KINETICS OF GROWTH AND DEATH IN ANABAENA FLOS-AQUAE (CYANOBACTERIA) UNDER LIGHT LIMITATION AND SUPERSATURATION

The kinetics of population growth and death were investigated in Anabaena flos-aquae (Lyngb.) Bréb grown at light intensities ranging from limitation to photoinhibition (5 W·m⁻² to 160 W·m⁻²) in a nutrient-replete turbidostat. Steady-state growth rate (μ, or dilution rate, D) increased with light intensity from 0.44·day⁻¹ at a light intensity of 5 W·m⁻² to 0.99·day⁻¹ at 20 W·m⁻² and started to decrease above about 22 W·m⁻², reaching 0.56·day⁻¹ at 160 W·m⁻². The Haldane function of enzyme inhibition fit the growth data poorly, largely because of the unusually narrow range of saturation intensity. However, it produced a good fit (P < 0.001) for growth under photoinhibition. Anabaena flos-aquae died at different specific death rates (γ) below and above the saturation intensity. When calculated as the slope of a v_x⁻¹ and D⁻¹ plot, where v_x and D are cell viability (or live cell fraction) and dilution rate, respectively; γ was 0.047·day⁻¹ in the range of light limitation and 0.103·day⁻¹ under photoinhibition. Live vegetative cells and heterocysts, either in numbers or as a percentage of the total cells, showed a peak at the saturation intensity and decreased at lower and higher intensities. The ratio of live heterocysts to live vegetative cells increased with intensity when light was limiting but decreased when light was supersaturating. In cells growing at the same growth rate, the ratio was significantly lower under light inhibition than under subsaturation and the cell N:C ratio was also lower under inhibition. The steady-state rate of dissolved organic carbon (DOC) production increased with light intensity. However, its production as a percentage of the total C fixation was lowest at the optimum intensity and increased as the irradiance decreased or increased. The rate and percentage was significantly higher under photoinhibition than limitation in cells growing at the same growth rate. About 22% of the total fixed carbon was released as DOC at the highest light intensity. No correlation was found between the number of dead cells and DOC.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

EFFECTS OF NITROGEN SOURCES ON P-LIMITED GROWTH OF ANABAENA FLOS-AQUAE1

The effects of N₂, nitrate and ammonia as N sources were investigated in P-limited and nutrient-sufficient cultures of Anabaena flos-aquae (Lyngb.) Bréb. The maximum growth rate (μ_m) was highest at 1.34 d^?1 with ammonia, compared to 1.18 with nitrate and 0.95 d^?1 with N₂. There was no difference in P requirement between N₂ and nitrate cultures. Under P-limited conditions, the increase in cell P with growth rate (μ) was identical. With N₂ as the N source, cell-N concentrations in P-limited cells increased with μ as did cell P, and the cellular N:P ratio remained the same (14) within the range of μ examined. With nitrate, however, cell N concentrations were high and independent of n, except at a low μ. It appears that this organism fixes atmospheric N₂ only at the minimum concentration required to maintain a μ. The acetylene reduction rate increased with μ in both N_2- and nitrate-grown cells, but the rate was lower in nitrate. Under P-limitation, there was no difference in net C-fixation rate per cell between N₂ and nitrate cultures at a given μ. However, the rate per unit of chlorophyll a (chl a) was higher in N₂ than in nitrate cultures, and the rate was independent of μ with N₂ but was a linear function of nitrate supplied. The maximum C-fixation rate in nutrient sufficient cells was highest with ammonia, followed by nitrate and N₂. The cellular chl a concentration was correlated with the total cell-N concentrations regardless of H and the source of N.     

COENOBIAL CELL NUMBER IN SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE) AS A FUNCTION OF GROWTH RATE IN NITRATE-LIMITED CHEMOSTATS1

The relative numbers of cells growing as coenobia of different cell number are functions of growth rate when Scenedesmus quadricauda (Turp.) Bréb. is grown axenically in nitrate-limited, steady-state chemostats in continuous light. Unicells decreased from a maximum fraction of 0.04–0.05 of the total number of cells at 0.1 day^?1 to 0.01 or less as growth rate increased. The fraction of cells that grew as two-celled coenobia decreased from about 0.2 to 0.01–0.02. The fraction that grew as 4-celled coenobia increased from about 0.7–0.8 at 0.1 day^?1 to near unity at 0.5–0.6 day^?1, and then decreased sharply. The fraction of 8-celled coenobial cells increased from very small values below 0.6 day^?1, to ca. 0.9 at 1.0 day^?1.     

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4?L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ?=?0.07?day^?1 versus 0.05?day^?1) and growth maximum (G _max, 169.10⁶ versus 41.10⁶ cells L^?1). Maximal gymnodimine concentration (1200?μg L^?1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell^?1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L^?1], though the yield [pg cell^?1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793?μg day^?1 over 58?days in continuous culture was achieved versus an average of 60?μg day^?1 [carboy over 40?days] or 249?μg day^?1 [batch mode] over 26?days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25?days, the yield of SPX-desMe-C (29?μg day^?1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day^?1 and 7.7?μg day^?1 respectively). Similarly 5.4?L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12?μg day^?1 versus 7?μg day^?1). When the bioreactor system was upscaled to 52?L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.     

Photoheterotrophic and chemoheterotrophic dinitrogen fixation and nitrate utilization by the cyanobacteriumAnabaena torulosa

Anabaena torulosa exhibited fructose-dependent growth, heterocyst differentiation and N₂ fixation in nitrate-free (diazotrophic) cultures in photoheterotrophic and chemoheterotrophic conditions. The incorporation of nitrate into such cultures inhibited the formation of heterocysts and N₂ fixation. The rate of NO ₃ ⁻ uptake byA. torulosa in photoautotrophic, photoheterotrophic and chemoheterotrophic conditions was similar but it increased by 100% in phototrophic conditions. The activity of glucose-6-phosphate dehydrogenase was found to be maximum in phototrophic and photoheterotrophic conditions. Ferredoxin-NADP⁺ reductase, nitrate reductase and glutamate-ammonia ligase activities suggest that nitrate utilization takes place in nonphotosynthetic conditions.     

Inorganic nitrogen control in a novel zero-water exchanged aquaculture system integrated with airlift-submerged fibrous nitrifying biofilters

This work examined the feasibility of applying shrimp diets to establish nitrification on submerged fibrous biofilters. It also investigated the performance of a proposed zero-water exchanged aquaculture system, which integrated growing of aquatic stocks and operation of acclimated biofilters in the same environment. Addition of shrimp diets fully established nitrification within 3 weeks as indicated by continuous increase of nitrate and trivial levels of ammonium and nitrite. A series of batch experiment revealed an average ammonium degradation rate of 24.1 mg N m^?2 day^?1. Zero-water discharged tilapia cultivation could be carried out in the proposed aquaculture system for at least 44 days when daily inorganic loadings increased from 1.24 to 10.78 mg N l^?1 day^?1. The corresponding daily growth rates of tilapia from the proposed aquaculture systems integrated with acclimated biofilters varied from 3.01 to 3.35 g day^?1, which was approximately 7–16% better than numbers from the systems using non-acclimated biofilters.     

PHYSIOLOGICAL CHARACTERIZATION OF SIX LIPID-PRODUCING DIATOMS FROM THE SOUTHEASTERN UNITED STATES1

Six strains of diatoms from intertidal waters were isolated as part of the SERI Aquatic Species Program collection and screening effort: Amphiprora hyalina Greville, Cyclotella cryptica Reimann, Lewin & Guillard, Navicula acceptata Hustedt (two strains, NAVIC6 and NAVIC8), Navicula saprophila Lange-Bertalot & Bonik, and Nitzschia dissipata (Kütz.) Grunow. Among numerous algal strains isolated as part of this collection effort, these six strains showed rapid growth and elevated lipid content in preliminary screening experiments and were chosen for further physiological characterization. N. dissipata grew most rapidly at 25°C, whereas the other five strains grew best at 30–35°C. Salinity tolerance varied among strains, with maximal growth occurring at the following conductivities: 10–60 mS.cm^?1 (A. hyalina and N. acceptata NAVIC8), 10–35 mS.cm^?1 (C. cryptica), 20–45 mS.cm^?1 (N. acceptata NAVIC6), 10 mS.cm^?1 (N. saprophila), and 20–35 mS.cm^?1 (N. dissipata). The diatoms also differed in their utilization of nitrogen sources with A. hyalina growing optimally with either nitrate or urea; N. acceptata NAVIC6, with either nitrate or ammonium; C. cryptica, N. acceptata NAVIC8, and N. dissipata, with nitrate; and N. saprophila, with urea. Under optimal conditions, A. hyalina grew at 2.0 doublings. day^?1; C. cryptica grew at 3.0 doublings. day^?1. Each Navicula strain had a growth rate of 3.8 doublings. day^?1, and N. dissipata grew at 2.6 doublings.day^?1. All six strains had lipid contents in excess of 37% ashfree dry weight (AFDW) under nutrient-limited conditions, with N. saprophila having the highest lipid content at 48% AFDW.     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.     

Nitrogen cycling in a flooded-soil ecosystem planted to rice (Oryza sativa L.)

¹⁵N studies of various aspects of the nitrogen cycle in a flooded rice ecosystem on Crowley silt loam soil in Louisiana were reviewed to construct a mass balance model of the nitrogen cycle for this system. Nitrogen transformations modeled included 1) net ammonification (0.22 mg NH₄ ⁺?N kg dry soil^?1 day^?1), 2) net nitrification (2.07 mg NO₃ ^??N kg^?1 dry soil^?1 day^?1), 3) denitrification (0.37 mg N kg dry soil^?1 day^?1), and 4) biological N₂ fixation (0.16 mg N kg dry soil^?1 day^?1). Nitrogen inputs included 1) application of fertilizers, 2) incorporation of crop residues, 3) biological N₂ fixation, and 4) deposition. Nitrogen outputs included 1) crop removal, 2) gaseous losses from NH₃ volatilization and simultaneous occurrence of nitrification-denitrification, and 3) leaching and runoff. Mass balance calculations indicated that 33% of the available inorganic nitrogen was recovered by rice, and the remaining nitrogen was lost from the system. Losses of N due to ammonia volatilization were minimal because fertilizer-N was incorporated into the soil. A significant portion of inorganic-N was lost by ammonium diffusion from the anaerobic layer to the aerobic layer in response to a concentration gradient and subsequent nitrification in the aerobic layer followed by nitrate diffusion into the anaerobic layer and denitrification into gaseous end products. Leaching and surface runoff losses were minimal.     

Toxicity of parathion-methyl to cells, akinetes and heterocysts of the cyanobacterium Cylindrospermum, sp. and the probit analysis of toxicity

The toxicity of a commercial formulation of the insecticide parathion‐methyl to the N2‐fixing filamentous cyanobacterium (blue‐green alga) Cylindrospermum, sp. was studied. A concentration of parathion‐methyl of 0.5 ppm caused growth increase in liquid growth media. The minimum inhibitory concentration of parathion‐methyl for both types (N₂, fixing and nitrate supplemented) of liquid and solid media was 1.0 ppm. LC₅₀ values were: 4.4 ppm (liquid, N₂, fixing), 5.5 ppm (liquid, nitrate supplemented), 3.3 ppm (agar, N₂‐fixing) and 4.0 ppm (agar, nitrate supplemented). LC100 values for N₂‐fixing liquid and both types of agar media were 10.0 ppm, while for the liquid nitrate supplemented medium the LC₁₀₀ was 12.0 ppm. Both akinete (spore) formation and germination were inhibited below the highest permissive concentration of 8.0 ppm, with the insecticide incorporated in the agar media. In soil, the LC50 and LC100 values for parathion‐methyl were 13.6 and 30 ppm, respectively. Both the dehydrogenase activity of heterocysts (monitored by 2,3,5‐triphenyl tetrazolium chloride reduction) and the nitrogen concentration of cultures (estimated by the micro‐Kjeldahl method) were affected by the insecticide, but the latter (N₂‐fixation) was more sensitive. The Kruskal‐Wallis H test on the numbers of vegetative cells in the filaments revealed that the insecticide significantly affected the division of vegetative cells. The cyanobacterium could detoxify the growth medium containing high levels (30 and 40 ppm) of the insecticide in short‐term exposures at the expense of cell viability.     

Nitrogen fixation by the diazotroph Cylindrospermopsis raciborskii (Cyanophyceae)

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen‐limited freshwater systems. However, it is unclear whether dinitrogen fixation (N₂ fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N₂ fixation is unknown. This study investigated N₂ fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 μmol  · L^?1) and N‐free conditions and then either received a single dose of 941 μmol  · L^?1 (N941), 118 μmol  · L^?1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N₂ fixation and up‐regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N₂ fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N₂ fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N₂ fixation only provides enough N to support relatively low growth under N‐limited conditions, and does not supplement available nitrate to increase growth rates.     

AUTOLYSIS KINETICS OF THE MARINE DIATOM DITYLUM BRIGHTWELLII (BACILLARIOPHYCEAE) UNDER NITROGEN AND PHOSPHORUS LIMITATION AND STARVATION1

Autolysis kinetics in axenic cultures of the diatom Ditylum brightwellii (West) Grunow were studied under nutrient limitation in continuous cultures and under nutrient starvation in batch-mode cultures obtained by switching off nutrient supply in the continuous cultures. Under N limitation, the specific algal autolysis rates (δ, day^?1) were found constant at 0.014 ± 0.002 day^?1over a broad range of specific dilution rates (D, day^?1) (0.09–0.56 day^?1), implying an intrinsic death factor independent of the physiologzc state of the algal cells. Under P limitation, 8 was inversely related to D and ranged between 0.067 and 0.005 day^?1 at D = 0.17–0.44 day^?1. Under conditions of nutrient stamation, the degree of algal nutrient deficiency prior to stamation affected autolysis rates (δ_b, day^?1) and subsequently survival of the algal cultures. Nitrogen-starved D. brightwellii showed highest δ_b (maximum, 0.10 day^?1) when precultured at the higher growth rates. Phosphorus stamation led to highest δ_b (maximum, 0.21 day^?1) in the cultures preconditioned at the lower steady state growth rates. The lower death rates for D. brightwellii under limitation and starvation of N compared to P suggest that D. brightwellii was better equipped to handle N than P deficiency. The present results showed that cell lysis induced by nutrient stress was a significant cause of mortality in D. brightwellii and provided more insight into the field distribution of this neritic diatom.     

Phytoplankton nitrogen demand and the significance of internal and external nitrogen sources in a large shallow lake (Lake Balaton,Hungary)

Since the middle of 1990s the trend of Lake Balaton towards an increasingly trophic status has been reversed, but N₂-fixing cyanobacteria are occasionally dominant, endangering water quality in summer. The sources of nitrogen and its uptake by growing phytoplankton were therefore studied. Experiments were carried out on samples collected from the middle of the Eastern (Siófok) and Western (Keszthely) basins between February and October 2001. Ammonium, urea and nitrate uptake and ammonium regeneration were measured in the upper 5-cm layer of sediment using the ¹⁵N-technique. Ammonium was determined by an improved microdiffusion assay. N₂ fixation rates were measured by the acetylene-reduction method. Ammonium regeneration rates in the sediment were similar in the two basins. They were relatively low in winter (0.13 and 0.16 μg N cm^?3 day^?1 in the Eastern and Western basin, respectively), increased slowly in the spring (0.38 and 0.45 μg N cm^?3 day^?1) and peaked in late summer (0.82 and 1.29 μg N cm^?3 day^?1, respectively). Ammonium uptake was predominant in spring in the Eastern basin and in summer in the Western basin, coincident with the cyanobacterial bloom. The amount of N₂ fixed was less than one third of the internal load during summer when external N loading was insignificant. Potentially, the phytoplankton N demand could be supported entirely by the internal N load via ammonium regeneration in the water column and sediment. However, the quantity of N from ammonium regeneration in the upper layer of sediment combined with that from the water column would limit the standing phytoplankton crop in spring in both basins and in late summer in the Western basin, especially when the algal biomass increases suddenly.     

LytM factor Alr3353 affects filament morphology and cell–cell communication in the multicellular cyanobacterium Anabaena sp. PCC 7120

Heterocyst‐forming cyanobacteria are organized as multicellular filaments of tightly interacting, functionally specialized cells. N₂‐fixing heterocysts differentiate from vegetative cells under nitrogen limitation in a semi‐regular pattern along the filament. Diazotrophic growth requires metabolite exchange between neighboring cells within the filament. This exchange occurs via cell–cell junction complexes that span the gap between the plasma membranes and thereby cross the septal peptidoglycan through an array of uniform nanopores formed by AmiC‐type cell wall hydrolases. We investigated how the lytic hydrolase AmiC1 (Alr0092) from Anabaena sp. PCC 7120, whose activity needs to be tightly controlled to avoid cell lysis, is regulated by the LytM factor Alr3353. Inactivation of alr3353 resulted in significantly fewer nanopores and as a consequence, a lower rate of fluorescent tracer exchange between cells. The mutant was not able to grow with N₂ as sole nitrogen source, although heterocysts were formed. Alr3353 localized mainly to fully developed intercellular septa of vegetative cells. The purified protein bound to peptidoglycan and enhanced the hydrolytic activity of AmiC1 in vitro. Our data show that the LytM factor Alr3353 regulates nanopore formation and cell–cell communication by directly interacting with AmiC1.     

Bacterial metabolism in the River Danube: parameters influencing bacterial production

1. Microbial parameters were determined at five sampling sites in the River Danube up-and downstream of Vienna, Austria, twice monthly over an annual cycle. Bacterial production (BP) was estimated from thymidine and leucine incorporations; additionally, the effect of turbulence on BP and the conversion factors for converting incorporation rates into bacterial cell production were determined using the cumulative approach. 2. BP under turbulent conditions was not significantly different from that under stagnant conditions. For thymidine, a mean annual conversion factor of 3.2 ± 10¹⁸ cells mol^?1 thymidine incorporated was calculated. For leucine, the corresponding factor was 0.07 ± 10¹⁸ cells mol^?1 leucine. Average annual BP calculated by thymidine incorporation was significantly higher than BP calculated from leucine incorporation and ranged from 47.2 to 77.5 μg C 1-^?1 day^?1 depending on the tracer and the conversion factor used. 3. Bacterial growth rates ranged from 0.1 day^?1 during winter to 1.7 day^?1 in the summer. A strong correlation was found between temperature as well as chlorophyll a and bacterial growth when temperature was greater than 5 °C; a major spring phytoplankton bloom at a temperature below 5 °C did not increase BP. 4. Dissolved organic carbon (DOC) concentrations varied between 2 and 7.2 mg C 1-^?1 and comprised between 50 and 92% of the total organic carbon pool in the River Danube, Based on the DOC concentration and an assumed bacterial growth yield of 20% we calculated mean DOC turnover times of around 60 days in the winter and less than 8 days during the summer.     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Small-scale experiments aimed at optimization of large-scale production of the microalga <Emphasis Type="Italic">Rhodomonas salina</Emphasis>

The cryptophyte Rhodomonas is an important feed item for live feed organisms in aquaculture and although large-scale cultivation of Rhodomonas in photobioreactors (PBRs) is feasible, the production needs to be optimized through further studies of specific factors. Through small-scale experiments, several factors relevant for an on-going large-scale production of Rhodomonas were studied and the results presented here provide a useful insight on factors that can help future large-scale production. The content of polyunsaturated fatty acids (PUFAs) and the temporal sedimentation was compared in five strains of Rhodomonas. Strain K-1487 (R. salina) was chosen as the most suitable for cultivation in PBRs due to a good biochemical content of PUFAs and low cell sedimentation. The f/2 growth medium used for cultivation was modified by excluding CoCl₂ which did not affect either growth rate or cell content of the PUFAs, DHA, EPA, and ARA. Furthermore, the growth medium was modified by adding the nitrogen source as ammonium (NH₄⁺), nitrate (NO₃^?), urea, or combinations of these, with NH₄⁺ yielding a significantly higher growth rate of 1.30?±?0.07 day^?1. The seawater used for cultivation was exposed to three types of treatments which gave no significant difference in the growth rate: (1) filtration (0.2 μm)?+?autoclaving, (2) filtration (0.2 μm)?+?UV-radiation, and (3) filtration (0.2 μm). Finally, the results for growth rates of inocula at initial densities ranging from 2000 to 200,000 cells mL^?1 showed that growth rate decreased with increasing density but a final density of 10⁶ cells mL^?1 was obtained fastest with the highest initial density. With the present findings, several barriers for effective cultivation of Rhodomonas are solved and future large-scale production has become a great step closer.     

Nonlinear response of N2O flux to incremental fertilizer addition in a continuous maize (Zea mays L.) cropping system

The relationship between nitrous oxide (N₂O) flux and N availability in agricultural ecosystems is usually assumed to be linear, with the same proportion of nitrogen lost as N₂O regardless of input level. We conducted a 3‐year, high‐resolution N fertilizer response study in southwest Michigan USA to test the hypothesis that N₂O fluxes increase mainly in response to N additions that exceed crop N needs. We added urea ammonium nitrate or granular urea at nine levels (0–292 kg N ha^?1) to four replicate plots of continuous maize. We measured N₂O fluxes and available soil N biweekly following fertilization and grain yields at the end of the growing season. From 2001 to 2003 N₂O fluxes were moderately low (ca. 20 g N₂O‐N ha^?1 day^?1) at levels of N addition to 101 kg N ha^?1, where grain yields were maximized, after which fluxes more than doubled (to >50 g N₂O‐N ha^?1 day^?1). This threshold N₂O response to N fertilization suggests that agricultural N₂O fluxes could be reduced with no or little yield penalty by reducing N fertilizer inputs to levels that just satisfy crop needs.