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The hydrolytic activity of different types of polyphosphates, and the induction of soluble pyrophosphatase (sPPase, EC 3.6.1.1) activity have been assessed in cell extracts of nutrient limited green seaweed Ulva pseudorotundata Cormaci, Furnari &amp, Alongi subjected to different phosphorus regimes. Following a long period of nutrient limitation, the addition of different types of (poly)phosphates to artificial seawater enhanced growth rates on fresh weight and area, but not on dry weight bases. Chlorophyll and internal P content were affected by P supply. In contrast, internal soluble reactive P was kept low and was little affected by P additions. Soluble protein content increased in all treatments, as ammonium was added to prevent N limitation. The C:N:P atomic ratio revealed great changes depending on the nutrient regime along the experiment. Cell extracts of U. pseudorotundata were capable of hydrolyzing polyphosphates of different chain lengths (pyro, tripoly, trimeta, and polyphosphates) at high rates. The sPPase activity was kept very low in P limited plants. Following N and different kind of P additions, sPPase activity was kept low in the control, but slightly stimulated after 3 days when expressed on a protein basis. The highest activities were found at the end of the experiment under pyro and polyphosphate additions (7 days). The importance of alternative P sources to phosphate and the potential role of internal soluble pyrophosphatases in macroalgae are discussed.

In Latin America, the energy potential from sugarcane straw, such as tops, secondary stalk, and leaves, has been debated, but the literature associated with organic crops and its implementation for energy purposes is limited. Sugarcane straw is either left in the field for soil recovery or used for animal feed. This paper presents an analysis of using organic sugarcane straw for energy generation. We have characterized the physicochemical properties of this organic sugarcane straw, including caloric value, moisture, carbon, and ash content, and have explored the environmental benefits of using biomass to replace coal as an energy source. The study showed that it is possible to achieve the replacement of coal using 16% of the residual biomass, with the benefit of generating lower CO2 emissions compared to coal combustion. Additionally, emissions derived from transporting the biomass from the field to the farm are 68% lower than those involved in transporting coal. The residual biomass from the organic crop showed similar properties relative to bagasse. The use of organic sugarcane straw biomass will result in the reduction of energy costs and will generate green energy with lower emission of CO2.

espanolEn la presente investigacion se evaluo la sintesis verde de nanoparticulas de oxido de zinc (ZnO) y su aplicacion como agente antibacteriano para sustratos textiles. El proceso de sintesis implico el uso de extractos de Azadirachta indica, Aloe vera, y acetato de zinc como precursor metalico. La sintesis de nanoparticulas se confirmo mediante espectrofotometria UV-Vis, espectroscopia de infrarrojo con transformada de Fourier (FTIR), difraccion de rayos X (DRX) y microscopia electronica de barrido. La eficiencia de las nanoparticulas como agentes antibacterianos en textiles se probo en uno de 100 % algodon funcionalizado por impregnacion. Este fue sometido a cinco y diez ciclos de lavado domestico para analizar la durabilidad de las nanoparticulas. La actividad antibacteriana del textil funcionalizado, con y sin ciclos de lavado, se determino mediante la norma ISO 20743:2007 contra la bacteria Staphylococcus aureus ATCC 6538. Se encontro que las nanoparticulas confieren una actividad antibacteriana fuerte al textil y, aunque hay una perdida importante del nanomaterial con los ciclos de lavado, la concentracion remanente es suficiente para mantener una actividad antibacteriana significativa hasta los diez ciclos de lavado. Se concluye que las nanoparticulas de ZnO son candidatas promisorias para ser empleadas como agentes antibacterianos en la industria textil, pues presentan actividad antibacteriana y son relativamente faciles de obtener por sintesis verde con extractos de A. indica y A. vera, material vegetal de facil acceso en el departamento de Antioquia. EnglishIn the present investigation, we evaluated the green synthesis of ZnO nanoparticles and their application as an antibacterial agent for textile substrates. The synthesis process involved the use of Azadirachta indica and Aloe vera extracts, and zinc acetate dihydrate as a metallic precursor. The synthesis of the nanoparticles was confirmed by UV-Vis spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (DRX), and Scanning Electron Microscopy (SEM). The efficiency of nanoparticles as a textile antibacterial agent was tested in a 100 % cotton textile functionalized by exhaustion. The textile was subjected to five and ten domestic washing cycles to analyze the durability of the nanoparticles. The antibacterial activity of the functionalized textile, with and without washing cycles, was determined by the ISO 20743:2007 standard against Staphylococcus aureus ATCC 6538 bacteria. We found that nanoparticles conferred strong antibacterial activity to the textile, and while there is a significant loss of nanoparticles with wash cycles, the remaining concentration is sufficient to maintain significant antibacterial activity up to ten cycles. We concluded that ZnO nanoparticles are promising candidates to be used as antibacterial agents in the textile industry, presenting antibacterial activity and being relatively easy to obtain by green synthesis using vegetal subtracts available in Antioquia.

The effects of light reduction [100%, 25%, 10% and 1% mean daily-integrated photon irradiance (I0)] by Ulva rigida C. Agardh canopies on carbon balance, sugar-related enzymes and proteolytic activities of the seagrass Zostera noltii Hornem. were investigated. Shaded plants showed negative net growth and starch was mobilized in both above- and below-ground tissues. Sucrose declined in below-ground parts under severe light deprivation (10% and 1% I0), but was accumulated in above-ground parts. Mobilization of the non-structural carbohydrates (sucrose and starch) was explained by changes in activities of sucrose synthase (SuSy, EC 2.4.1.13) and sucrose-phosphate synthase (SPS, EC 2.4.2.24). Under severe light reduction, the capacity of above-ground tissues for sucrose formation and export declined, indicated by the lowest SPS activity. In contrast, severe light reduction increased the 'sink strength' of below-ground tissues, demonstrated by the highest SuSy activities, and diminished the capacity for sucrose resynthesis from starch breakdown, as the lowest SPS activity was observed under low light. These results suggest a cessation of sucrose transport throughout the plant under extreme light limitation, the carbon supply being dependent on the starch breakdown in each tissue. The response of Z. noltii to gradual light reduction was co-ordinated at the whole-plant level, since an enhancement of proteolytic activities induced by carbon starvation in both above- and below-ground tissues was also recorded during prolonged light deprivation. Therefore, carbon mobilization was accompanied by enhanced protein turnover and changes in metabolic pathways.

Stigmasterol is a phytosterol contained in Kraft mill effluent that is able to increase over 100% after aerobic biological treatment. This compound can act as an endocrine disrupter as its structure is similar to that of cholesterol. The aim of this study was to evaluate the removal of stigmasterol from Kraft mill effluents treated by a moving bed biofilm reactor (MBBR) with steroidal metabolite detection. The MBBR was operated for 145 days, with a hydraulic retention time of 2 days. Stigmasterol and steroidal metabolites were detected by gas chromatography with a flame ionization detector during MBBR operation. The results show that the MBBR removed 87.4% of biological oxygen demand (BOD5), 61.5% of chemical oxygen demand (COD), 24.5% of phenol and 31.5% of lignin, expressed in average values. The MBBR system successfully removed 100% of the stigmasterol contained in the influent (33 µg L(-1)) after 5 weeks of operation. In that case, the organic load rate was 0.343 kg COD m(-3) d(-1). Furthermore, different steroidal compounds (e.g., testosterone propionate, stigmast-4-en-3-one, 5α-pregnan-12-one-20α-hydroxy, 5α-pregnane-3,11,20-trione and 3α-hydroxy-5α-androstane-11,17-dione were detected in the Kraft mill effluent as potential products of phytosterol biotransformation.

Climate change intensifies the frequency and intensity of rainfall events, which increases the discharge of freshwater and nutrients to coastal areas. This may lower salinity and increase nutrient availability and, thus, affect estuarine eelgrass populations. We studied the interactive effect of increasing NH4 + levels and low salinity on estuarine eelgrass Zostera marina, grown in microcosm at various combinations of NH4 + enrichment (0, 10 and 25 μM) and salinity (5, 12.5 and 20). Increasing NH4 + had a positive effect on eelgrass performance as long as salinity was kept at ambient level (20). N enrichment was followed by an increase in pigments, photosynthesis and various growth variables and a decrease in stored carbon concentrations (sucrose and starch). Low salinity had an overall negative effect on plant fitness; pigment concentration, photosynthesis and growth were reduced while mortality increased. Exposure to low salinity was also followed by a decrease in sucrose, suggesting that it was used as an osmolyte and/or that photosynthesis could not cover energy requirements needed for osmoregulation or repairing processes. Concomitant exposure to high NH4 + and low salinity turned the positive effect of NH4 + into a strong, negative synergistic effect. Several growth-related variables were affected significantly and mortality increased substantially. We suggest that this simultaneous exposure intensified competition for energy and C skeletons affecting other metabolic processes (e.g. growth, repair processes) negatively. Our results suggest that climate change driven alterations in precipitation and NH4 + loading might seriously impact estuarine eelgrass communities.

Global change, such as warming and ocean acidification, and local anthropogenic disturbances, such as eutrophication, can have profound impacts on marine organisms. However, we are far from being able to predict the outcome of multiple interacting disturbances on seagrass communities. Herbivores are key in determining plant community structure and the transfer of energy up the food web. Global and local disturbances may alter the ecological role of herbivory by modifying leaf palatability (i.e. leaf traits) and consequently, the feeding patterns of herbivores. This study evaluates the main and interactive effects of factors related to global change (i.e. elevated temperature, lower pH levels and associated ocean acidification) and local disturbance (i.e. eutrophication through ammonium enrichment) on a broad spectrum of leaf traits using the temperate seagrass Cymodocea nodosa, including structural, nutritional, biomechanical and chemical traits. The effect of these traits on the consumption rates of the generalist herbivore Paracentrotus lividus (purple sea urchin) is evaluated. The three disturbances of warming, low pH level and eutrophication, alone and in combination, increased the consumption rate of seagrass by modifying all leaf traits. Leaf nutritional quality, measured as nitrogen content, was positively correlated to consumption rate. In contrast, a negative correlation was found between feeding decisions by sea urchins and structural, biomechanical and chemical leaf traits. In addition, a notable accomplishment of this work is the identification of phenolic compounds not previously reported for C. nodosa. Our results suggest that global and local disturbances may trigger a major shift in the herbivory of seagrass communities, with important implications for the resilience of seagrass ecosystems.

Global (e.g. climate change) and local factors (e.g. nutrient enrichment) act together in nature strongly hammering coastal ecosystems, where seagrasses play a critical ecological role. This experiment explores the combined effects of warming, acidification and ammonium enrichment on the seagrass Cymodocea nodosa under a full factorial mesocosm design. Warming increased plant production but at the expense of reducing carbon reserves. Meanwhile, acidification had not effects on plant production but increased slightly carbon reserves, while a slight stimulation of net production and a slight decrease on carbon reserves under ammonium supply were recorded. When all the factors were combined together improved the production and carbon reserves of Cymodocea nodosa, indicating that acidification improved ammonium assimilation and buffered the enhanced respiration promoted by temperature. Therefore, it could indicate that this temperate species may benefit under the simulated future scenarios, but indirect effects (e.g. herbivory, mechanical stress, etc.) may counteract this balance.

The effects of light and ammonium levels on net production, fluorescence parameters and non-structural carbohydrates of the seagrass Zostera noltii under different phosphate conditions were studied. A fully factorial design was used with light (low/high levels), ammonium supply and phosphate preculture conditions of the plants as the experimental variables. Both ammonium supply and low light caused negative and synergistic effects on net production, while ammonium toxicity was more severe at high light levels; in this case, it was independent of the non-structural carbohydrate (sucrose and starch) content. Preculturing of plant with added phosphate alleviated the ammonium toxicity, and also attenuated the negative production balance of plants grown at low light levels. The results indicated that phosphate preculture ameliorated the plant's short-term response against the assayed stressors (low light, high ammonium) significantly. An overall consumption of non-structural carbohydrates in response to environmental stressors was recorded throughout the experiment, indicating the importance of carbon and phosphorus reserves to cope with adverse conditions. ln addition, phosphate deficiency increased the vulnerability of plants, which could have negative ecological consequences for seagrass species thriving under phosphate deficiency conditions, or in developing seagrass transplantation programs. Marie Curie fellowship [MEIF-CT-2005-515071]; European Reintegration Grant [MERG-CT-2007-205675]; Ministerio de Educacion y Ciencia; Spanish Project [CTM2005-00395/MAR]

Changes in biomass of several macroalgae [Ulva rotundata Bliding; Gracilariopsis longissima (S. G. Gmel.) Steentoft, L. M. Irvine et Farnham; Ulva intestinalis L.; and Cladophora sp.] and marine plants (Zostera noltii and Ruppia cirrhosa) growing naturally in earthen ponds of a fish farm (Acuinova, San Fernando, Southern Spain) were recorded during a year. The farm is mainly devoted to the culture of gilthered seabream (Sparus aurata). The most conspicuous algal species thriving in the ponds was U. rotundata, which reached densities up to 600 g dry mass · m(-2) and produced up to 20.45 g C · m(-2) · d(-1) . Dissolved nutrients (phosphate and ammonium), tissue nutrient content, and growth rates of this species were estimated during 2001 and 2002. Evidence of natural biomitigation by U. rotundata when water circulates throughout the fish farm is presented. Due to the fish cultivation, both phosphate and ammonium increased as water circulated from the preculture ponds to the postculture ponds. As a consequence, U. rotundata tissue nitrogen (N) and phosphorus (P) increased from algae growing in preculture ponds to algae growing in the outflow channel, so that mean C:N:P ratio varied from 773:57:1 in preculture ponds to 567:64:1 in the outflow channel. Phosphorus limited growth of U. rotundata during the spring. As growth rates increased as a function of tissue P, data were fitted to the Droop equation. From this equation, the estimated maximal growth rate was 0.295 ± 0.041 d(-1) , the subsistence quota was 0.05 ± 0.01% P of dry mass, and the critical quota was 0.215% P of dry mass. The results suggest that management of the fish farm based on a large-scale integrated mariculture system of fish and macroalgae may increase the total ecological and economic benefits, both for the farm and for the environment.

Conversion of limonene in its derivatives has been an interesting process due to its high value for cosmetic, pharmaceutical and food industries. The use of molecular sieves based on transition metals in heterogeneous catalysis, and specifically in oxidative processes, has shown good results. Oxidation of limonene has been studied using zinc based catalyst, hydrogen peroxide as oxidative agent and t-butanol or tetrahydrofurane as solvents at different reaction times and temperatures. 1,2-Epoxi-limonene, carvone, carveol and limonene-1,2-diol were obtained as main products.

Plants of the seagrass Zostera noltii were cultured in the laboratory (mesocosms) for two weeks to assess the effect of above:below-ground (AG/BG) biomass ratios and light on growth, photosynthesis and chemical composition. Experimental plant units (EPUs) with different proportions between AG and BG biomass were obtained from plants of the same size (containing 6 shoots and 5 internodes) by excising 0–5 shoots. The EPUs maintained the proportions in AG/BG biomass ratios during the experiment. While growth rate was unaffected by biomass partitioning at high light, maximum growth at low light was recorded in plants with low AG/BG ratios. The production of shoots and rhizomes showed a compensatory morphological response depending on the initial AG/BG proportions regardless of the light level. While shoot production, estimated as shoot appearance rate, was high at low AG/BG ratios and minimal under high AG/BG values, rhizome production, estimated as internode appearance rate and internode elongation rate, was maximal under high AG/BG proportions and decreased towards lower AG/BG ratios. This rhizomatic response was observed for secondary rhizomes and not for primary ones. In contrast to morphological response, no significant differences were detected in maximum electron transport rates (ETRm) among the different shoots in the plant. However, mean values of ETRm in plants were affected by biomass partitioning and light. EPUs grown in low light increased the sucrose stored in shoots as the AG/BG biomass ratios decreased; however, EPUs grown at high light showed no effect of biomass partitioning on sucrose levels. In conclusion, shoots excision by experimental manipulation caused a compensatory morphological response in plants while photosynthetic performance remained almost unaffected.

P align=justify>Cymodocea nodosa plants were dark incubated for four months. The potential of reactivating photosynthesis was tested in an experiment in which half of the plants were reilluminated (HL) while the other half were grown under very low irradiance levels (LL). Photosynthesis was measured using PAM fluorescence and tissue nutrient and carbohydrate contents were analysed. Photosynthetic efficiency (Fv/Fm) in HL plants increased from 0 to 0.58, whereas LL plants remained inactive. Photosynthetic parameters also increased, resulting in a final Ik of 97.5 µmol m-2 s-1. Leaf shedding led to a negative mean RGR in HL plants. Tissue C and N dropped considerably during dark incubation in both rhizomes and shoots. Starch content was nearly equal for rhizomes and shoots (4.3 mg /g DW) and was not affected by dark incubation. In contrast, sucrose content dropped from 40.0 mg /g DW to zero in shoots and from 240 to 40.0 mg /g DW in rhizomes in HL plants. We conclude that C. nodosa is capable of recovering photosynthetic activity after four months darkness, which is considerably longer than the 80 d recorded so far for a seagrass. Stored carbohydrates, more specifically sucrose, play an important role in both survival and reactivation.

A kinetic analysis of the photosynthesis inhibition by buffers allowed quantification of some components of the carbon concentrating mechanism (CCM) of the brown macroalga Laminaria saccharina. The CCM was based on the presence of acid regions outside the plasma membrane that increased the CO(2) concentration available for photosynthesis by 10-20 times above that of the bulk medium at alkaline pH. Furthermore, the results suggested that the CCM is located mainly on the cell membrane and not in the chloroplast, as suggested for most macroalgae. The degree of dissipation of the acid regions by a buffer was related to the buffer anion concentration (B(-)), estimated from the titration of the buffer from bulk medium pH to a pH endpoint value close to the first pK (a) of the carbonic acid system. A kinetic model describing the relationship between inhibition of photosynthesis by a buffer and B(-) was developed suggesting that buffers act as competitive inhibitors with IC(50) (the concentration of the buffer anion which reduces the reaction velocity by half) of 5.0 mol m(-3). This model can be used to estimate the inhibitory effect of any buffer on the photosynthesis of L. saccharina. Nevertheless, some buffers tested showed a lower effect than that predicted from the hyperbolic model suggesting that their strength as inhibitors depended on: (1) the pK (a) in relation to the first pK (a) of the carbonic acid system and (2) its molecular weight (i.e. its mobility).

The potential of the red agarophyte Gracilariopsis longissima as biofilter for phosphate and ammonium in effluents outflowing intensive marine fish cultures was assessed at different scales. Previous studies showed that both laboratory (microscale level) and outdoor cultivation (mesoscale level) were feasible, with a maximum sustainable yield of 270 g fresh wt m − 2 day − 1 approximately, at a biomass higher than that predicted in a logistic model, a deviation attributable to an improvement of the culture conditions during the monitoring period. At a mesoscale level, a 34-h cycle suggested that the nitrification rate on the seaweed fronds showed diel fluctuations, with rates peaking early in the morning, when ammonium uptake rates were negligible. Mean nitrification rates were similar to ammonium uptake rates, suggesting that nitrifyers outcompete G. longissima for the use of ammonium; especially when mean biofiltering efficiencies were less than 15% during the 34-h period. G. longissima thrives naturally in different earthen ponds of a fish farm in Cadiz Bay Natural Park, Southern Spain, especially in the outflowing reservoir earthen ponds, where biomass reached values up to 278 g dry wt m − 2 during the spring. A field cultivation system for G. longissima (macroscale level) was designed to find the best scenario in terms of earthen pond, season or current conditions. The best cultivation method was the growth of vegetative cuttings on suspended braided nylon ropes. The highest growth rates (up to 6% day − 1 ) and biomass (up to 10 g fresh wt cm − 1 rope) were obtained in ponds receiving outflow waters, suggesting a nutrient effect. The net P production reached 24.9 μg P cm − 1 rope day −1 and was also higher on braided nylon suspended ropes placed at the outflowing reservoir earthen ponds. A similar result was found regarding net N production. However, in this case, mean production (≈ 170 μg N cm − 1 rope day − 1 ) was similar in the different earthen ponds and channels. The increase in P and N biomass suggested that G. longissima was biofiltering efficiently nutrient wastes from the fish farm. The results pointed out the high potential ability of G. longissima to biofilter waste waters from a fish farm, encouraging a large scale cultivation of this species. Future practices using this macroalgae may be implemented in local fish farms, resulting in both environmental and economic advantages.

An integrated outdoor cultivation of two macroalgal species: Ulva rotundata (Chlorophyta) and Gracilariopsis longissima (Rhodophyta) was designed. The macroalgae were cultured in effluents from an intensive marine culture (growout phase) of gilthead seabream Sparus aurata. The biomass evolution of the algal tanks followed a logistic curve, where the approach to the maximum stocking density of seaweeds was governed by thalli self-shading, as nutrient limitation in the cultivation tank was unlikely. The maximum stocking density of the system was approximately 27.8 g U. rotundata L−1 (16.7 Kg m−2) and 11.9 g G. longissima L−1 (7.12 Kg m−2). Yield was more than 3 times higher in U. rotundata than in G. longissima. Overall, U. rotundata removed a greater percentage of phosphate (8.9%) and total dissolved inorganic nitrogen (54%) flowing into the algal tanks than G. longissima. The latter species biofiltered approximately 3.2% of phosphate and 17% of the total dissolved inorganic nitrogen input. However, mean nutrient uptake rates on wet weight basis were usually higher in G. longissima than in U. rotundata. The production of total oxidised nitrogen in the algal tanks, considered as being the nitrification rate occurring on the algal fronds by nitrifying bacteria, was less than half of the ammonium uptake by the macroalgae, suggesting that seaweeds competed efficiently for ammonia against the nitrifyers. The biofiltration during a diel cycle showed that mean phosphate biofiltration was lower than 4.5% in the two species whereas ammonium was biofiltered efficiently (up to 67%), especially in U. rotundata. The metal and heavy metal content in the algal tissue at the end of the monitoring period suggested no metal contamination of tissues so that both macroalgal species could be used in the food industry. The study reveals the value of ecological engineering techniques in reducing the dissolved nutrient content in effluents from the fish farm, with the prospect of a better management practises, based on integrated mariculture designs, being developed by the local farmers.

Proteases are essential components of cells and participate in processes ranging from photoacclimation and nutrient acquisition to development and stress responses. Virtually nothing is known about this diverse group of enzymes in macroalgae. Methods to measure proteases developed for phytoplankton (caseinolysis, leucine aminopeptidase (LAP) activity and casein zymograms) have been modified to allow use with spectrophotometers as well as spectrofluorometers, so making them accessible to a wider number of users. Applying the methods to a wide range of macroalgae from intertidal zones in south-western Spain and Northern Ireland detected proteases in all species examined. Protease activities differed among species but there was little systematic variation with taxonomic group. Moreover, similar species in Spain and Northern Ireland often showed differences. With the exception of LAP activities in brown algae, protease activities were one to two orders of magnitude greater in macroalgae than those previously measured in phytoplankton, when expressed per unit protein. However, scaling of activities to protein was complicated by significant interferences. Copper-based protein assays gave erroneously high results when applied to the brown algae, and partial purification of proteins by trichloroacetic acid precipitation did not overcome the problem. The higher levels of protease activity suggest that proteolysis plays a more significant role in multicellular than in unicellular algae. Proteases were characterized in terms of activities at different pH and the effect of protease inhibitors. In common with microalgae but in contrast to findings for animals and higher plants, proteases tended to show higher activities in the neutral and alkaline ranges. Many commonly used protease inhibitors (e.g. leupeptin and phenylmethylsulphonyl fluoride) had relatively little effect, which may explain why biochemical work with macroalgal species has traditionally been difficult. Our results suggest that macroalgal proteases are easily measurable but highly variable. A major source of variability that has not been assessed is differing environmental conditions. If this is correct, measurements of proteolytic enzymes may provide a valuable tool for examining biologically relevant changes in environments. Controlled laboratory experiments and seasonal monitoring are the next logical steps towards this goal.

Three estuarine macroalgae (Ulva rotundata,Enteromorpha intestinalis, Gracilariagracilis) of economic potential were cultivated in the laboratory toassess their biofiltering capacities for ammonium in waste effluents from a seabass (Dicentrarchus labrax) cultivation tank. The studywasdeveloped to investigate the functioning of N nutrition of the three species.Atlow water flow (< 2 volumes d−1) the three species strippedefficiently the ammonium dissolved in the waste water from the fish tank, withaminimum biofiltering efficiency estimate of 61% in unstarved cultures ofG. gracilis at a water flow of 2 volumesd−1. Maximum velocity for ammonium uptake (89.0 μmolNH4 + g−1 dry wth−1) was found in U. rotundata,whereas G. gracilis showed the highest affinity for thisnutrient. The net ammonium uptake rate was significantly affected by the waterflow, being greatest at the highest flow assayed (2 volumesd−1). Variations of tissue N and C:N ratios during aflow-through experiment suggested that N was not limiting macroalgal growth.However, when ammonium was supplied at a flow rate of 0.5 volumesd−1, specially in a three-stage design, the marked reductionintissue N and the biomass C:N:P ratios suggested a more general nutrientdeficiency. A significant correlation was found between growth rates and the Nbiomass gained in the cultures. The three-stage design under low water flow(0.5volumes d−1) showed that the highest ammonium uptake rates (upto 80.9 μmol NH4 + g−1 dry wtd−1 in U. rotundata) were found inthe first stage, with decreasing rates in the following ones. As a result, lowincrements or even losses of total N biomass in these stages were found,suggesting that ammonium was excreted from the algae. We conclude that thesespecies present a potential ability to biofilter the ammonium dissolved inwastewater from a D. labrax cultivation tank, suggesting thatscaling up the biofiltration designs, future practises using these macroalgaemay be implemented in the local fish farms, resulting in both environmental andeconomical advantages.

Photosynthetic acclimation and the interactions between carbon (C) and nitrogen (N) metabolism have been studied in the red macroalga Gracilaria sp. from Cadiz, Spain, cultured under different inorganic C and N levels. The use of chemostats and buffered medium allowed continuous restoration of the alkaline reserve and constancy of pH during the experiments. The N:C ratios and phycobiliprotein, chlorophyll a and soluble protein contents decreased when Gracilaria sp. was grown at low N levels. Algae grown in a high inorganic C concentration (5% CO2) displayed a higher soluble carbohydrate concentration and maximum photosynthesis rates but a lower photosynthesic affinity for inorganic C, and lower phycobiliprotein and Rubisco contents, than those cultured at low inorganic C levels (air CO2). The inorganic C enrichment also affected the N uptake and assimilation in Gracilaria sp., causing a decrease in the N uptake rate even under conditions of N sufficiency. These results reflect the significant influence of...

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO3− use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O2 evolution rates with those theoretically supported by CO2, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO3− use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO3− exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O2 evolution after addition of CO2 and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO3− use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO2 compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO3− use. The potential contribution to HCO3− acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO3− use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO3− use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations.

The diel variation and regulation of the enzyme nitrate reductase (NR) were examined in the diatom Thalassiosira weissflogii (Gru.) Fryxell et Hasle. NR was purified, and polyclonal antibodies were raised to a 98-kD polypeptide. The antibodies cross-reacted only with proteins from closely related diatom species, suggesting significant epitopic variation of this enzyme within algal divisions. Neither NR enzymatic activity nor protein was detected in cells grown with ammonium as the sole nitrogen source; the addition of ammonium to cells growing on nitrate decreased both protein levels and enzyme activity by 40% within 2 h. In cells grown on a 12:12 h LD cycle, NR activity and NR protein levels were highly correlated, with a peak at midday, a decrease toward the end of the photoperiod, and an increase in activity beginning near the end of the dark period. The addition of actinomycin D (an inhibitor of RNA synthesis) and cycloheximide (an inhibitor of protein synthesis) affected NR activity and NR protein levels identically, strongly suggesting that this nuclearencoded protein is regulated primarily at a transcriptional level. The diel pattern of NR protein and activity ceased immediately following transfer to continuous light, indicating that the periodicity is not directly controlled by a circadian rhythm. Time-lagged cross-correlation analysis revealed a 6-h phased difference between the minimum enzyme activity or protein levels and the maximum cellular carbon pool. On the basis of the experimental results, we develop a model proposing that (1) NR activity is regulated primarily by transcriptional regulation of NR synthesis and that (2) the level of expression of the enzyme during a given day is correlated with the integrated pool of organic carbon accumulated during the preceding photoperiod.

Morphology and polysaccharide characterization of Gelidium sesquipedale (Clem.) Bornet et Thuret were studied in cultures grown under various light qualities. White light (WL), blue light (BL) and red light (RL) (all at photon fluence rate of 40 μmol m-2 s-1) were used for the study of morphological characteristics, and in addition yellow light (YL) for polysaccharide characterization. RL and BL induced a proliferating growth, which resulted in bushy plants under RL. Cortical cells of BL-grown plants were smaller and presented a higher density per unit area, whereas those of WL- and RL-grown alga were larger. Medullary cells followed the inverse pattern. Light quality also affected polysaccharide yield and composition, with the yield being higher under BL, RL or YL than WL. Most of the polysaccharide was extracted in distilled water at 100 °C, while a low amount was solubilized at 22 °C and 120 °C. Extracts from BL-grown alga presented the highest galactan content. The starch concentration was lower in extracts from RL-, BL- and YL-cultivated alga than in those from the initial plants. The degree of substitution with methoxyl groups and precursor was very low in all the agar fractions, but fractions extracted from BL- and WL-grown alga were more substituted by precursor. The highest sulfate content was reached under BL (about 9% w/w) and the highest 2-O-methyl-3,6-anhydro-L-galactose and 6-O-methyl-D-galactose content were found in extracts from alga grown under YL.

During the last decade, the Palmones River estuary has undergone severe eutrophication followed by a green tide episode; two species of Ulva, rotundata Blid. and Ulva curvata (Kutz.) De Toni, were the main macroalgae responsible for this bloom. From November 1993 to December 1994, we followed the biomass, the growth dynamics, and tissue elemental composition (C:N:P)of Ulva species, as well as some physicochemical variables in the estuary. Maximum biomass (up to 375 g dry wt·m−2 in some spots, corresponding to a thallus area index of nearly 17 m2Ulva·m−2 sediment) were observed in June and December. However, the biomass varied among the sampling stations. Water nitrate, ammonia, and phosphate showed high concentrations throughout the year, with extremely high transient pulses, sustaining the high growth rates observed. Growth rates were estimated directly in the field. The rates were generally higher in Ulva discs maintained in net cages than those estimated by changes in biomass standing stock between two consecutive samplings. The difference between both estimates was used to quantify the importance of the processes causing loss of biomass, which were attributable to grazing, exported biomass, and thallus decomposition under anaerobic conditions resulting from extreme self-shading. Maximum chlorophyll content was found in winter, whereas the minimum was in spring. Atomic N:P ratios were generally higher in the algae than in the water. However, the absolute concentrations of tissue N and P were always higher than the critical levels for maximum growth, which suggests that growth was not limited by inorganic N or P availability. The results suggested that the increase in nutrient loading in the river may have triggered the massive development of green algae and that light limitation and temperature stress in summer seem to be the main factors controlling the abundance of Ulva in the estuary. In addition to light availability and thermal stress, the different loss processes may have a decisive role in the dynamics of Ulva biomass.

The effects of photoacclimation on growth, photosynthesis, pigment content and elemental composition of Ulva curvata and Ulva rotundata, which grow together in eutrophic areas of southern Spain, were investigated. Cultures were grown for 6 days at different photon fluence rates (PFR) ranging from darkness up to 200 µmolm-2 s-1 under nutrient-sufficient conditions (artificial seawater supplemented with ammonium and phosphate). Growth rates were not light-saturated (up to 200 µmolm-2s-1), reaching a value close to 0·2d-1. Growth rates based on mass, area or C content were equivalent, except in darkness and very low light levels (2 µmolm-2s-1), where thallus expansion occurred by diluting internal biomass and C. Chlorophyll and absorptance showed a bell-shaped PFR-response curve, with maxima at 30–60 µmolm-2s-1 and lower values at light saturation and under light-limiting conditions. Although net NH4 + incorporation was not affected by growth-PFR, there was inefficient assimilation of N at low light levels, ...

We studied the effect of ecologically relevant ammonium concentrations and light on several morphological and physiological properties, nitrogen metabolism and carbon reserves of eelgrass Zostera marina L. Eelgrass was grown under mesocosm conditions at 3 levels of ammonium enrichment (target concentrations of 0, 10 and 25 μM) and 2 levels of light (low and high light). High ammonium supply combined with low light had a negative effect on several morphological and physiological response parameters, while no such effects were found when ammonium was supplied under high light. N enrichment caused an increase in the content of total N, intracellular ammonium, free amino acids and residual N in the plants and this response was more pronounced under low-light conditions than under high light. The soluble proteins content de - crea sed, in contrast with external ammonium enrichment. The accumulation of free amino acids and residual N in NH4 +-enriched plants was followed by a substantial drop in carbohydrate reserves (sucrose and starch), which was larger in plants grown under low-light conditions. Our results indicate that N enrichment increases the demand for C skeletons and energy, and that photosynthesis cannot supply enough C and energy to cover that demand under low-light conditions. Eelgrass plants exposed to reduced light conditions, for example close to their depth limit or when covered by drift macroalgae, may thus be especially susceptible to enhanced ammonium concentrations. Our study demonstrates that ammonium toxicity may explain why eelgrass and other seagrasses deteriorate under nutrient-rich, low-light conditions.

Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta, Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var. liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate plots FL3 vs. FSC and FL3 us. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5±9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein.

Eutrophication affects seagrasses negatively by increasing light attenuation through stimulation of biomass of fast-growing, bloom-forming algae and because high concentrations of ammonium in the water can be toxic to higher plants. We hypothesized nevertheless, that moderate amounts of nitrophilic macroalgae that coexists with seagrasses under eutrophic conditions, can alleviate the harmful effects of eutrophication on seagrasses by reducing ammonium concentrations in the seawater to non-toxic levels because such algae have a very large capacity to take up inorganic nutrients. We studied therefore how combinations of different ammonium concentrations (0, 25 and 50 μM) and different standing stocks of macroalgae (i.e. 0, 1 and 6 layers of Ulva sp.) affected survival, growth and net production of the seagrass Zostera noltei. In the absence of Ulva sp., increasing ammonium concentrations had a negative influence on the performance of Z. noltei. The presence of Ulva sp. without ammonium supply had a similar, but slightly smaller, negative effect on seagrass fitness due to light attenuation. When ammonium enrichment was combined with presence of Ulva sp., Ulva sp. ameliorated some of negative effects caused by high ammonium availability although Ulva sp. lowered the availability of light. Benthic microalgae, which increased in biomass during the experiment, seemed to play a similar role as Ulva sp.--they contributed to remove ammonium from the water, and thus, aided to keep the ammonium concentrations experienced by Z. noltei at relatively non-toxic levels. Our findings show that moderate amounts of drift macroalgae, eventually combined with increasing stocks of benthic microalgae, may aid seagrasses to alleviate toxic effects of ammonium under eutrophic conditions, which highlights the importance of high functional diversity for ecosystem resistance to anthropogenic disturbance.

Received 8 October 2010; returned 9 November 2010; revised 2 December 2010; accepted 6 December 2010Objectives: The impact of oseltamivir on mortality in critically ill patients with 2009 pandemic influenza A(2009 H1N1) is not clear. The main objective of this study was to investigate the relationship between thetiming of antiviral administration and intensive care unit (ICU) outcomes.Methods: Prospective, observational study of a cohort of ICU patients with confirmed 2009 H1N1 infection.Clinical data, treatment and outcome were compared between patients receiving early treatment (ET) withoseltamivir, initiated within 2 days, and patients administered late treatment (LT), initiated after this timepoint.Multivariate analysis and propensity score were used to determine the effect of oseltamivir on ICU mortality.Results: Six hundred and fifty-seven patients were enrolled. Four hundred and four (61.5%) patients requiredmechanical ventilation (MV; mortality 32.6%). Among them, 385 received effective antiviral therapy andwere included in the study group. All patients received oseltamivir for a median duration of 10 days (interquar-tile range 8–14 days). Seventy-nine (20.5%) ET patients were compared with 306 LT patients. The two groupswere comparable in terms of main clinical variables. ICU length of stay (22.7+16.7 versus 18.4+14.2 days;P¼0.03), hospital length of stay (34.0+20.3 versus 27.2+18.2 days; P¼0.001) and MV days (17.4+15.2versus 14.0+12.4; P¼0.04) were higher in the LT group. ICU mortality was also higher in LT (34.3%) than inET (21.5%; OR¼1.9; 95% CI 1.06–3.41). A multivariate model identified ET (OR¼0.44; 95% CI 0.21–0.87) asan independent variable associated with reduced ICU mortality. These results were confirmed by propensityscore analysis (OR¼0.44; 95% CI 0.22–0.90; P,0.001).Conclusions: Our findings suggest that early oseltamivir administration was associated with favourableoutcomes among critically ill ventilated patients with 2009 H1N1 virus infection.Keywords: antiviral treatment, prognosis, pneumonia

Short-term (5-h) phycobiliprotein photoacclimation was a NO3−, dependent process in the red alga Corallina elongata Ellis et Soland. At low irradianre levels, phycobiliprotein synthesis (both r-phycocyacin and r-phycocyanin) took place when N supply was sufficient but was restricted by N limitation. Exposure to saturating irradiance resulted in pigment degradation under N limitation; however, under N-sufficient conditions a partial r-phycoerythrin synthesis was observed, despite the repressing role of high photon flux densities on phycobiliprotein synthesis. Soluble protein was less affected than phycobiliprotein by N limitation at low photon flux densities indicating that N limitation stimulates the flow of internal N metabolites toward the synthesis of nonpigmented proteins rather than pigmented proteins. The addition of protein synthesis inhibitors revealed that new phycobiliprotein synthesis occurs in response to sufficient N conditions. When protein synthesis was blocked in the chloroplast and cytoplasm simultaneously (addition of chloramphemcol and cycloheximide), both pigmented and nonpigmented protein synthesis was inhibited. Howeever, when protein systhesis was blocked in the chloroplast, only phycobiliprotein synthesis was clearly inhibited, whereas nonpigmented protein was less affected, indicating that phycobiliprotein is the main fraction of protein synthesized in the chloroplast at low photon flux densities when external N is available. This inhibition of phycobiliprotein synthesis was consistent with a maximal increase in metabolites of protein synthesis (internal NH4+ and amino acids). Our results suggest that phycobiliproteins may be an important N reservoir to meet internal N demands during N limitations in C. elongata. Moreover, r-phycoerythrin, synthesized even at saturating irradiance levels, and the major constituent of the phycobiliprotein pigments, may be more sensitive to changes in N supply than r-phycocyanin. The influence of limited irradiance levels on N assimilation and the effects of repressing protein synthesis on internal N accumulation are also discussed.

Light reduction in the water column and enhanced organic matter (OM) load into the sediments are two main consequences of eutrophication in marine coastal areas. This study addresses the combined effects of light, OM, and clonal traits in the seagrass Zostera noltii. Large Z. noltii plants were grown in sand with or without the addition of OM and under two light levels (high light and low light). Whereas some complete plant replicates were grown under homogeneous light and/or OM conditions, other replicates were grown under contrasting light and/or OM levels between the apical and the distal parts of the same plant. The three-way factorial design (light, OM load, and apex position) allowed us to determine the harmful effect of light reduction and OM enrichment on the growth, photosynthetic performance, and biochemical composition of Z. noltii. The addition of OM to the sediment promoted a decrease, or even an inhibition, in net plant growth regardless of the light level when the whole plants were grown under homogeneous light conditions. However, the results differed when plants were grown under contrasting light and/or OM conditions between apical and distal parts. In this case, the harmful effect of OM load was alleviated when apical parts were grown under high light conditions. OM loads also negatively affected the photosynthetic performance, evaluated as leaf fluorescence. The results indicate the importance of clonal traits in the response of Z. noltii growth to light conditions and OM enrichment.

The effect of nitrogen (N) load and irradiance on morphology, growth and photosynthetic performance was studied in the green macroalga Caulerpa prolifera (Forsskal) J. V. Lamouroux from the Gulf of Cddiz (south Spain). Constant growth rates were obtained for thalli growing at different N loads, which could be fitted to tissue N using the Droop equation, rendering a maximum growth rate of 0.09 d(-1), a minimum tissue N level of 1.71 % dry weight (DW) and a critical tissue N of 5.2 % DW. N limitation had no effects on F-v/F-m (maximum quantum yield of chlorophyll a fluorescence). Stolon production was significantly highest at low N loads; a reverse trend was observed for assimilator production. In a second experiment, algae were subjected to combinations of high and low N loads (HN and LN) and irradiance (HL and LL) levels. Highest growth rates were observed in the HNLL treatment, whereas the reverse combination rendered the lowest growth rate. High irradiance and high N load both led to increased biomass allocation to assimilators; at low N, the bulk of the biomass (> 75 % in the HLLN treatment) was allocated to the stolons. HN had a positive effect on F-v/F-m and HL had a negative effect. HL algae had a higher capacity for non-photochemical quenching. Despite its prolific nature, C. prolifera should be characterised as a slow-growing, but highly nitrophilic alga which has the capacity to forage for nutrients by allocating biomass to the stolons. REN2002-0746/MAR MERG-CT-2004-006385 EVK3-CT-2000-50003

Two species of agarophytes, Gelidium sesquipedale (Clem.) Born. et Thur. and Gracilaria tenuistipitata var. liui (Zhang & Xia), have been cultured in chemostat systems under different light qualities to study the response of growth and polysaccharide yield and composition to certain wavelengths. The yield of galactans, methoxyl groups and sulphate content in the agar as well as the starch concentration of these species are controlled by light quality in a different way, presumably because of their patterns of growth. The polysaccharide characteristics will be discussed in relation to the growth and metabolism of those algae.

The presence of different carbonic anhydrase (EC 4.2.1.1) activities has been investigated in the intertidal macroalgae Gracilaria sp. and Enteromorpha intestinalis (L.) Nees by using fractionation techniques. Activities, measured potentiometrically, were recorded for all fractions in both species, including those containing proteins associated with chloroplast membranes. In Gracilaria sp., most of the total activity was present in the soluble fraction, while similar activities were obtained for all fractions in E. intestinalis. By using inhibitors with a different capacity to enter the cell (acetazolamide and 6-ethoxyzolamide, inhibitors of external and total activity, respectively), a surface-accessible location was indicated for a high proportion of the soluble activity obtained in Gracilaria sp. In E. intestinalis, the inhibitor assays showed a substantial dependence of photosynthesis on intracellular activity. The short-term regulation of the extracellular activity in response to inorganic carbon availability was also examined in both macroalgae. Rapid repression (after 2 h) of the activity was recorded when Gracilaria sp. was transferred from limited to replete carbon conditions, while a fairly constant activity was recorded for E. intestinalis. In contrast, an increase of external activity was obtained for both macroalgae after being transferred to carbon-limited conditions, this response being more pronounced in E. intestinalis. Our results suggest the occurrence of a species-specific carbonic anhydrase system.

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.

A motile, lamellipodium-like structure, the ridge, forms as amoeboflagellate cells of Physarum polycephalum release from a substratum and begin swimming in fluid. Actin microfilaments form a distinct laminar core within the ridge; they are seen as a sparse, disordered meshwork in cytoskeletons prepared by conventional methods using uranyl acetate negative staining [10]. Preservation and visualization of these filaments and their arrangements improved considerably when cytoskeletons were imaged with phosphotungstic acid buffered with ammonium hydroxide (PTA(NH 4 )). Microfilaments within ridge cytoskeletons were found to form loose bundles and criss-crossing, ‘meshwork’ arrays several layers deep. Differences could be detected in morphology and detailed arrangement of microfilaments within cytoskeletons prepared in the presence of phalloidin. PTA(NH 4 ) may be useful for studies of cytoskeletal elements and their rearrangements in dynamic, motile regions of cells.

Surface membranes of mouse urinary bladder have associated with them a hexagonal structure with a unit cell side of 160 A containing subunits about 50 A in diameter packed on a p6 lattice. The structure has been confirmed and refined by analysis of electron micrographs of negatively-stained specimens using an optical diffractometer.

The effects of macroalgae blooms on seagrasses were assessed by shading Zostera noltii Hornem. with Ulva rigida C. Agardh mats under laboratory and field conditions. In the laboratory, where there was no direct contact between U. rigida and Z. noltii, leaf, rhizome, and root elongation rates, as well as gross production, declined as a function of U. rigida layers, causing a mobilization of non-structural carbohydrates in both above- and belowground tissues to meet carbon demands. However, when shading was performed in the field, where direct contact exists between Z. noltii and U. rigida, Z. noltii responses were not proportional to the number of Ulva layers. Elongation rates and gross production were reduced by U. rigida shading, with the lowest values under 2 Ulva layers, while there were no significant differences between controls and 4 U. rigida layers. This suggests another Ulva effect occurs besides shading. To test the likely effect of dissolved organic carbon (DOC) derived from U. rigida, Z. noltii plants were cultured under light limitation with radioactive dissolved organic carbon (DO 14 C) released by U. rigida. Plants cultured under a full DO 14 C load showed a significant enhancement of growth. The DO 14 C disappeared from the culture medium dur- ing the first 4 d of culture as a linear function of external DO 14 C concentration. This was coupled to a linear increase of radioactive particulate organic carbon (PO 14 C) in aboveground tissues, while a sub- stantial part of this PO 14 C was allocated in belowground tissues. Overall, the PO 14 C incorporated in Z. noltii plants represented ca. 20 to 25% of the DO 14 C which had disappeared. Therefore, a net transfer of DO 14 C from U. rigida to Z. noltii has been documented. Other additional possibilities, such as a light quality effect or other kind of signals (i.e. growth factors), are discussed.

The present study assessed the effect of seawater ammonium enrichment on survival and growth of the seagrass Zostera noltii Hornem. Ammonium enrichment had an inhibitory effect on shoot, rhizome and root elongation rates, as well as on primary production. The inhibitory effect was partially alleviated by phosphate addition. The frequency of ammonium pulses also affected growth negatively, with a maximum effect at both low and high pulse frequencies. Similar results were obtained when the experiment was run in the field during a winter trial. However, when the field experiment was repeated in spring at higher mean temperature and irradiance levels, opposite results were obtained, with ammonium enrichment causing a substantial increase in growth rates and productivity. These responses were related to the internal balance of non-structural carbo- hydrates (especially sucrose) with respect to C and N cell metabolism. Thus, there was a mobilisation of sucrose in both above- and belowground tissues to meet C increased demands arising from am- monium assimilation in winter, with sucrose concentrations reaching critical levels in relation to the total internal C pool. In contrast, sucrose accumulated in the tissues when ammonium pulses were applied in spring, indicating an enhanced C turnover that was able to meet the increased demands arising from ammonium assimilation into organic N compounds.