reduced. However, acclimation of the culture at 6 °C resulted in better agreement of the rotifer associated flora and that in water. Enrichment of the cultures induced a shift in the bacterial composition from Cytophaga/Flavobacterium dominance to Pseudomonas/Alcaligenes dominance. The bacterial flora of the rotifer cultures are dominated by presumably opportunistic species after enrichment, which may have detrimental effects when rotifers are fed as live food to marine fish larvae.     

Diurnal vertical distribution of rotifers (Rotifera) in the Chara zone of Budzyńskie Lake,Poland

Diurnal vertical distribution of rotifers was investigated in the Chara bed and the water immediately above it in the shallow region (ca. 1 m depth) of Budzyskie Lake (Wielkopolski National Park, Poland) in early September 1998. Eighty one rotifer species were identified – 71 among Chara and 59 in the open water. Significant differences in rotifer densities were observed in the Chara, with highest numbers during the day (2316 ind. l^–1) and lowest numbers early morning (521 ind. l^–1) and at dusk (610 ind. l^–1). Above the Chara, the numbers of rotifers did not change significantly (615–956 ind. l^–1). Littoral- or limnetic-forms differed in their diel vertical distribution between both zones. One group of littoral species was characterized by increased densities in the Chara in the daytime, while a second group increased in density during the night. The densities of limnetic species, which were much higher in open water, decreased in the morning or daytime in this zone. These differences in the diel behaviour of particular groups of rotifers may be dependent on microhabitat and may also be related to different kinds of predation, the exploitative competition for shared food resources between rotifers and crustaceans, as well as typical adaptation to littoral or limnetic life.     

Use of swimming speed and egg ratio as predictors of the status of rotifer cultures in aquaculture

This study evaluated the use of egg ratio (eggs rotifer^–1) and swimming speed (mm min^–1) as prediction criteria for production and culture quality in mass cultures of the rotifer Brachionus plicatilis. Egg ratio was determined to be a suitable predictor of rotifer growth and production in the cultures. Low egg ratios (i.e., 0–0.17 eggs rotifer^–1) indicate reduced rotifer population over time (i.e., negative net population growth rates). However, at this time egg ratio dynamics are not suitably understood to predict in advance a sudden population collapse.Swimming speed of reproductive, egg-carrying females in the exponential growth phase was 40–45 mm min^–1. During exponential growth swimming speed was independent of the food used. Lower swimming speeds were obtained in late stationary phase (10–25 mm min^–1) when yeast was used as a food source. Both environmental factors (e.g., accumulating metabolites) and changes in nutritional state of the rotifers may have affected the swimming speed, but environmental factors appear to be the most important. We believe that swimming speed has the potential of becoming an accurate predictor of culture quality in mass cultures of rotifers.     

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.     

Population growth rate of the parasitic rotifer Proales gigantea,and susceptibility to parasitization in the snail Lymnaea acuminata at different stages of embryonic development

Developing eggs of the host snail Lymnaea acuminata were experimentally parasitized with the parasitic rotifer Proales gigantea to study the population growth rate of the parasite within the snail egg capsule and the susceptibility of the host eggs at different stages of embryonic development. The population growth rate of P. gigantea was 0.46 ± 0.07 individual^–1 day^–1 at the ambient temperature of 18–22 °C. Snail eggs were most susceptible to rotifer attack during the initial stages of development, becoming progressively more resistant after the hippo stage. Yet, regardless of the stage of development, the host embryo was doomed to die without hatching even if one individual rotifer gained entry inside the egg capsule. The presence of P. gigantea within the parasitized egg capsules or in the mucilage had no effect on the developmental rates and hatching success of non-parasitized eggs within the same egg mass.     

Biofiltering efficiency in removal of dissolved nutrients by three species of estuarine macroalgae cultivated with sea bass (Dicentrarchus labrax) waste waters 1. Phosphate

The potential of three estuarine macroalgae (Ulvarotundata, Enteromorpa intestinalis andGracilaria gracilis) as biofilters for phosphate ineffluents of a sea bass (Dicentrarchus labrax) cultivationtank was studied. These seaweeds thrive in Cádiz Bay and were alsoselected because of their economic potential, so that environmental andeconomicadvantages may be achieved by future integrated aquaculture practices in thelocal fish farms. The study was designed to investigate the functioning of Pnutrition of the selected species. Maximum velocity of phosphate uptake (2.86mol PO₄ g^–1 dry wth^–1) was found in U. rotundata.This species also showed the highest affinity for this nutrient. At low flowrates (< 2 volumes d^–1), the three species efficientlyfiltered the phosphate dissolved in the waste water, with a minimum efficiencyof 60.7% in U. rotundata. Net phosphate uptake rate wassignificantly affected by the water flow, being greatest at the highest rateassayed (2 volumes d^–1). The marked decrease in tissue P shownby the three species during a flow-through experiment suggested that growth wasP limited. However, due to the increase in biomass, total P biomass increasedinthe cultures. A significant correlation was found between growth rates and thenet P biomass gained in the cultures. A three-stage design under low water flow(0.5 volumes d^–1) showed that the highest growth rates (up to0.14 d^–1) and integrated phosphate uptake rates(up to 5.8 mol PO₄ ^3– g^–1dry wt d^–1) were found in E.intestinalis in the first stage, with decreasing rates in thefollowing ones. As a result, phosphate become limiting and low increments oreven losses of total P biomass in these stages were found suggesting thatphosphate was excreted from the algae. The results show the potential abilityofthe three species to reduce substantially, at low water flow, the phosphateconcentration in waste waters from a D. labrax cultivationtank, and thus the quality of effluents from intensive aquaculture practices.     

Modelling continuous culture of Rhodospirillum rubrum in photobioreactor under light limited conditions

Rhodospirillum rubrum was grown continuously and photoheterotrophically under light limitation using a cylindrical photobioreactor in which the steady state biomass concentration was varied between 0.4 to 4 kg m^–3 at a constant radiant incident flux of 100 W m^–2. Kinetic and stoichiometric models for the growth are proposed. The biomass productivities, acetate consumption rate and the CO₂ production rate can be quantitatively predicted to a high level of accuracy by the proposed model calculations. Nomenclature: C _X, biomass concentration (kg m^–3) D, dilution rate (h^–1) Ea, mean mass absorption coefficient (m² kg^–1) I , total available radiant light energy (W m^–2) K, half saturation constant for light (W m^–2) R _W, boundary radius defining the working illuminated volume (m) r _X, local biomass volumetric rate (kg m^–3 h^–1) <r _X>, mean volumetric growth rate (kg m^–3 h^–1) V _W, illuminated working volume in the PBR (m^–3). Greek letters: , working illuminated fraction (–) _M, maximum quantum yield (–) bar, mean energetic yield (kg J^–1).     

Utilization of cyanobacteria by Brachionus calyciflorus: Anabaena flos-aquae (NRC-44-1) as a sole or complementary food source

The rotifer Brachionus calyciflorus can utilize the cyanobacterium Anabaena flos-aquae as either a sole or supplementary food source in laboratory culture. Positive population growth rates accompany food densities of 10 or 100 µg dry weight ml^–1, but slightly negative rates are found at a lower density (1.0 µg ml^–1). These results are consistent for rotifers feeding on two strains of A. flos-aquae, UTEX-1444 and NRC-44-1, with slightly enhanced survivorship and reproduction with the latter food. A 1:1 mixture (by dry weight) of Euglena gracilis and A. flos-aquae (NRC-44-1) produces survivorship comparable to that of control rotifer cohorts fed E. gracilis alone, but elicits significantly greater fecundity and population growth rates than found with the control food suspension at the same biomass density.     

Cultivation ofMonostroma nitidum (Chlorophyta) in a river estuary,southern Japan

The green seaweedMonostroma nitidum Wittrock is cultivated in brackish waters of southern Japan and an ecological survey of its cultivation was carried out in the estuary of R. Shimanto over three years. Artificial seed culture began by collecting many gametes in April. The zygotes adhered to a plastic settlement board (20–30 cm long and 10 cm wide). The cultivated zygotes in the indoor tank increased gradually in size from 8 to 40m till early September. Maturation of the zygote was promoted by providing dark conditions for 2–3 weeks. The production of a concentrated zoospore solution was achieved by adding freshwater 2–3°C above that of the zygote culture tank and a photon flux density of 100mol m^–2 s^–1).The culture nets were set our horizontally at a level exposed for 4 h and were harvested 3–4 times till March. The total production was approximately 6–10 kg dry weight per net during culture periods. The total production of nets harvested 3–4 times per year was greater than that of nets harvested only once.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Fermentation ofCandida utilis for uricase production

Summary Conditions for the production of microbial uricase byCandida utilis were studied. For the selected strain, hypoxanthine proved to be the most effective inducer of uricase formation. The highest values of biomass as well as uricase activity in the mechanically agitated fermentor were obtained under the following conditions: 50 h, rotation impeller speed 7 s^–1, air flow rate 1.25×10^–5 m³s^–1, concentration of inducer 0.1%.List of symbols b width of baffle, m - c length of baffle, m - D diameter of cylindrical fermentor, m - d diameter of impeller, m - d ₁ diameter of impeller disc, m - Fr _m impeller Froud number - g gravitional acceleration, ms^–2 - H height of batch surface above bottom, m - H ₂ height of impeller disc above bottom, m - h height of impeller blade, m - Kp _g flow rate number - L length of impeller blade, m - N rotational speed of impeller, s^–1 - Re _m impeller Reynolds number - T time, h - V volume of batch, m³ - V _g air (gas) flow rate, m³s^–1 - x mass fraction of the dry matter of cells - x ₀ initial value of the mass fraction of the dry matter of cells - _r volume fraction of the dry matter of cells - <eta<₁ viscosity of pure liquid, Pa s - viscosity of batch (suspension of microbial suspension), Pa s - density of batch, kg m^–3     

The potential for population growth of Ascomorpha ecaudis

The intrinsic rate of natural increase (r_m) of the rotifer Ascomorpha ecaudis was found to be a hyperbolic function of food concentration. The threshold food concentration and r_max/2-food concentration determined for this species were significantly lower than values predicted from allometric models. These growth characteristics may be related to the mucus house in which Ascomorpha lives and/or the symbiotic algae living in its body tissues. The maximum rate of population growth recorded (0.71 d^–1) was similar to that of other soft-bodied rotifers of similar body mass. These population growth characteristics and the resistance of this species to invertebrate predation should allow it to become a dominant member of freshwater zooplankton communities. However, field observations suggest that it is not. Reasons for this are suggested.     

In situ primary production in young Antarctic sea ice

An in situ incubation technique used successfully to measure the photosynthetic carbon assimilation of internal algal assemblages within thick multiyear Arctic ice was developed and improved to measure the photosynthetic carbon assimilation within young sea ice only 50 cm thick (Eastern Weddell Sea, Antarctica). The light transmission was improved by the construction of a cylindrical frame instead of using a transparent acrylic-glass barrel. The new device enabled some of the first precise measurements of in situ photosynthetic carbon assimilation in newly formed Antarctic sea ice, which is an important component in the sea ice ecosystem of the Antarctic Ocean. The rates of carbon assimilation of the interior algal assemblage (top to 5 cm from bottom) was 0.25 mg C m^–2 d^–1 whereas the bottom algal community (lowest 5 cm) attained only 0.02 mg C m^–2 d^–1. Chl a specific production rates (P^Chl) for bottom algae (0.020 – 0.056 g C g chl a ^–1 h^–1) revealed strong light limitation, whereas the interior algae (P^Chl = 0.7 – 1.2 g C g chl a ^–1 h^–1) were probably more limited by low temperatures (< –5 °C) and high brine salinities.