A Simple, High-Precision, High-Sensitivity Tracer Assay for N(inf2) Fixation

We describe a simple, precise, and sensitive experimental protocol for direct measurement of N(inf2) fixation using the conversion of (sup15)N(inf2) to organic N. Our protocol greatly reduces the limit of detection for N(inf2) fixation by taking advantage of the high sensitivity of a modern, multiple-collector isotope ratio mass spectrometer. This instrument allowed measurement of N(inf2) fixation by natural assemblages of plankton in incubations lasting several hours in the presence of relatively low-level (ca. 10 atom%) tracer additions of (sup15)N(inf2) to the ambient pool of N(inf2). The sensitivity and precision of this tracer method are comparable to or better than those associated with the C(inf2)H(inf2) reduction assay. Data obtained in a series of experiments in the Gotland Basin of the Baltic Sea showed excellent agreement between (sup15)N(inf2) tracer and C(inf2)H(inf2) reduction measurements, with the largest discrepancies between the methods occurring at very low fixation rates. The ratio of C(inf2)H(inf2) reduced to N(inf2) fixed was 4.68 (plusmn) 0.11 (mean (plusmn) standard error, n = 39). In these experiments, the rate of C(inf2)H(inf2) reduction was relatively insensitive to assay volume. Our results, the first for planktonic diazotroph populations of the Baltic, confirm the validity of the C(inf2)H(inf2) reduction method as a quantitative measure of N(inf2) fixation in this system. Our (sup15)N(inf2) protocols are comparable to standard C(inf2)H(inf2) reduction procedures, which should promote use of direct (sup15)N(inf2) fixation measurements in other systems.     

Methodological Underestimation of Oceanic Nitrogen Fixation Rates

The two commonly applied methods to assess dinitrogen (N₂) fixation rates are the ¹⁵N₂-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N₂ fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N₂. Here we demonstrate that the ¹⁵N₂-tracer addition method underestimates N₂ fixation rates significantly when the ¹⁵N₂ tracer is introduced as a gas bubble. The injected ¹⁵N₂ gas bubble does not attain equilibrium with the surrounding water leading to a ¹⁵N₂ concentration lower than assumed by the method used to calculate ¹⁵N₂-fixation rates. The resulting magnitude of underestimation varies with the incubation time, to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N₂ fixation patterns. Here, we propose and test a modified ¹⁵N₂ tracer method based on the addition of ¹⁵N₂-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N₂ fixation rates for both field and laboratory studies. We hypothesise that application of N₂ fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.     

The sources of carbon and nitrogen supplying leaf growth. Assessment of the role of stores with compartmental models

Patterns of synthesis and breakdown of carbon (C) and nitrogen (N) stores are relatively well known. But the role of mobilized stores as substrates for growth remains less clear. In this article, a novel approach to estimate C and N import into leaf growth zones was coupled with steady-state labeling of photosynthesis ((13)CO(2)/(12)CO(2)) and N uptake ((15)NO(3)(-)/(14)NO(3)(-)) and compartmental modeling of tracer fluxes. The contributions of current C assimilation/N uptake and mobilization from stores to the substrate pool supplying leaf growth were then quantified in plants of a C(3) (Lolium perenne) and C(4) grass (Paspalum dilatatum Poir.) manipulated thus to have contrasting C assimilation and N uptake rates. In all cases, leaf growth relied largely on photoassimilates delivered either directly after fixation or short-term storage (turnover rate = 1.6-3.3 d(-1)). Long-term C stores (turnover rate < 0.09 d(-1)) were generally of limited relevance. Hence, no link was found between the role of stores and C acquisition rate. Short-term (turnover rate = 0.29-0.90 d(-1)) and long-term (turnover rate < 0.04 d(-1)) stores supplied most N used in leaf growth. Compared to dominant (well-lit) plants, subordinate (shaded) plants relied more on mobilization from long-term N stores to support leaf growth. These differences correlated well with the C-to-N ratio of growth substrates and were associated with responses in N uptake. Based on this, we argue that internal regulation of N uptake acts as a main determinant of the importance of mobilized long-term stores as a source of N for leaf growth.     

Anoxygenic Photosynthesis and Nitrogen Fixation by a Microbial Mat Community in a Bahamian Hypersaline Lagoon

Simultaneous measurements of photosynthesis (both oxygenic and anoxygenic) and N(inf2) fixation were conducted to discern the relationships between photosynthesis, N(inf2) fixation, and environmental factors potentially regulating these processes in microbial mats in a tropical hypersaline lagoon (Salt Pond, San Salvador Island, Bahamas). Major photoautotrophs included cyanobacteria, purple phototrophic bacteria, and diatoms. Chemosystematic photopigments were used as indicators of the relative abundance of mat phototrophs. Experimental manipulations consisted of light and dark incubations of intact mat samples exposed to the photosystem II inhibitor DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a dissolved organic carbon source (D-glucose), and normal seawater (37(permil)). Photosynthetic rates were measured by both O(inf2) and (sup14)C methods, and nitrogenase activity (NA) was estimated by the acetylene reduction assay. Moderate reductions in salinity (from 74 to 37(permil)) had no measurable effect on photosynthesis, O(inf2) consumption, or NA. CO(inf2) fixation in DCMU-amended samples was (symbl)25% of that in the control (nonamended) samples and demonstrated photosynthetic activity by anoxygenic phototrophs. NA in DCMU-amended samples, which was consistently higher (by a factor of 2 to 3) than the other (light and dark) treatments, was also attributed to purple phototrophic bacteria. The ecological implication is that N(inf2) fixation by anoxygenic phototrophs (purple phototrophic bacteria and possibly cyanobacteria) may be regulated by the activity of oxygenic phototrophs (cyanobacteria and diatoms). Consortial interactions that enhance the physiological plasticity of the mat community may be a key for optimizing production, N(inf2) fixation, and persistence in these extreme environments.     

Nitrogen fixation (acetylene reduction) associated with duckweed (lemnaceae) mats

Duckweed (Lemnaceae) mats in Texas and Florida were investigated, using the acetylene reduction assay, to determine whether nitrogen fixation occurred in these floating aquatic macrophyte communities. N(2)-fixing microorganisms were enumerated by plating or most-probable-number techniques, using appropriate N-free media. Results of the investigations indicated that substantial N(2)-fixation (C(2)H(2)) was associated with duckweed mats in Texas and Florida. Acetylene reduction values ranged from 1 to 18 mumol of C(2)H(4) g (dry weight) day for samples incubated aerobically in light. Dark N(2) fixation was always two- to fivefold lower. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (7 to 10 muM) reduced acetylene reduction to levels intermediate between light and dark incubation. Acetylene reduction was generally greatest for samples incubated anaerobically in the light. It was estimated that 15 to 20% of the N requirement of the duckweed could be supplied through biological nitrogen fixation. N(2)-fixing heterotrophic bacteria (10 cells g [wet weight] and cyanobacteria (10 propagules g [wet weight] were associated with the duckweed mats. Azotobacter sp. was not detected in these investigations. One diazotrophic isolate was classified as Klebsiella.     

Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-atoms of N m(-2) h(-1)). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Nitrogen fixation and primary production in Western Mediterranean

Nitrogen fixation, nitrate assimilation and primary production ((13)C/(15)N method) were investigated during one year and half in the northwestern Mediterranean Sea. Nitrogen fixation was detectable all over the year with rates ranged from 2 to 17 nmol N l(-1) d(-1)(d). Highest values being obtained during spring associated with the phytoplankton bloom. High rates (4-8 nmol N l(-1) d(-1)(d)) were also measured during summer, when primary productivity was very low. Then, diazotrophy process supplies significant new nitrogen during summer oligotrophic periods. This new nitrogen input can balance the annual nitrogen biogeochemical budget in the Mediterranean Sea and should explain the high nitrate/phosphate ratio observed in deep waters.     

Biological nitrogen fixation in acidic high-temperature geothermal springs in Yellowstone National Park, Wyoming

The near ubiquitous distribution of nifH genes in sediments sampled from 14 high-temperature (48.0-89.0°C) and acidic (pH 1.90-5.02) geothermal springs in Yellowstone National Park suggested a role for the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) (e.g. nitrogen fixation or diazotrophy) in these environments. nifH genes from these environments formed three unique phylotypes that were distantly related to acidiphilic, mesophilic diazotrophs. Acetylene reduction assays and (15) N(2) tracer studies in microcosms containing sediments sampled from acidic and high-temperature environments where nifH genes were detected confirmed the potential for biological N(2) reduction in these environments. Rates of acetylene reduction by sediment-associated populations were positively correlated with the concentration of NH(4)(+), suggesting a potential relationship between NH(4)(+) consumption and N(2) fixation activity. Amendment of microcosms with NH(4)(+) resulted in increased lag times in acetylene reduction assays. Manipulation of incubation temperature and pH in acetylene reduction assays indicated that diazotrophic populations are specifically adapted to local conditions. Incubation of sediments in the presence of a N(2) headspace yielded a highly enriched culture containing a single nifH phylotype. This phylotype was detected in all 14 geothermal spring sediments examined and its abundance ranged from ≈ 780 to ≈ 6800 copies (g dry weight sediment)(-1), suggesting that this organism may contribute N to the ecosystems. Collectively, these results for the first time demonstrate thermoacidiphilic N(2) fixation in the natural environment and extend the upper temperature for biological N(2) fixation in terrestrial systems.     

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.     

Tracer studies of nitrogen assimilation in yeast

By using N(15) as a tracer the assimilation of ammonia by the yeast, Torulopsis utilis, has been studied. It has been shown that: 1. There was no measurable incorporation of N in the protein or polynucleotide purine of carbon-starved yeast. 2. When ammonia is added to nitrogen-starved yeast there is a long lag period before division begins during which the yeast rapidly synthesizes protein, this process being accompanied by a turnover of polynucleotide purine. There was no significant dilution of the N(15)H(4) (+) of the medium by ordinary NH(4) (+). 3. When yeast containing N(15) is allowed to divide and grow in ordinary ammonia, the total amount of N(15) in the yeast remains constant. The dicarboxylic amino acids are most diluted, while arginine and nucleic acid guanine are not diluted at all.     

Incorporation of Different N Sources and Light Response Curves of Nitrogenase and Photosynthesis by Cyanobacterial Blooms from Rice Fields

In this work, we estimate the contributions of the different sources of N incorporated by two N₂-fixing cyanobacterial blooms (Anabaena sp. and Microchaete sp.) in the rice fields of Valencia (Spain) during the crop cycles of 1999 and 2000, and evaluate the response of nitrogenase and C assimilation activities to changing irradiances. Our results show that, far from the generally assumed idea that the largest part of the N incorporated by N₂-fixing cyanobacterial blooms in rice fields comes from N₂ fixation, both cyanobacterial blooms incorporated about three times more N from dissolved combined compounds than from N₂ fixation (only about 33–41% of the N incorporated came from N₂ fixation). Our results on the photodependence of C and N₂ fixation indicate that in both cyanobacterial blooms, N₂ fixation showed a steeper initial slope (α) and was saturated with less irradiance than C fixation, suggesting that N₂ fixation was more efficient than photosynthesis under conditions of light limitation. At saturating light, N₂ fixation and C fixation differed depending on the bloom and on the environmental conditions created by rice plant growth. Carbon assimilation but not nitrogenase activity appeared photoinhibited in the Anabaena but not in the Microchaete bloom in August 1999, when the plants were tall and the canopy was important, and there was no limitation of dissolved inorganic carbon. The opposite was found in the Microchaete bloom of June 2000, when plants were small and produced little shade, and dissolved inorganic carbon was very low.     

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.     

Biological dinitrogen fixation (acetylene reduction) associated with Florida mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C(2)H(4)/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C(2)H(2)) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.Higher rates of nitrogenase activity (15 to 53 nmol of C(2)H(4)/g [wet weight] per h were associated with washed excised roots of three Florida mangrove species [Rhizophora mangle L., Avicennia germinans (L) Stern, and Laguncularia racemosa Gaertn.] as well as with isolated root systems of intact plants (11 to 58 mug of N/g [dry weight] per h). Following a short lag period, root-associated activity was linear and did not exhibit a marked response to glucose amendment. It appears that dinitrogen-fixing bacteria in the mangrove rhizoplane are able to use root exudates and/or sloughed cell debris as energy sources for dinitrogen fixation.     

Denitrification and nitrogen fixation dynamics in the area surrounding an individual ghost shrimp (Neotrypaea californiensis) burrow system

Bioturbated sediments are thought of as areas of increased denitrification or fixed-nitrogen (N) loss; however, recent studies have suggested that not all N may be lost from these environments, with some N returning to the system via microbial dinitrogen (N(2)) fixation. We investigated denitrification and N(2) fixation in an intertidal lagoon (Catalina Harbor, CA), an environment characterized by bioturbation by thalassinidean shrimp (Neotrypaea californiensis). Field studies were combined with detailed measurements of denitrification and N(2) fixation surrounding a single ghost shrimp burrow system in a narrow aquarium (15 cm by 20 cm by 5 cm). Simultaneous measurements of both activities were performed on samples taken within a 1.5-cm grid for a two-dimensional illustration of their intensity and distribution. These findings were then compared with rate measurements performed on bulk environmental sediment samples collected from the lagoon. Results for the aquarium indicated that both denitrification and N(2) fixation have a patchy distribution surrounding the burrow, with no clear correlation to each other, sediment depth, or distance from the burrow. Field denitrification rates were, on average, lower in a bioturbated region than in a seemingly nonbioturbated region; however, replicates showed very high variability. A comparison of denitrification field results with previously reported N(2) fixation rates from the same lagoon showed that in the nonbioturbated region, depth-integrated (10 cm) denitrification rates were higher than integrated N(2) fixation rates (～9 to 50 times). In contrast, in the bioturbated sediments, depending on the year and bioturbation intensity, some (～6.2%) to all of the N lost via denitrification might be accounted for via N(2) fixation.     

In vivo gas exchange measurement of the site and dynamics of nitrate reduction in soybean

A gas analysis system was built to study the relationship between the reductant cost of NO(3)(-) assimilation and the measured rate of CO(2) and O(2) exchange in roots, leaves, and stems+ petioles of soybean (Glycine max L. Merr. cv Maple glen) plants. The measurements were used to calculate the diverted reductant utilization rate (DRUR = 4*[measured rate of CO(2) + measured rate of O(2)], in moles of high-energy electron [e(-)] per gram per hour) in plants in the presence (N+) and absence (N-) of NO(3)(-). The differences in DRUR between the N+ and N- treatments provided a measure of the NO(3)(-)-coupled DRUR of 25-d-old plants, whereas a (15)NO(3)(-)-enriched nutrient solution was used to obtain an independent measure of the rate of NO(3)(-) assimilation. The measured reductant cost for the whole plant was 9.6 e(-) per N assimilated, a value within the theoretical range of four to 10 e(-) per N assimilated. The results predicted that shoots accounted for about 55% of the whole-plant NO(3)(-) assimilation over the entire day, with shoots dominating in the light, and roots in the dark. The gas analysis approach described here holds promise as a powerful, noninvasive tool to study the regulation of NO(3)(-) assimilation in plant tissue.     

Crystal structures of ethyl 3-azido-2,3-dideoxy-D-arabino-hexopyranoside anomers

The structures of the title alpha (1) and beta (2) anomers of ethyl 3-azido-2,3-dideoxy-d-arabino-exopyranoside (C(8)H(15)N(3)O(4)) are reported. The single-crystal structures of C(8)H(15)N(3)O(4) were determined by X-ray crystallography at 293K. It has been found that both title compounds crystallize in the orthorhombic space group. In both cases, the unit cell contains four asymmetric molecules. From intensity measurements, it has been shown that each of these molecules adopts a (4)C(1) chair conformation. The packing arrangement in the unit cell displays a stratified structure. Moreover, medium strength O-H...O hydrogen bonds in both crystal lattices can be observed.     

Contribution of diazotrophy to nitrogen inputs supporting Karenia brevis blooms in the Gulf of Mexico

Blooms of Karenia brevis plague the West Florida Shelf (WFS) region in the Gulf of Mexico (GOM) where they exert harmful effects on aquatic biota and humans. Because productivity on the WFS is N limited, new N inputs into the region are thought to trigger blooms of K. brevis. Here we examine the potential for new N inputs via N₂ fixation by Trichodesmium and other diazotrophic plankton to contribute to the N demand of K. brevis. Because of possible methodological biases, we also compared N₂ fixation rates by cultured Trichodesmium using the ¹⁵N₂ bubble addition method and the ¹⁵N₂ saturated seawater. Both methods yielded identical results in 12 and 24 h incubations; however, there was more variability in rate estimates made using the bubble addition method. Pelagic N₂ fixation rates by other planktonic diazotrophs ranged from 0 to 13.6 nmol N L⁻¹ d⁻¹, comparable to or higher than rates observed in oligotrophic gyres. These rates should be considered conservative estimates because they were made using the bubble addition method. Integrating over our study area, we estimate that new inputs of N to the WFS via N₂ fixation are on the order of 0.011 Tmol N annually. Further, we measured directly the trophic transfer of recently fixed N₂ to co-occurring plankton that included K. brevis and found that up to 47% of N₂ fixed was transferred to non-diazotrophic plankton even in short (<6 h) incubations where N₂ fixation was likely underestimated.     

Manipulation of the Dissolved Organic Carbon Pool in an Agricultural Stream: Responses in Microbial Community Structure, Denitrification, and Assimilatory Nitrogen Uptake

Carbon (C) and nitrogen (N) are strongly coupled across ecosystems due to stoichiometrically balanced assimilatory demand as well as dissimilatory processes such as denitrification. Microorganisms mediate these biogeochemical cycles, but how microbial communities respond to environmental changes, such as dissolved organic carbon (DOC) availability, and how those responses impact coupled biogeochemical cycles in streams is not clear. We enriched a stream in central Indiana with labile DOC for 5?days to investigate coupled C and N cycling. Before, and on day 5 of the enrichment, we examined assimilatory uptake and denitrification using whole-stream ¹⁵N-nitrate tracer additions and short-term nitrate releases. Concurrently, we measured bacterial and denitrifier abundance and community structure. We predicted N assimilation and denitrification would be stimulated by the addition of labile C and would be mediated by increases in bacterial activity, abundance, and a shift in community structure. In response to the twofold increase in DOC concentrations in the water column, N assimilation increased throughout the enrichment. Community respiration doubled during the enrichment and was associated with a change in bacterial community structure (based on terminal restriction fragment length polymorphisms of the 16S rRNA gene). In contrast, there was little response in denitrification or denitrifier community structure, likely because labile C was assimilated by heterotrophic communities on the stream bed prior to reaching denitrifiers within the sediments. Our results suggest that coupling between C and N in streams involves potentially complex interactions with sediment texture and organic matter, microbial community structure, and possibly indirect biogeochemical pathways.     

The photoproduction of H2 and NH4 fixed from N2 by a derepressed mutant of Rhodospirillum rubrum.

A mutant of Rhodospirillum rubrum has been isolated, after mutagenesis with nitrosoguanidine, which is characterized by its inability to grow in the light on malate-minimal media with exogenous ammonia or alanine, poor growth on glutamine and vigorous growth on glutamate. This mutant produces low levels of a key NH+4 assimilation enzyme, glutamate synthase (NADPH-dependent). It also exhibits significant derepression of nitrogenase biosynthesis in the presence of ammonia or alanine, being 15% derepressed for the former and about 70% derepressed for the latter. Some of this mutant's fixed N2 is excreted into the medium as NH+4 (1 mumol NH+4 per mg cell protein in 50 h). Nitrogenase-mediated H2 production by this strain is considerable (42 mumol H2 per mg cell protein in 50 h), approximately twice that of the wild type assayed under similar conditions. These results demonstrate that genetic alteration of the photosynthetic N2-fixer's NH+4 assimilation system disrupts the tight coupling of N2 fixation and NH+4 assimilation normally observed in these organisms, enabling photochemical conversion steps to be utilized for the photoproduction of NH+4 and H2.     

The Symbiosis between Lophelia pertusa and Eunice norvegica Stimulates Coral Calcification and Worm Assimilation

We investigated the interactions between the cold-water coral Lophelia pertusa and its associated polychaete Eunice norvegica by quantifying carbon (C) and nitrogen (N) budgets of tissue assimilation, food partitioning, calcification and respiration using ¹³C and ¹⁵N enriched algae and zooplankton as food sources. During incubations both species were kept either together or in separate chambers to study the net outcome of their interaction on the above mentioned processes. The stable isotope approach also allowed us to follow metabolically derived tracer C further into the coral skeleton and therefore estimate the effect of the interaction on coral calcification. Results showed that food assimilation by the coral was not significantly elevated in presence of E. norvegica but food assimilation by the polychaete was up to 2 to 4 times higher in the presence of the coral. The corals kept assimilation constant by increasing the consumption of smaller algae particles less favored by the polychaete while the assimilation of Artemia was unaffected by the interaction. Total respiration of tracer C did not differ among incubations, although E. norvegica enhanced coral calcification up to 4 times. These results together with the reported high abundance of E. norvegica in cold-water coral reefs, indicate that the interactions between L. pertusa and E. norvegica can be of high importance for ecosystem functioning.