Heterotrophic Carbon Dioxide Fixation Products of Euglena: EFFECTS OF AMMONIUM

The metabolic products of heterotrophic (dark) CO₂ fixation by Euglena gracilis Klebs strain Z Pringsheim were separated and identified. They consisted of amino acids, phosphorylated compounds, tricarboxylic acid cycle intermediates, and nucleotides. Exposure of the cells to NH₄⁺ after a period of NH₄⁺ deprivation stimulated heterotrophic CO₂ fixation almost 4-fold, modifying the spectrum of the fixation products. In particular, the NH₄⁺ treatment stimulated fixation of CO₂ into glutamine, glycine, alanine, and serine.     

EFFECTS OF NH4+ ASSIMILATION ON DARK CARBON FIXATION AND β-1,3-GLUCAN METABOLISM IN THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

The effects of NH₄⁺ assimilation on dark carbon fixation and β-1,3-glucan metabolism in the N-limited marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) were investigated by chemical analysis of cell components and incorporation of ¹⁴C-bicarbonate. The diatom was grown in pH-regulated batch cultures with a 14:10 h LD cycle until N depletion. The cells were then incubated in the dark with ¹⁴C-bicarbonate, but without a source of N for 2 h, then in the dark with 63 μmol·L⁻¹ NH₄⁺ for 3 h. Without N, the cellular concentration of free amino acids was almost constant (∼4.5 fmol·cell⁻¹). Added NH₄⁺ was assimilated at a rate of 12 fmol·cell⁻¹·h⁻¹, and the cellular amino acid pool increased rapidly (doubled in <1 h, tripled in <3 h). The glutamine level increased steeply (45× within 3 h), and the Gln/ Glu ratio increased from 0.1 to 2.4 within 3 h. The rate of dark C fixation during N depletion was only 1.0 fmol·cell⁻¹·h⁻¹. The addition of NH₄⁺ strongly stimulated dark C fixation, leading to an assimilation rate of 4.0 fmol·cell⁻¹·h⁻¹, corresponding to a molar C/N uptake ratio of 0.33. Biochemical fractionation of organic ¹⁴C showed no significant ¹⁴C fixation into amino acids during N depletion, but during the first 1–2 h of NH₄⁺ assimilation, amino acids were rapidly radiolabeled, accounting for virtually all net ¹⁴C fixation. These results indicate that anaplerotic β-carboxylation is activated during NH₄⁺ assimilation to provide C₄ intermediates for amino acid biosynthesis. The level of cellular β-1,3-d-glucan was constant (16.5 pg·cell⁻¹) during N depletion, but NH₄⁺ assimilation activated a mobilization of 28% of the reserve glucan within 3 h. The results indicate that β-1,3-glucan in diatoms is the ultimate substrate for β-carboxylation, providing precursors for amino acid biosynthesis in addition to energy from respiration.     

Inorganic carbon uptake by phytoplankton in Vineyard Sound,Massachusetts. II. Comparative primary productivity and nutritional status of winter and summer assemblages

Using time-course, natural-light incubations, we assessed the rate of carbon uptake at a range of light intensities, the effect of supplemental additions of nitrogen (as NH₄⁺ or urea) on light and dark carbon uptake, and the rates of uptake of NH₄⁺ and urea by phytoplankton from Vineyard Sound, Massachusetts from February through August 1982. During the winter, photoinhibition was severe, becoming manifested shortly after the start of an incubation, whereas during the summer, there was little to no evidence of photoinhibition during the first several hours after the start of an incubation. At light levels which were neither photoinhibiting nor light limiting, rates of carbon uptake normalized per liter were high and approximately equal during winter and summer (22–23 μg C·l^?1 · h^?1), and low during spring (<10 μgC·l^?1· h^?1). In contrast, on a chlorophyll a basis, rates of carbon fixation were as high during spring (15–20μg C·μg Chl a^?1·h^?1), when concentrations of chlorophyll a were at the yearly minimum (<0.5 μg · l^?1) as during the summer, when chlorophyll a concentrations were substantially higher (0.8–1.3 μg · l^?1). Highest rates of NH₄⁺ and urea uptake were observed during summer, and at no time of the year was there evidence for severe nitrogen deficiency, although moderate nitrogen nutritional stress was apparent during the summer months.     

Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle : Evidence for Partial Oxidative and Reductive Pathways during Dark Ammonium Assimilation

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH₄⁺ in the dark under anaerobic conditions. Addition of NH₄⁺ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO₂ efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H¹⁴CO₃⁻ to anaerobic cells assimilating NH₄⁺ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH₄⁺ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH₄⁺ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH₄⁺ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH₄⁺ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.     

Regulation of non-autotrophic carbon dioxide assimilation by ammonia in cultured cells of Acer pseudoplatanus L

The assimilation of ¹⁴CO₂ by Acer pseudoplatanus cells in the dark was stimulated by the addition of either NH₄Cl or methylamine. Results were obtained demonstrating that methylamine was not metabolised to any appreciable extent by Acer cells. This suggests that the mechanism of stimulation of dark fixation by methylamine does not involve metabolism via the glutamine synthetase reaction. NH₄⁺ stimulation of CO₂ fixation also occurred in cells pretreated with the glutamine-synthetase inhibitor methionine sulfoximine. This further supports the conclusion that neither NH₄⁺ nor methylamine exerts its effect on CO₂-assimilation via a mechanism that depends upon the assimilation of NH₃ by glutamine synthetase.     

Effects of host nutritional status and seasonality on the nitrogen status of zooxanthellae in the temperate coral Plesiastrea versipora (Lamarck)

The nitrogen status of endosymbiotic dinoflagellates (zooxanthellae) in the temperate coral Plesiastrea versipora (Lamarck) was determined by measuring the extent to which ammonium (40 μM NH₄⁺) enhanced the rate of zooxanthellar dark carbon fixation above that seen in filtered seawater (FSW) alone; the enhancement ratio was expressed as [dark NH₄⁺ rate/dark FSW rate]. V_D′/V_L, a further index of nitrogen status, was also calculated where V_D′ = [dark NH₄⁺ rate − dark FSW rate] and V_L = rate of carbon fixation in the light. When corals were starved for 2-8 weeks, zooxanthellar nitrogen deficiency became apparent at ≥ 4 weeks, with NH₄⁺/FSW and V_D′/V_L averaging up to 2.08 and 0.0061, respectively. A decrease in light-saturated photosynthesis per zooxanthella also occurred, with the photosynthetic rate after 4-6 weeks being just 81% of that seen prior to starvation. In comparison, when corals were fed 5 times per week for 8 weeks the addition of ammonium had little effect, indicating nitrogen sufficiency; NH₄⁺/FSW and V_D′/V_L were 1.03 and 0.0003, respectively. Photosynthetic rates of zooxanthellae from well-fed corals were up to 1.7 times greater than those of zooxanthellae from starved corals. The nitrogen status of zooxanthellae from corals in the field exhibited seasonal differences. Autumn samples were nitrogen sufficient, with NH₄⁺/FSW = 1.003 and V_D′/V_L = 0.0001. In contrast, a small degree of nitrogen deficiency was seen in winter and spring, when NH₄⁺/FSW averaged 1.075 and 1.249, and V_D′/V_L averaged 0.0013 and 0.0014, respectively. The greatest degree of nitrogen deficiency was observed in summer, when NH₄⁺/FSW averaged 1.318 and V_D′/V_L averaged 0.0036. Given the clear links between food supply and nitrogen status seen under experimental conditions, and the likelihood that the zooxanthellae are also able to take up nutrients directly from the seawater, the fluctuations in nitrogen status may reflect temporal fluctuations in seawater nutrient concentrations and plankton abundance. The nutrient status of these temperate zooxanthellae in the field is in contrast to the marked nitrogen deficiency seen in zooxanthellae from nutrient-poor coral reef waters, and raises the possibility that temperate zooxanthellae can store nitrogen for use when exogenous nutrients and food are less readily available. This, in turn, may contribute to the considerable stability of temperate zooxanthellar populations under highly variable environmental conditions.     

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO₃⁻ or NH₄⁺ induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH₄⁺ addition resulted in O₂ consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO₃⁻ or NH₄⁺ resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O₂ evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.     

Regulation by Ammonium of Variation in Heterotrophic CO2 Fixation by Euglena gracilis During Growth Cycles*

SYNOPSIS Heterotrophic (dark) CO₂ fixation by Euglena gracilis strain Z varies with phase of batch culture growth and mode of nutrition. Increases in the fixation during growth cycles correlate closely with the depletion of exogenous NH₄* from the medium during growth. It is demonstrated that exogenous NH₄⁺ regulates a component of heterotrophic CO₂ fixation and that another component is independent of NH₄⁺. This is true for cells grown heterotrophically (glucose, dark), autotrophically (CO₂, light) and for a permanently bleached strain (E. gracilis SB3). Some kinetics of the NH₄⁺ regulation are presented.     

Phytoplankton nutrient deficiency in lakes of the McMurdo dry valleys, Antarctica

1. The influence of inorganic nitrogen and phosphorus enrichment on phytoplankton photosynthesis was investigated in Lakes Bonney (east and west lobes), Hoare, Fryxell and Vanda, which lie in the ablation valleys adjacent to McMurdo Sound, Antarctica. Bioassay experiments were conducted during the austral summer on phytoplankton populations just beneath the permanent ice cover in all lakes and on populations forming deep-chlorophyll maxima in the east and west lobes of Lake Bonney. 2. Phytoplankton photosynthesis in surface and mid-depth (13 m) samples from both lobes of Lake Bonney were stimulated significantly (P < 0.01) by phosphorus enrichment (2 μM) with further stimulation by simultaneous phosphorus plus NH₄⁺ (20 μM) enrichment. Similar trends were observed in deeper waters (18 m) from the east lobe of Lake Bonney, although they were not statistically significant at P < 0.05. Photosynthesis in this lake was never enhanced by the addition of 20 μM NH₄⁺ alone. Simultaneous addition of phosphorus plus nitrogen stimulated photosynthesis significantly (P < 0.01) in both Lake Hoare and Lake Fryxell. No nutrient response occurred in Lake Vanda, where activity in nutrient-enriched samples was below unamended controls; results from Lake Vanda are suspect owing to excessively long sample storage in the field resulting from logistic constraints. 3. Ambient dissolved inorganic nitrogen (DIN) (NH4⁺+ NO₂^?+ NO₃^?): soluble reactive phosphorus (SRP) ratios partially support results from bioassay experiments indicating strong phosphorus deficiency in Lake Bonney and nitrogen deficiency in Lakes Hoare and Fryxell. DIN : SRP ratios also imply phosphorus deficiency in Lake Vanda, although not as strong as in Lake Bonney. Particulate carbon (PC): particulate nitrogen (PN) ratios all exceed published ratios for balanced phytoplankton growth, indicative of nitrogen deficiency. 4. Vertical nutrient profiles in concert with low advective flux, indicate that new (sensu Dugdale & Goering, 1967) phytoplankton production in these lakes is supported by upward diffusion of nutrients from deep nutrient pools. This contention was tested by computing upward DIN : SRP flux ratios across horizontal planes located immediately beneath each chlorophyll maximum and about 2 m beneath the ice (to examine flux to the phytoplankton immediately below the ice cover). These flux ratios further corroborated nutrient bioassay results and bulk DIN : SRP ratios indicating phosphorus deficiency in Lakes Bonney and Vanda and potential nitrogen deficiency in Lakes Hoare and Fryxell. 5. Neither biochemical reactions nor physical processes appear to be responsible for differences in nutrient deficiency among the study lakes. The differences may instead be related to conditions which existed before or during the evolution of the lakes.     

Effects of Methane Metabolism on Nitrification and Nitrous Oxide Production in Polluted Freshwater Sediment

We report the effect of CH₄ and of CH₄ oxidation on nitrification in freshwater sediment from Hamilton Harbour, Ontario, Canada, a highly polluted ecosystem. Aerobic slurry experiments showed a high potential for aerobic N₂O production in some sites. It was suppressed by C₂H₂, correlated to NO₃^- production, and stimulated by NH₄⁺ concentration, supporting the hypothesis of a nitrification-dependent source for this N₂O production. Diluted sediment slurries supplemented with CH₄ (1 to 24 μM) showed earlier and enhanced nitrification and N₂O production compared with unsupplemented slurries (≤1 μM CH₄). This suggests that nitrification by methanotrophs may be significant in freshwater sediment under certain conditions. Suppression of nitrification was observed at CH₄ concentrations of 84 μM and greater, possibly through competition for O₂ between methanotrophs and NH₄⁺ -oxidizing bacteria and/or competition for mineral N between these two groups of organisms. In Hamilton Harbour sediment, the very high CH₄ concentrations (1.02 to 6.83 mM) which exist would probably suppress nitrification and favor NH₄⁺ accumulation in the pore water. Indeed, NH₄⁺ concentrations in Hamilton Harbour sediment are higher than those found in other lakes. We conclude that the impact of CH₄ metabolism on N cycling processes in freshwater ecosystems should be given more attention.     

Influence of phytoplankton on the response of bacterioplankton growth to nutrient enrichment

1. Laboratory experiments were conducted to test the effect of nutrient enrichment on bacterioplankton growth in the presence and absence of phytoplankton. 2. In one series of experiments, bacterioplankton growth in terms of specific activity [³H-thymidine incorporation (cell number)^?1] was greater in whole lake water samples than in samples from which phytoplankton had been removed by filtration (1.0 μm), regardless of the nutrient enrichments (control, NH⁺₄ plus PO^3-₄ and mannitol). Organic C enhanced bacterioplankton growth in both whole and filtered lake water. 3. In another series of experiments (with the same nutrient enrichments as in the first experiment except that glucose replaced mannitol), bacterioplankton growth in whole lake water enriched with PO^3-₄ plus NH⁺₄ and incubated in the light was greater than in two treatments designed to inhibit photosynthetic activity (+DCMU and dark). Bacterioplankton response to nutrient addition was greatest in the PO^3-₄ plus NH⁺₄ enrichment under all three conditions (light +DCMU, and dark). 4. These results indicate that bacterioplankton growth could be directly limited by inorganic P and N when these elements are in short supply. Enhancement of bacterioplankton growth by phytoplankton occurs only under PO^3-₄ and NH⁺₄ replete environments.     

Responses of Two Coastal Algae (Skeletonema costatum and Chlorella vulgaris) to Changes in Light and Iron Levels1

Iron (Fe) is essential for phytoplankton growth and photosynthesis, and is proposed to be an important factor regulating algal blooms under replete major nutrients in coastal environments. Here, Skeletonema costatum, a typical red-tide diatom species, and Chlorella vulgaris, a widely distributed Chlorella, were chosen to examine carbon fixation and Fe uptake by coastal algae under dark and light conditions with different Fe levels. The cellular carbon fixation and intracellular Fe uptake were measured via ¹⁴C and ⁵⁵Fe tracer assay, respectively. Cell growth, cell size, and chlorophyll-α concentration were measured to investigate the algal physiological variation in different treatments. Our results showed that cellular Fe uptake proceeds under dark and the uptake rates were comparable to or even higher than those in the light for both algal species. Fe requirements per unit carbon fixation were also higher in the dark resulting in higher Fe: C ratios. During the experimental period, high Fe addition significantly enhanced cellular carbon fixation and Fe uptake. Compared to C. vulgaris, S. costatum was the common dominant bloom species because of its lower Fe demand but higher Fe uptake rate. This study provides some of the first measurements of Fe quotas in coastal phytoplankton cells, and implies that light and Fe concentrations may influence the phytoplankton community succession when blooms occur in coastal ecosystems.     

Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.     

MOBILIZATION OF β-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

Mobilization of the reserve β-1,3-glucan (chrysolaminaran) in N-limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH-regulated batch cultures with a 14:10-h light:dark cycle until N depletion. In a pulse-chase experiment, the cells were first incubated in high light (200 μmol photons·m ^{− 2}·s ^{− 1}) with ¹⁴C-bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m ^{− 2}·s ^{− 1}) with additions of 40 μM NH₄ ⁺ . In the ¹⁴C pulse phase with high light and N depletion, β-1,3-glucan accumulated and accounted for 85% of incorporated ¹⁴C. In the subsequent ¹⁴C chase phases, added NH₄ ⁺ was assimilated at an N-specific rate of 0.11 h ^{− 1} in both the dark and low light, and in both cases it caused a significant mobilization of β-1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic ¹⁴C showed that free amino acids were most rapidly labeled in the early stage of NH₄ ⁺ assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH₄ ⁺ was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N-limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH₄ ⁺ under inorganic carbon limitation. Added NH₄ ⁺ was assimilated at an N-specific rate of 0.023 h ^{− 1}, and cellular β-1,3-glucan decreased by 15% within 6 h. Hence, β-1,3-glucan was mobilized during NH₄ ⁺ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β-1,3-glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation.     

THE PHYSIOLOGICAL ECOLOGY OF A FRESHWATER DINOFLAGELLATE BLOOM POPULATION: VERTICAL MIGRATION,NITROGEN LIMITATION,AND NUTRIENT UPTAKE KINETICS1,2

The motile freshwater dinoflagellate Gymnodinium bogoriense Klebs., which forms dense blooms in Jezre'el Valley water reservoirs (Israel) appears to be physiologically suited to exploit stratified environments, where it outcompetes all other phytoplankton types. The dense summer blooms (“red tides”) were found to be nitrogen-limited. The algae's competitive advantage, however, cannot result from superior uptake capabilities: its K_s (μmol NH₄·L^?1) for NH₄ was higher and its V_maxμmol NH₄·mg chlorophyll a^?1·h^?1) was lower than other phytoplankton types commonly occurring in the region. The competitive advantage of G. bogoriense probably stems from other physiological capabilities: dark ammonia and phosphorus assimilation and the ability to undertake diel vertical migration cycles between the upper photic water layers during the day and nutrient-rich deeper layers at night. These findings confirm the vertical nutrient retrieval hypothesis in migrating phytoplankton.     

Role of nitrification and denitrification for NO metabolism in soil

Release and uptake of NO was measured in a slightly alkaline (pH 7.8) and an acidic (pH 4.7) cambisol. In the alkaline soil under aerobic conditions, NO release was stimulated by ammonium and inhibited by nitrapyrin. Nitrate accumulated simultaneously and was also inhibited by nitrapyrin.¹⁵NO was released after fertilization with¹⁵NH₄NO₃ but not with NH₄ ¹⁵NO₃. The results indicate that in aerobic alkaline cambisol NO was mainly produced during nitrification of ammonium. The results were different under anaerobic conditions and also in the acidic cambisol. There, NO release was stimulated by nitrate and not by ammonium, and was inhibited by chlorate and not by nitrapyrin indicating that NO production was exclusively due to reduction of nitrate. The results were confirmed by¹⁵NO being released mainly from NH₄ ¹⁵NO₃ rather than from¹⁵NH₄NO₃. The observed patterns of NO release were explained by the NO production processes being stimulated by either ammonium or nitrate in the two different soils, whereas the NO consumption processes being only stimulated by nitrate. NO release was larger than N₂O release, but both were small compared to changes in concentrations of soil ammonium or nitrate.(*request for offprints)     

Effects of carbon dioxide and oxygen on the regulation of photosynthetic carbon metabolism by ammonia in spinach mesophyll cells

Photosynthetic carbon metabolism of isolated spinach mesophyll cells was characterized under conditions favoring photorespiratory (PR; 0.04% CO₂ and 20% O₂) and nonphotorespiratory (NPR; 0.2% CO₂ and 2% O₂) metabolism, as well as intermediate conditions. Comparisons were made between the metabolic effects of extracellularly supplied NH₄⁺ and intracellular NH₄⁺, produced primarily via PR metabolism. The metabolic effects of ¹⁴CO₂ fixation under PR conditions were similar to perturbations of photosynthetic metabolism brought about by externally supplied NH₄⁺; both increased labeling and intracellular concentrations of glutamine at the expense of glutamate and increased anaplerotic synthesis through α-ketoglutarate. The metabolic effects of added NH₄⁺ during NPR fixation were greater than those during PR fixation, presumably due to lower initial NH₄⁺ levels during NPR fixation. During PR fixation, addition of ammonia caused decreased pools and labeling of glutamate and serine and increased glycolate, glyoxylate, and glycine labeling. The glycolate pathway was thus affected by increased rates of carbon flow and decreased glutamate availability for glyoxylate transamination, resulting in increased usage of serine for transamination. Sucrose labeling decreased with NH₄⁺ addition only during PR fixation, suggesting that higher photosynthetic rates under NPR conditions can accommodate the increased drain of carbon toward amino acid synthesis while maintaining sucrose synthesis.     

Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH ₄ ⁺ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH ₄ ⁺ and prevented the growth of algae by rapidly assimilating all NH ₄ ⁺ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH ₄ ⁺ , thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.     

Free Ammonia Inhibition of Algal Photosynthesis in Intensive Cultures

The effect of free NH₃ inhibition on short-term photosynthesis was investigated in three microalgal species: the freshwater chlorophyte Scenedesmus obliquus, the marine diatom Phaeodactylum tricornutum and the marine chlorophyte Dunaliella tertiolecta. By performing a series of assays at various concentrations of added NH₄Cl and culture pH, we demonstrated that the inhibitory compound was free NH₃ and that pH played no role in determining the magnitude of inhibition, other than in establishing the degree of dissociation of nontoxic NH₄⁺ to toxic NH₃. When corrections were made for pH, all three species displayed the same sigmoidal response curve to free NH₃ concentration; 1.2 mM NH₃ led to 50% reduction in photoassimilation of ¹⁴C. Based on literature values, some marine phytoplankton appear to be significantly more sensitive to free NH₃ than were the test species, which are noted for their excellent growth characteristics. However, the combination of low algal biomass and strong pH buffering commonly found in most marine and many freshwater environments probably limits the possibilities for NH₃ toxicity to low alkalinity freshwaters and intensive algal cultures in which NH₄⁺ is the main source of N. Such conditions occur commonly in algal wastewater treatment systems.     

Length of Incubation for Enumerating Nitrifying Bacteria Present in Various Environments

The effect of incubation time on most-probable-number estimates of autotrophic nitrifying bacteria was investigated by using waters, rooted aquatic plants, sediments, and slimes as inoculum sources. Maximum most probable numbers of the NH₄⁺-oxidizing group were attained in 20 to 55 days (median, 25). Estimates of NO₂^- oxidizers were highest at termination (103 to 113) days.