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     

Ammonium uptake by Chondrus crispus Stackhouse (Gigartinales,Rhodophyta) in culture

Cultivated Chondrus crispus was used in N-NH₄ uptake experiments in the laboratory. An elevation of temperature increased the apparent rate of uptake, especially up to 11 °C. Uptake in the dark was found to be 83 % of that in the light. The apparent uptake decreased with increasing internal N pool; rates were 26.5, 22.2 and 20.2 µg N g dry wt^–1 min^–1 for internal N pools of 2.7, 3.5 and 4.6%, respectively. Apparent uptake increased with the substrate N concentration. The resulting curve has two components: an active uptake and a diffusion component at high (> 5000 µg N L^–1) external N levels. Ks and V _max were calculated by deducting the diffusion component from the uptake curve: these were of 497 µg N L ^–1 and 14.4 µg N g dry wt^–1 min^–1. respectively, and reflect a low substrate affinity. This could be the result of 10 years of continuous culture of C. crispus. Uptake was similarly followed in the culture tanks and showed comparable results; nighttime would be the most appropriate time to supply nutrients.     

The mass culture of Dunaliella viridis (Volvocales,Chlorophyta) for oxygenated carotenoids : laboratory and pilot plant studies

The biology, and hence the mass culture, of Dunaliella viridis closely follows that of Dunaliella salina, which is successfully mass cultured for the production of -carotene. Both algae can grow at extremely high salinities and light intensities. They co-exist in the coastal salt lake, Hutt Lagoon, Western Australia. In contrast to D. salina, D. viridis does not accumulate large amounts of -carotene, producing only up to 0.7% of mixed carotenoids (lutein, zeaxathin, other oxygenated carotenoids and -carotene), compared to D. salina's ca 10% dry wt of mainly -carotene. However, in laboratory experiments, D. viridisgrew much faster and to much higher cell densities than D. salina, and attained levels of mixed carotenoids similar to those of D. salina (ca 13 mg L^–1 carotenoid). Preliminary experiments in outdoor ponds were much less promising. Harvesting by chemical flocculation was as effective as with D. salina, but extraction of carotenoids directly into vegetable oil proved inefficient. When incorporated into feed, caretonoids derived from D. viridis pigmented hen eggs acceptably. Extrapolating from laboratory results, and using costing derived from D. salina technology, the cost of production of mixed oxygenated carotenoids from D. viridis was similar to that for the production of -carotene from D. salina, at ca $A500 kg^–1.     

Atypical development of Chaetomorpha antennina in culture (Cladophorales,Chlorophyta)

The green seaweed genus Chaetomorpha is characterized by unbranched filaments. Molecular phylogenetic data indicate that Chaetomorpha forms a clade that is nested in a paraphyletic assemblage of branched species (Cladophora). It follows that the unbranched condition is evolutionarily conserved and likely evolved early in the evolution of this clade. In this study we show that under laboratory culture conditions, the filaments of C. antennina frequently produce lateral branches, similar to Cladophora. Our results thus indicate that the unbranched thallus architecture is not entirely genetically constrained, but at least in part subject to morphological plasticity. Additionally, culture observations of C. antennina allowed a detailed study of rhizoidal development, which seems unique among Cladophorales.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

Ammonium (methylammonium) uptake by Alcaligenes eutrophus H16

The uptake of the radioactive ammoniumanalogue ¹⁴C-methylammonium¹ was measured in heterotrophically grown cells of Alcaligenes eutrophus H16 in order to study the mechanism of NH ₄ ⁺ uptake. MA gradients of up to 200 were built up by a substrate-specific and energy-dependent system which showed a K _m of 35–111 M and a V _max of 0.4–1.8 nmol MA/min per mg protein. The involved carrier exhibited a higher affinity towards NH ₄ ⁺ than towards CH₃NH ₃ ⁺ indicating that ammonium rather than MA was its natural substrate. Cold shock with hypotonic but not with hypertonic solutions caused the efflux of almost the entire accumulated MA. Osmotic shock did not affect the uptake reaction, suggesting that no periplasmic binding proteins were involved. Indirect observations indicate the membrane potential as driving force for MA uptake. High rates of uptake were observed in cells grown under nitrogen deficiency or with nitrate as nitrogen source. The uptake rate decreased during growth at high ammonium concentrations indicating that biosynthesis of nitrogenous compounds was supported by passive diffusion of NH₃. The data suggest that the formation of the carrier is subject to nitrogen control.Non-standard abbreviations CCCP Carbonylcyanide-m-chlorphe-nylhydazone - MA methylammonium - pCMB para-chlormercuribenzoate     

A survey of Polytomella (Chlorophyceae,Chlorophyta) strains in public culture collections

Polytomella is a genus of colorless green algae in the Reinhardtinia clade of the Chlamydomonadales, which has proven useful for a broad range of studies particularly those exploring the evolutionary loss of photosynthesis and mitochondrial genomics/biochemistry. Although 13 Polytomella strain accessions are currently available from public culture collections, the taxonomic status and redundancy of many of these strains is not clear because of possible mix‐ups, deficient historical records, and incomplete molecular data. This study therefore considers previously available and/or new cox1 and mitochondrial DNA telomere sequences from all 13 Polytomella strain accessions. Among four of these, namely P. parva SAG 63‐3, P. piriformis SAG 63‐10, P. capuana SAG 63‐5, and P. magna SAG 63‐9, cox1 and mitochondrial telomere regions are both highly divergent between strains. All of the remaining nine Polytomella strain accessions have cox1 sequences that are identical to that of P. parva SAG 63‐3 and although five of these have a mitochondrial telomere haplotype that is identical to that of P. parva SAG 63‐3, the remaining four have one of three different haplotypes. Among the 10 strains with identical cox1 sequences, we suggest that three of the telomere haplotypes are associated with distinct geographical isolates of Polytomella and the fourth evolved from one of these isolates during 50 years of active culture.     

Baculoviral mid-gut gland necrosis (BMN) of kuruma shrimp(Penaeus japonicus) larvae in Japanese intensive culture systems

In many shrimp farms in the Kyushu and Chugoku areas of Japan, the so-called mid-gut gland cloudy disease of kuruma shrimp larvae(Penaeus japonicus) has occurred since 1971. The pathological changes associated with this baculoviral mid-gut gland necrosis (BMN) are extensive cellular necrosis, collapse of mid-gut gland cells, nuclear hypertrophy and finally karyorrhexis. Electron microscopic examination revealed the presence of virions and virogenic stages in the affected nuclei. Average length and diameter of the virions detected was 310 and 72 nm, respectively; nucleocapsids were 250 nm in size. Virions enclosing 2 nucleocapsids within a single envelope were rarely found. The spirally arranged capsomeres were at an angle of 37 to 38° to a horizontal line meeting at right angles with the long axis of the virion. Infectivity trials resulted in high mortality of healthy mysis and juveniles (2nd post-larval stage). Juveniles at the 9th post-larval stage showed no mortality, although they could be infected easily by the agent. Hypertrophied nuclei in squashed and stained preparations of the affected gland cells can be considered to be of reliable presumptive diagnostic character, and fluorescent antibody staining can be employed to confirm the diagnosis of BMN.     

Biodiversity and fish farming intensification in French fishpond systems

Fishponds are artificial ecosystems in which biodiversity may be strongly affected by fish farming management. We studied biodiversity variation along a gradient of fish farming intensification within a 180-pond sample of a French region to establish whether biodiversity primarily depended on extensive management with the alternative hypothesis that some habitat characteristics could compensate for the effect of intensification. We compared the relationships of three biodiversity indicators (breeding birds, protected plants, macrophytes) to an index of intensification and to habitat characteristics likely to influence each indicator. In all selected indicators, species richness did not vary according to the composite intensification gradient. Protected plant richness was best explained by periodic drainage, which could thus benefit biodiversity in intensified fishponds provided that shallow littoral areas are still present. Bird richness was linked to macrophyte cover and probably to reed bed areas. Macrophyte richness and coverage were negatively influenced by low water transparency and high fish biomass which seemed likely to affect bird habitat above 350–400 kg/ha. Aquatic vegetation, which may reflect interactive effects of environmental factors and fish farming management, may then contribute to assessment of the ecological status of fishponds under the E.U. Water Framework Directive.     

Ammonium uptake in alpine streams in the High Tatra Mountains (Slovakia)

Uptake of NH _inf4 ^sup+ -N by streambed biota of mountain brooks was studied in the alpine zone of the High Tatra Mountains. Experiments were performed involving in situ dosing of ammonium directly to the acidified stream and incubations of ammonium and streambed bryophytes in enclosures within a range of pH from 4.45 to 8.10.NH _inf4 ^sup+ -N uptake length decreased with decreasing stream discharge, while comparable values of discharge-normalized uptake lengths were found during two in situ experiments.Maximum uptake rates of NH _inf4 ^sup+ -N obtained during the incubation of bryophytes (6 to 11 mg m^–2 h^–1) were comparable with results of two in situ experiments (8 and 12 mg m^–2 h^–1). The average NH _inf4 ^sup+ -N uptake rates observed during incubations lasting 3 to 5 hours (4.3 mg m^–2 h^–1) were not related to the pH of stream water. Nitrification of about 50% of the NH _inf4 ^sup+ -N added was observed in non-acidified streams, but was negligible in acidified streams. Significant photoinhibition of nitrification was observed in non-acidified streams during enclosure experiments.     

Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.     

Seasonal variation in productivity,chemical composition and nutrient uptake of Ulva spp. (Chlorophyta) strains

Journal of Applied Phycology - Applications of seaweeds require the supply of uniform biomass, yet performance of Ulva is generally characterised by a wide variation across study sites and seasons,...     

Release and uptake of minerals during decomposition of plant litter in fishpond littoral

Changes in chemical composition of autochthonous plant litter undergoing decomposition in litter mesh-bags exposed in a transect across four vegetational zones of a fishpond littoral were estimated during the spring, summer and winter season. The highest decomposition rate was found in all four zones in spring, the lowest one in winter. The decomposition was faster in the two aquatic than in the two terrestrial zones in the spring and the summer season. The litter was enriched with nitrogen and released potassium in all locelities and seasons. The uptake or release of other mineral constituents appeared to be specific for each zone and to depend on the season of the year.     

Microalgal mass culture systems and methods: Their limitation and potential

Cultivation of microalgae using natural and man-made open-ponds istechnologically simple, but not necessary cheap due to the high downstream processing cost. Products of microalgae cultured in open-pondscould only be marketed as value-added health food supplements, specialityfeed and reagents for research. The need to achieve higher productivityand to maintain monoculture of algae led to the development of enclosedtubular and flat plate photobioreactors. Despite higher biomassconcentration and better control of culture parameters, data accumulatedin the past 25 years have shown that the illuminated areal, volumetricproductivity and cost of production in these enclosed photobioreactors arenot better than those achievable in open-pond cultures. The technicaldifficulty in sterilizing these photobioreactors has hindered their applicationfor the production of high value pharmaceutical products. The alternativeof cultivating microalgae in heterotrophic mode in sterilizable fermentorshas achieved some commercial success. The maximum specific growth ratesof heterotrophic algal cultures are in general slower than those measured inphotosynthetic cultures. The biomass productivity of heterotrophic algalcultures has yet to achieve a level that is comparable to industrialproduction of yeast and other heterotrophic microrganisms. Mixotrophiccultivation of microalage takes advantage of their ability to utilise organicenergy and carbon substrates and perform photosynthesis concurrently. Moreover, production of some algal metabolites is light regulated. Futuredesign of sterilizable bioreactors for mixotrophic cultivation of microalgaemay have to consider the organic substrate the main source of energy andlight the supplemental source of energy, a change in mindset.     

Molecular phylogeny of the Siphonocladales (Chlorophyta: Cladophorophyceae)

The Siphonocladales are tropical to warm-temperate, marine green macro-algae characterized by a wide variety of thallus morphologies, ranging from branched filaments to pseudo-parenchymatous plants. Phylogenetic analyses of partial large subunit (LSU) rDNA sequences sampled from 166 isolates revealed nine well-supported siphonocladalean clades. Analyses of a concatenated dataset of small subunit (SSU) and partial LSU rDNA sequences greatly clarified the phylogeny of the Siphonocladales. However, the position of the root of the Siphonocladales could not be determined unambiguously, as outgroup rooting and molecular clock rooting resulted in a different root placement. Different phylogenetic methods (likelihood, parsimony and distance) yielded similar tree topologies with comparable internal node resolution. Likewise, analyses under more realistic models of sequence evolution, taking into account differences in evolution between stem and loop regions of rRNA, did not differ markedly from analyses using standard four-state models. The molecular phylogeny revealed that all siphonocladalean architectures may be derived from a single Cladophora-like ancestor. Parallel and convergent evolution of various morphological characters (including those traditionally employed to circumscribe the families and genera) have occurred in the Siphonocladales. Consequently, incongruence with traditional classifications, including non-monophyly in all families and most genera, was shown.     

Colorimetric protein determination in microalgae (Chlorophyta): association of milling and SDS treatment for total protein extraction

The use of colorimetric methods for protein quantification in microalgae is hindered by their elevated amounts of membrane‐embedded intracellular proteins. In this work, the protein content of three species of microalgae was determined by the Lowry method after the cells were dried, ball‐milled, and treated with the detergent sodium dodecyl sulfate (SDS). Results demonstrated that the association of milling and SDS treatment resulted in a 3‐ to 7‐fold increase in protein quantification. Milling promoted microalgal disaggregation and cell wall disruption enabling access of the SDS detergent to the microalgal intracellular membrane proteins and their efficient solubilization and quantification.     

Nutrient removal and biomass production in land treatment systems receiving domestic effluent

Land application of pre-treated wastewater is increasingly practiced to achieve both treatment and beneficial reuse of applied effluent. Vegetation is an important component of these systems, affecting hydraulic loading and nutrient uptake and hence treatment efficiency. This work investigated the effect of plant species (Acacia cyanophylla, Eucalyptus camaldulensis, Populus nigra and Arundo donax), on water requirements, nutrient removal, water use efficiency (WUE) and biomass production in land treatment systems (LTS) in which pre-treated wastewater was applied at rates to meet crop evapotranspiration. Vegetation had a strong effect on all the parameters monitored during this trial. A. cyanophylla produced the greatest amount of biomass and showed the highest irrigation requirements and WUE, followed by E. camaldulensis, A. donax and P. nigra. In addition, A. cyanophylla and A. donax achieved a higher leaf-N content compared to other species. As a result of the differences in tissue nutrient content and biomass, A. cyanophylla accumulated 23, 20, and 70% more N in hypergeous biomass than E. camaldulensis, A. donax and P. nigra, respectively. A. cyanophylla and E. camaldulensis accumulated 57 and 53% respectively more P than did P. nigra and A. donax. Therefore substantial improvement of the performance of the LTS in terms of nutrient removal can be achieved through the selection of appropriate plant species. Despite the enhanced growth rates observed in the study nutrient recovery by vegetation did not exceed 31 and 35% of the applied N and P, respectively. The relatively low percentages of removal are attributed to increased concentration of nutrients in effluent and the high ET rates prevailing in the study area. These findings suggest that additional practices are required to mitigate environmental impacts arising from excessive nutrient loads when effluent is applied at rates to meet crop water requirements.     

Sugar uptake in soybean (Glycine max) cells in suspension culture

In chlorophylkras soybean ( Glycine max L.) cell suspensioo cultures glucose uptake has been studied using the analogue 3-O-methyIglucose. Uptake could be distinguished into: a) a high affinity phase with K_m= 0.06 m M and b) a low affinity phase with K_m 2.0 m M . The uptake of glucose was accompanied by H⁺-cotransport with a stoichiometry of 0.3 H⁺ per molecule 3-O-methylglucose. Experiments in which sugar uptake was measured in the presence of various inhibitors of respiration and photosynthesis demonstrated that the glucose uptake system was dependent on energy metabolism and the ATP-content of the cells. Efflux experiments in the presence of the uncoupler dinitrophenol confirmed this energy dependency. Glucose uptake did not decrease before the ATP-content of the cells had decreased considerably.     

Sustained high yields of Gracilaria (Rhodophyta) grown in intensive large-scale culture

Gracilaria ferox J. Agardh was grown continuously in large, outdoor tanks under a pulse-fed nutrient regime for four years. Productivity ranged from 21.4 to 59.2 g d. wt m⁻² d⁻¹ with a mean of 39.7 g d. wt m⁻² d⁻¹ over the entire study period. Because the cultures were maintained under non-nutrient limiting conditions, productivity was regulated primarily by seasonal changes in light and temperature, which accounted for 75% of the variability of growth in algal yields. Salinity ranged from 31.0 to 36.5‰ and had insignificant effects on growth within this range. The original vegetative strain was maintained over the entire study without the need for additional supplementation from field-collected stock. Because of the pulse-fed nutrient supply, epiphytic growth on the target species was negligible (< 3% total biomass) throughout the study. The yields attained in this study rank among the highest reported for any intensively managed photosynthetic crop and demonstrate the feasibility of growing red macroalgae like Gracilaria at a sustained high yield in a large-scale, land-based culture system. This revised version was published online in July 2006 with corrections to the Cover Date.