INTERACTIONS BETWEEN NITRATE UPTAKE AND NITROGEN FIXATION IN CONTINUOUS CULTURES OF THE MARINE DIAZOTROPH TRICHODESMIUM (CYANOBACTERIA)1

Diazotrophic cyanobacteria can take up combined nitrogen (nitrate, ammonium, amino acids, dissolved organic nitrogen) from solution, but the interaction between N₂ fixation and uptake of combined nitrogen is not well understood. We studied the effects of combined nitrogen ) additions on N₂ fixation rates in the cyanobacterium Trichodesmium erythraeum (IMS‐101) maintained in continuous culture in an N‐free medium (YBCII) and a 12:12‐h light:dark cycle. We measured acetylene reduction rates, nutrient concentrations, and biomass throughout the 12 h of illumination after the addition of nitrate (0.5–20 μM) at the start of the light period. Compared with unamended controls, Trichodesmium showed strong inhibition of acetylene reduction (up to 70%) in the presence of , with apparent saturation of the inhibition effect at an initial concentration of approximately 10 μM. The inhibition of acetylene reduction persisted through much of the light period as concentration in the culture vessel decreased. Recovery of N₂ fixation was observed late in the light period in cultures amended with low concentrations of (<5 μM) when ambient concentrations had decreased to 0.3–0.4 μM in the culture vessel. Nitrate uptake accounted for as much as 86% of total N uptake and, at the higher treatment concentrations, more than made up for the observed decrease in N₂ fixation rates. We conclude that Trichodesmium can obtain significant quantities of N through uptake of nitrate and does so in preference to N₂ fixation when sufficient is available.     

THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10^?7 M Fe for 15 days. Chlorophyll-a, cell counts, and total cell volume were two or three times higher in medium with 10^?7 M Fe than with no added Fe. Oxygen production rate per chlorophyll-a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m^?2· s^?1. Nitrogen fixation rate, estimated from acetylene reduction, for 10^?7 and 10^?8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O₂ production and N₂ fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.     

NUTRIENT CONTROLS ON NITROGEN UPTAKE AND METABOLISM BY NATURAL POPULATIONS AND CULTURES OF TRICHODESMIUM (CYANOBACTERIA)

The effects of inorganic nutrient (ammonium [NH₄ ⁺ ] and nitrate [NO₃ ⁻ ]) and amino acid (glutamate [glu] and glutamine [gln]) additions on rates of N₂ fixation, N uptake, glutamine synthetase (GS) activity, and concentrations of intracellular pools of gln and glu were examined in natural and cultured populations of Trichodesmium. Additions of 1 μM glu, gln, NO₃ ⁻ , or NH₄ ⁺ did not affect short-term rates of N₂ fixation. This may be an important factor that allows for continued N₂ fixation in oligotrophic areas where recycling processes are active. N₂ fixation rates decreased when nutrients were supplied at higher concentrations (e.g. 10 μM). Uptake of combined N (NH₄ ⁺ , NO₃ ⁻ , and amino acids) by Trichodesmium was stimulated by increased concentrations. For NO₃ ⁻ , proportional increases in NO₃ ⁻ uptake and decreases in N₂ fixation were observed when additions were made to cultures before the onset of the light period. GS activity did not change much in response to the addition of NH₄ ⁺ , NO₃ ⁻ , glu, or gln. GS is necessary for N metabolism, and the bulk of this enzyme pool may be conserved. Intracellular pools of glu and gln varied in response to 10 μM additions of NH₄ ⁺ , glu, or gln. Cells incubated with NH₄ ⁺ became depleted in intracellular glu and enriched with intracellular gln. The increase in the gln/glu ratio corresponded to a decrease in the rate of N₂ fixation. Although the gln/glu ratio decreased in cells exposed to the amino acids, there was only a corresponding decrease in N₂ fixation after the gln addition. The results presented here suggest that combined N concentrations on the order of 1 μM do not affect rates of N₂ fixation and metabolism, although higher concentrations (e.g. 10 μM) can. Moreover, these effects are exerted through products of NH₄ ⁺ assimilation rather than exogenous N, as has been suggested for other species. These results may help explain how cultures of Trichodesmium are able to simultaneously fix N₂ and take up NH₄ ⁺ and how natural populations continue to fix N₂ once combined N concentrations increase within a bloom.     

DIAZOTROPHIC GROWTH OF THE MARINE CYANOBACTERIUM TRICHODESMIUM IMS101 IN CONTINUOUS CULTURE: EFFECTS OF GROWTH RATE ON N2‐FIXATION RATE,BIOMASS, AND C:N:P STOICHIOMETRY1

Trichodesmium N₂ fixation has been studied for decades in situ and, recently, in controlled laboratory conditions; yet N₂‐fixation rate estimates still vary widely. This variance has made it difficult to accurately estimate the input of new nitrogen (N) by Trichodesmium to the oligotrophic gyres of the world ocean. Field and culture studies demonstrate that trace metal limitation, phosphate availability, the preferential uptake of combined N, light intensity, and temperature may all affect N₂ fixation, but the interactions between growth rate and N₂ fixation have not been well characterized in this marine diazotroph. To determine the effects of growth rate on N₂ fixation, we established phosphorus (P)–limited continuous cultures of Trichodesmium, which we maintained at nine steady‐state growth rates ranging from 0.27 to 0.67 d^?1. As growth rate increased, biomass (measured as particulate N) decreased, and N₂‐fixation rate increased linearly. The carbon to nitrogen ratio (C:N) varied from 5.5 to 6.2, with a mean of 5.8 ± 0.2 (mean ± SD, N = 9), and decreased significantly with growth rate. The N:P ratio varied from 23.4 to 45.9, with a mean of 30.5 ± 6.6 (mean ± SD, N = 9), and remained relatively constant over the range of growth rates studied. Relative constancy of C:N:P ratios suggests a tight coupling between the uptake of these three macronutrients and steady‐state growth across the range of growth rates. Our work demonstrates that growth rate must be considered when planning studies of the effects of environmental factors on N₂ fixation and when modeling the impact of Trichodesmium as a source of new N to oligotrophic regions of the ocean.     

GAS VACUOLE COLLAPSE IN MARINE OSCILLATORIA (TRICHODESMIUM) THIEBAUTII (CYANOPHYTA) AND THE EFFECT ON NITROGENASE ACTIVITY AND PHOTOSYNTHESIS1

Photosynthesis and nitrogenase activity (acetylene reduction) in Oscillatoria (Trichodesmium) thiebautii were significantly reduced after its gas vesicles were collapsed by pressurizing to 6·2 MPa (62 bars). The reduction in nitrogenase activity was observed with both intact and disrupted colonies, and under both aerobic and microaerobic conditions. It is unlikely, therefore, that the reduction in either process results solely from loss of gas diffusion pathways, and may simply originate from disturbance of the cellular architecture as gas vesicles are destroyed. Since nitrogenase activity of the samples was affected by gas vesicle collapse, the process presumably resides in the gas vacuolate organism (Oscillatoria) rather than in any associated bacteria which might be present.     

A REVISED ESTIMATE OF THE IRON USE EFFICIENCY OF NITROGEN FIXATION,WITH SPECIAL REFERENCE TO THE MARINE CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOPHYTA)1

Estimates of the iron use efficiency (IUE) for diazotrophic plant growth have been used to suggest iron limitation of marine N₂ fixation. However, in the course of these inferences, neither the physiological complexity of these estimates nor the specific physiological parameters of marine diazotrophs were evaluated. Here, a semiempirical prediction of the IUE of diazotrophic growth for Trichodesmium was computed from considerations of the Fe content and reaction rates of the nitrogenase complex and PSI:PSII ratios, as well as field measurements of Mehler activity, cellular Fe‐superoxide dismutase activity, and diel variability in C and N₂ fixation. With a PSI:PSII ratio of 1 and 48% Mehler activity, the instantaneous IUE (0.33 mol C fixed·mol cellular Fe ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. ·s ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. ) was only 4‐fold lower than that calculated for a phytoplankter growing on reduced N. We computed a range of daily integrated IUE values from 2900 to 7700 mol C·mol Fe ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. ·d ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. , accounting for the diel variability in C and N₂ fixation as well as the uncertainties in cyanobacterial nitrogenase biochemistry and PSI:II ratios of field‐collected Trichodesmium. The lowest observed Fe‐superoxide dismutase:C quota of 2.9 (μmol:mol) suggests a maintenance requirement for this enzyme. The maintenance Fe:C requirement of 13.5 μmol:mol (derived from cultures of Trichodesmium IMS 101) and values of the IUE yielded an Fe requirement ranging from 27 to 48 Fe:C (μmol:mol) to achieve a diazotrophic growth rate of 0.1 d ^{? 1} 1 Received 16 August 2001. Accepted 7 October 2002. . Based on these predicted requirements, the Fe:C contents of Caribbean Sea and most North Atlantic Ocean populations sampled thus far exceed that required to support the observed rates of N₂ fixation.     

ALKALINE PHOSPHATASE ACTIVITIES AMONG POPULATIONS OF THE COLONY-FORMING DIAZOTROPHIC CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOBACTERIA) IN THE RED SEA

Alkaline phosphatase activities of the diazotrophic marine cyanobacterium Trichodesmium were studied among natural populations in the northern Red Sea and in laboratory cultures of Trichodesmium sp. strain WH9601. Open-water tuft-shaped colonies of Trichodesmium showed high alkaline phosphatase activities with 2.4–11.7 μmol p-nitrophenylphosphate (PNPP) hydrolyzed·μg chl a ^{− 1}·h ^{− 1}, irrespective of date or origin of the sample. Coastal populations of the Trichodesmium tuft colonies had low alkaline phosphatase activities with 0.2–0.5 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. An exception was the Trichodesmium fall maximum, when both tuft colonies and the plankton community (<100 μm) had alkaline phosphatase activities of 0.6–7.4 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. Likewise, the more rare puff and bow-tie colonies of Trichodesmium spp. in coastal waters had elevated alkaline phosphatase activities (0.8–1.6 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}) as compared with tuft colonies coinhabiting the same waters. Intact filaments of tuft-forming Trichodesmium sp. strain WH9601 from phosphate-replete cultures had a base alkaline phosphatase activity of 0.5 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. This activity underwent a 10-fold increase in phosphate-deplete cultures and in cultures supplied with glycerophosphate as the sole P source. The elevated level of alkaline phosphatase activity was sustained in P-deplete cultures, but it declined in cultures with glycerophosphate. The decline is suggested to result from feedback repression of alkaline phosphatase synthesis by the phosphate generated in the glycerophosphate hydrolysis. The enhanced alkaline phosphatase activities of Trichodesmium spp. populations provide evidence that P stress is an important factor in the ecology of Trichodesmium in the northern Red Sea.     

OSCILLATORIA (TRICHODESMIUM) THIEBAUTII (CYANOPHYTA) IN THE CENTRAL NORTH ATLANTIC OCEAN12

Physiological rate measurements were made with Oscillatoria thiebautii (Gom.) Geitler in the subtropical north Atlantic Ocean between Spain and Bermuda during May and June of 1975. The near surface C:N fixation ratios averaged 6.5, and the cellular composition ratio was 6.2, suggesting that N₂ fixation is the major path of nitrogenous nutrition for this alga. Compared to other oceanic phytoplankters, it has a low affinity for orthophosphate at oceanic concentrations (k_s= 9.0); however, it has a high potential for utilizing phosphomonoesters (170–300 ng atoms P ·μg chl a^?1· h^?1). Maximal photosynthesis occurred at 450–700 μ Einstein · m^?2· s^?1, and was inhibited by full sunlight. Calculated cell division rates (ca. 180 days) suggest that relative to other phytoplankters in this oceanic region, O. thiebautii must be subjected to negligible grazing pressure. No major differences in C, N, chl a or ATP were observed between the tuft (fusiform) and puff (spherical) colonies. ATP concentrations relative to other cellular constituents varied greatly between colonies, suggesting a general inter-colony physiological variability in the open Atlantic. With increasing depth in the euphotic zone, there was no evidence for chromatic adaption. The observations that O. thiebautii represents only a small fraction of total phytoplankton biomass and that its growth rate is 10–100 times slower than that of the other indigenous phytoplankton, strongly suggest that N₂ fixation by this alga is a virtually insignificant component of the nitrogenous nutrition for the phytoplankton of the North Atlantic central gyre in late Spring.     

SPATIAL SEGREGATION OF CO2 FIXATION IN TRICHODESMIUM SPP.: LINKAGE TO N2 FIXATION POTENTIAL1

The aggregate-forming, nonheterocystous, filamentous blue-green alga (cyanobacteria) Trichodesmium spp. is a widespread and important planktonic N₂ fixer and primary producer in tropical and subtropical oceans. It is unique among nonheterocystous genera because it conducts N₂ and CO₂ fixation (O₂ evolution) simultaneously; a notable achievement, because O₂ is a potent inhibitor of N₂ fixation. Spatial and temporal CO₂ fixation patterns were examined in trichomes and aggregates from natural and cultured populations, utilizing microautoradiographic detection of ¹⁴CO₂ incorporation. Parallel N₂ fixation (acetylene reduction) measurements were also made. Diel N₂ and CO₂ fixation patterns were similar, with co-optimization of both processes near midday. Microautoradiographs revealed several trichome-level ¹⁴CO₂ incorporation patterns: 1)uniform, heavy labeling, 2)uniform, light labeling, 3) heavier labeling in distal as opposed, to proximal regions, and 4) virtually no labeling throughout. Similar patterns were observed in natural and cultured populations. Given previous immunochemical findings that N₂ fixation potential is widespread in Trichodesmium spp. trichomes and aggregates, current results suggest a high degree of individuality, and possibly a “division of labor” in terms of CO₂ fixation, among trichomes comprising active N₂-fixing aggregates. Segregation of photosynthesis within and among trichomes facilitates simultaneous N₂ and CO₂ fixation in Trichodesmium spp. trichomes and aggregates.     

IRON STIMULATION OF PHOTOSYNTHESIS AND NITROGEN FIXATION IN ANABAENA 7120 AND TRICHODESMIUM (CYANOPHYCEAE)1

Iron availability may limit carbon and nitrogen fixation in the oceans. The freshwater cyanobacterium, Anabaena, was used as a laboratory model for the biochemical and physiological effects of iron. Increased iron nutrition, in the range of 10^?8 M to 10^?6 M resulted in increases of approximately four fold in carbon and nitrogen fixation rates. Chlorophyll concentration increased, and the relative amount of in vivo fluorescence was reduced with more iron. Natural samples of Trichodesmium, collected off Barbados and incubated with increased iron for two days, showed similar effects. Trichodesmium responded to iron additions indicating that it may be Fe limited in its natural environment. These responses to iron are consistent with the biochemical roles of iron in photosynthesis and nitrogen fixation. The results are discussed in the geochemical context of the sporadic total iron input to tropical oceans and possible implications to spatial and temporal patterns of productivity.     

INTERACTIVE EFFECTS OF IRRADIANCE AND CO2 ON CO2 FIXATION AND N2 FIXATION IN THE DIAZOTROPH TRICHODESMIUM ERYTHRAEUM (CYANOBACTERIA)1

The diazotrophic cyanobacteria Trichodesmium spp. contribute approximately half of the known marine dinitrogen (N₂) fixation. Rapidly changing environmental factors such as the rising atmospheric partial pressure of carbon dioxide (pCO₂) and shallower mixed layers (higher light intensities) are likely to affect N₂‐fixation rates in the future ocean. Several studies have documented that N₂ fixation in laboratory cultures of T. erythraeum increased when pCO₂ was doubled from present‐day atmospheric concentrations (～380 ppm) to projected future levels (～750 ppm). We examined the interactive effects of light and pCO₂ on two strains of T. erythraeum Ehrenb. (GBRTRLI101 and IMS101) in laboratory semicontinuous cultures. Elevated pCO₂ stimulated gross N₂‐fixation rates in cultures growing at 38 μmol quanta · m^?2 · s^?1 (GBRTRLI101 and IMS101) and 100 μmol quanta · m^?2 · s^?1 (IMS101), but this effect was reduced in both strains growing at 220 μmol quanta · m^?2 · s^?1. Conversely, CO₂‐fixation rates increased significantly (P < 0.05) in response to high pCO₂ under mid‐ and high irradiances only. These data imply that the stimulatory effect of elevated pCO₂ on CO₂ fixation and N₂ fixation by T. erythraeum is correlated with light. The ratio of gross:net N₂ fixation was also correlated with light and trichome length in IMS101. Our study suggests that elevated pCO₂ may have a strong positive effect on Trichodesmium gross N₂ fixation in intermediate and bottom layers of the euphotic zone, but perhaps not in light‐saturated surface layers. Climate change models must consider the interactive effects of multiple environmental variables on phytoplankton and the biogeochemical cycles they mediate.     

GENOTYPIC RELATIONSHIPS IN TRICHODESMIUM (CYANOPHYCEAE) BASED ON nifH SEQUENCE COMPARISONS1

The DNA sequence of a fragment of nifH was compared to natural populations of the marine cyanobacteria Trichodesmium thiebautii and T. erythraeum from the Caribbean Sea and the unialgal culture Trichodesmium sp. NIBB 1067, which was isolated from the Kuroshio waters (Japan). Through replication Of amplification, cloning, and sequencing, four nucleotides in a 359-bp fragment were identified that were identical in sequence to Trichodesmium sp. NIBB 1067 and natural populations of T. erythraeum but were distinctly different in sequence from T. thiebautii. The data indicate that Trichodesmium sp. NIBB 1067 is more closely related to T. erythraeum than to T. thiebautii.     

DEPRESSION OF MASTIGOCLADUS LAMINOSUS (CYANOPHYTA) NITROGENASE ACTIVITY UNDER NORMAL SUNLIGHT INTENSITIES1

The effect of light intensity (PAR) on the nitrogenase activity of Mastigocladus laminosus Cohn was studied by the acetylene reduction technique. Benthic mat from a thermal stream, Hot River, in Yellowstone National Park was used in both experimental and in situ incubations. This hot spring maintained a mean pH of 7.0, was essentially isothermal (ca. 50°C), and had virtually no upstream to downstream physicochemical gradients (P > 0.05). Two surveys of the stream showed that nitrogenase of the M. laminosus mat was significantly more active (P > 0.02) under low light intensities than under high intensities, 252 and 712 μE · m^?2· s^?1, respectively. Maximum activity of Hot River Mastigocladus (268 nmol C₂H₄· mg Chl a^?1· h^?1) occurred at 50% full midday light intensities; the rates at low light (mean = 247 nmol C₂H₄· mg Chl a^?1· h^?1) were significantly (P > 0.001) greater than those at high light (mean = 106). The results indicate that M. laminosus nitrogenase activity is low light adapted and suggest that the temporal pattern for nitrogen fixation might be significantly different from that of thermophilic Calothrix.     

BACTERIAL ASSOCIATIONS WITH MARINE OSCILLATORIA SP. (TRICHODESMIUM SP.) POPULATIONS: ECOPHYSIOLOGICAL IMPLICATIONS1

On three separate occasions we investigated morphological and physiological aspects of bacterial associations with planktonic aggregates of the ubiquitous marine N₂ fixing cyanobacterium Trichodesmium sp. Close associations generally characterized Trichodesmium blooms; associations were present during day- and night-time. Colonization by both rod-shaped and filamentous heterotrophic bacteria occurred on Trichodesmiun aggregates actively fixing N₂ (acetylene reduction). Scanning electron and optical microscopy showed bacteria located both around and within aggregates. Microautoradiography demonstrated that associated bacteria largely mediated utilization of trace additions of ³H-labeled carbohydrates (fructose, glucose, mannitol) and amino acids, whereas Trichodesmium utilized amino acids only. Oxygen measurements using microelectrodes revealed high localized oxygen consumption among aggregates, with rapid (within a minute) changes from supersaturated to subsaturated oxygen following the transition from photosynthetic illuminated to dark periods. Stab culturing techniques confirmed the presence of heterotrophic N₂ fixers among aggregate-associated bacteria. Parallel deployment of oxygen microelectrodes, the tetrazolium salt 2,3,5 triphenyl tetrazolium chloride (TTC) and acetylene reduction assays demonstrated microaerophilic requirements for expression of nitrogenase activity among cultured bacteria. Trichodesmium aggregates are characterized by dynamic nutrient and oxygen regimes, which promote and maintain simultaneous and contiguous oxygenic photosynthesis and N₂ fixation. In part, the above-mentioned consortial interactions with a variety of heterotrophic bacteria facilitate Trichodesmium biomass production and bloom formation in nitrogen depleted, oligotrophic tropical/subtropical waters.     

NITROGEN FIXATION,HYDROGEN CYCLING,AND ELECTRON TRANSPORT KINETICS IN TRICHODESMIUM ERYTHRAEUM (CYANOBACTERIA) STRAIN IMS1011

This study describes the relationships between dinitrogen (N₂) fixation, dihydrogen (H₂) production, and electron transport associated with photosynthesis and respiration in the marine cyanobacterium Trichodesmium erythraeum Ehrenb. strain IMS101. The ratio of H₂ produced:N₂ fixed (H₂:N₂) was controlled by the light intensity and by the light spectral composition and was affected by the growth irradiance level. For Trichodesmium cells grown at 50 μmol photons · m^?2 · s^?1, the rate of N₂ fixation, as measured by acetylene reduction, saturated at light intensities of 200 μmol photons · m^?2 · s^?1. In contrast, net H₂ production continued to increase with light levels up to 1,000 μmol photons · m^?2 · s^?1. The H₂:N₂ ratios increased monotonically with irradiance, and the variable fluorescence measured using a fast repetition rate fluorometer (FRRF) revealed that this increase was accompanied by a progressive reduction of the plastoquinone (PQ) pool. Additions of 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone (DBMIB), an inhibitor of electron transport from PQ pool to PSI, diminished both N₂ fixation and net H₂ production, while the H₂:N₂ ratio increased with increasing level of PQ pool reduction. In the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), nitrogenase activity declined but could be prolonged by increasing the light intensity and by removing the oxygen supply. These results on the coupling of N₂ fixation and H₂ cycling in Trichodesmium indicate how light intensity and light spectral quality of the open ocean can influence the H₂:N₂ ratio and modulate net H₂ production.     

NITROGENASE CONFINED TO RANDOMLY DISTRIBUTED TRICHOMES IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Nitrogenase reductase (Fe-protein) was detected in the marine planktonic cyanobacterium Trichodesmium. The molecular weight was about 38 kD, as shown by western blotting using anti -Rhodospirillum rubrum nitrogenase reductase antiserum. The enzyme was confined to a limited number (ca. 10–40%) of randomly distributed trichomes in the Trichodesmium colonies, as shown by immunogold localization and transmission electron microscopy. Associated microorganisms had little or no nitrogenase. Nitrogenase showed a diel cycle in localization: present throughout the cytoplasm of cells in N₂-fixing (daytime) colonies but at the periphery of non-N₂-fixing (nighttime) colonies. This structural arrangement of N₂-fixing trichomes and nitrogenase is novel and different from the previously held paradigm for this and other diazotrophic cyanobacteria.     

GROWTH AND CARBON CONTENT OF THREE DIFFERENT‐SIZED DIAZOTROPHIC CYANOBACTERIA OBSERVED IN THE SUBTROPICAL NORTH PACIFIC1

To develop tools for modeling diazotrophic growth in the open ocean, we determined the maximum growth rate and carbon content for three diazotrophic cyanobacteria commonly observed at Station ALOHA (A Long‐term Oligotrophic Habitat Assessment) in the subtropical North Pacific: filamentous nonheterocyst‐forming Trichodesmium and unicellular Groups A and B. Growth‐irradiance responses of Trichodesmium erythraeum Ehrenb. strain IMS101 and Crocosphaera watsonii J. Waterbury strain WH8501 were measured in the laboratory. No significant differences were detected between their fitted parameters (±CI) for maximum growth rate (0.51 ± 0.09 vs. 0.49 ± 0.17 d^?1), half‐light saturation (73 ± 29 vs. 66 ± 37 μmol quanta · m^?2 · s^?1), and photoinhibition (0 and 0.00043 ± 0.00087 [μmol quanta · m^?2 · s^?1]^?1). Maximum growth rates and carbon contents of Trichodesmium and Crocosphaera cultures conformed to published allometric relationships, demonstrating that these relationships apply to oceanic diazotrophic microorganisms. This agreement promoted the use of allometric models to approximate unknown parameters of maximum growth rate (0.77 d^?1) and carbon content (480 fg C · μm^?3) for the uncultivated, unicellular Group A cyanobacteria. The size of Group A was characterized from samples from the North Pacific Ocean using fluorescence‐activated cell sorting and real‐time quantitative PCR techniques. Knowledge of growth and carbon content properties of these organisms facilitates the incorporation of different types of cyanobacteria in modeling efforts aimed at assessing the relative importance of filamentous and unicellular diazotrophs to carbon and nitrogen cycling in the open ocean.     

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA: EVIDENCE FOR A CIRCADIAN RHYTHM1

Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert-Bebout and colleagues and maintained in seawater-based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater-based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.     

COMPARISON OF CULTURED TRICHODESMIUM (CYANOPHYCEAE) WITH SPECIES CHARACTERIZED FROM THE FIELD1

The filamentous, colonial cyanobacterium Trichodesmium has six well‐described species, but many more names. Traditional classification was based on field samples using morphological characteristics such as cell width and length, gas vesicle distribution, and colony morphology. We used the Woods Hole Trichodesmium culture collection to identify 21 cultured strains to species using cell morphology; phycobiliprotein absorption spectra; and sequences of the 16S rRNA gene, the 16S–23S internal transcribed spacer (ITS), and the heterocyst differentiation gene hetR. We compared our results to previous studies of field specimens and found similar clades, though not all phylogenetic groups were represented in culture. Our culture collection represented two of the four major clades of Trichodesmium: clade I, made up of Trichodesmium thiebautii Gomont, Trichodesmium tenue Wille, Katagnymene spiralis Lemmerm., and Trichodesmium hildebrandtii Gomont; and clade III, consisting of Trichodesmium erythraeum Ehrenb. and Trichodesmium contortum Wille. These clades were genetically coherent with similar phycobiliprotein composition, but morphologically diverse. In the continual revision of cyanobacterial taxonomy, genetic and biochemical information is useful and informative complements to morphology for the development of a functional classification scheme.     

CYTOMORPHOLOGICAL CHARACTERIZATION OF THE PLANKTONIC DIAZOTROPHIC CYANOBACTERIA TRICHODESMIUM SPP. FROM THE INDIAN OCEAN AND CARIBBEAN AND SARGASSO SEAS1

Trichodesmium Ehrenberg species were collected in the Caribbean Sea, Sargasso Sea, and coastal areas of Tanzania (Indian Ocean). The specimens were divided into five species on the basis of morphometric characters such as cell dimensions and colony formation: T. tenue Wille, T. erythraeum Ehrenberg, T. thiebautii Gomont, T. hildebrandtii Gomont, and T. contortum Wille. In addition, Trichodesmium sp., a spherical colony of uncertain taxonomic position was examined. The cell structure of each species was investigated by means of light, scanning electron, and transvnission electron microscopy. Particular attention was paid to the presence and ultrastructural arrangement of gas vacuoles and glycogen fiber clusters (GFCs). This resulted in identification of two major groups of species: 1) T. tenue, Trichodesmium sp. with spherical-shaped colonies, and T. erythraeum with GFCs and more or less localized gas vacuoles; and 2) T. thiebautii, T. hildebrandtii, and T. contortum lacking GFCs and with gas vacuoles spread at random. The species within each group were further characterized with respect to the dimension of the gas vesicles, cylindrical bodies, scroll bodies, and a new cellular inclusion body, Differences in colony formation and cell dimensions correlated with specific ultrastructural characters in five of the six forms. This is the first ultrastructural study comparing different forms of Trichodesmium sampled at geographically remote areas and shows that one species appears identical regardless of the sampling site. Some of the species had not been investigated earlier, and probably more species are to be identified and analyzed.