SPECIFIC ASSOCIATIONS OF THE BLUEGREEN ALGAE ANABAENA AND APHANIZOMENON WITH BACTERIA IN FRESHWATER BLOOMS1

Scanning electron micrographs and autoradiographs of Anabaena circinalis Rabenh. and Aphanizomenon flos-aquae (L.) Ralfs in samples from fresh-water communities show that bacteria are attached specifically at the polar region of heterocysts of these known N₂ fixers. This algal-bacterial association occurs most frequently during bloom conditions. The possible roles of this association in maintaining nuisance bloom conditions are discussed.     

OCCURRENCE OF TRI-LAMELLAR BODIES IN ISOLATES OF NOSTOC (CYANOPHYCEAE)1

Tri-lamellar bodies were observed in eight of 29 isolates of Nostoc examined. They appeared identical to the previously described bodies in various species of Anabaena. The bodies consist of three discoid lamellae each ca. 0.3 μm diam and 8 nm thick. The outer lamella (closest to the plasma membrane) is separated from the middle lamella by a 12 nm space whereas the middle and inner lamellae are ca. 8 nm apart. Osmiophilic striations 3 nm wide were generally observed running between the lamellae. Osmiophilic β granules were usually associated with the inner lamella. The bodies were most always located close to the plasma membrane along the longitudinal wall near the junction of the cross and longitudinal walls. In three isolates the bodies located near the cross walls were associated with gas vesicles and possessed a slightly different morphology. These tri-lamellar bodies consisted of three discoid lamellae, each ca. 2 nm thick, ca. 25 nm apart with electron dense material between the inner and middle lamellae. Pores 20 nm diam and ca. 60 nm apart were observed in layer 2 of the cell wall adjacent to the tri-lamellar bodies. These wall pores were also observed in isolates lacking tri-lamellar bodies.     

STORAGE OF PHOSPHORUS IN NITROGEN-FIXING ANABAENA FLOS-AQUAE (CYANOPHYCEAE)1

P accumulation and metabolic pathway in N₂-fixing Anabaena flos-aquae (Lyngb.) Bréb were investigated in P-sufficient (20 μMP) and P-limited (2 μMP) turbidostats in combined N-free medium. The cyanobacterium grew at its maximum rate (μ_max, 1.13 d^?1) at the high P concentration and at 65% of μ_max under P limitation, with total cell P concentrations (Q_P) at steady states of 12.0 and 5.2 fmol·cell^?1, respectively. At steady state, polyphosphates (PP_i) accounted for only 3% of Q_P (0.4 fmol·cell^?1) in P-rich cells. Its concentration in P-limited cells was 5.8% (0.3 fmol·cell^?1). On the other hand, sugar P was very high at 22% of Q_P in P-rich cells and was undetectable in P-limited cells. Pulse chase experiments with ³²P showed that P-rich cells initially incorporated the labeled P into the acid-soluble PP_i fraction within the first few minutes and to a lesser extent into nucleotide P. Radioactivity in the PP_i then declined rapidly with concomitant increases in sugar P and nucleotide P fractions. In contrast, in P-limited cells, no radiolabel was detected in acid-soluble PP_i, and ³²P was initially incorporated into nucleotide P, sugar P, and ortho P fractions. The latter two fractions then subsequently declined. Therefore, under N₂-fixing conditions the cyanobacteria appeared to store P as sugar P and also utilize P through different pathways under P-rich and -limited conditions. When nitrate was supplied as the N source under P-sufficient conditions, PP_i accounted for about 15% of steady-state Q_P, but no sugar P was detected. Therefore, the same organism stored P in different cell P fractions depending on its N sources.     

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.     

TRANSFER OF N2 AND CO2 FIXATION PRODUCTS FROM ANABAENA OSCILLARIOIDES TO ASSOCIATED BACTERIA DURING INORGANIC CARBON SUFFICIENCY AND DEFICIENCY1

Transfer of N₂ and CO₂ fixation products from the bloom forming blue-green alga, Anabaena oscillarioides Bory, to attached and free swimming bacteria is common during active growth of the former. Incubation with ¹⁵N₂ and ¹⁴CO₂ followed by size fractionation filtration reveals that: i) magnitudes of fixed N and C excretion, relative to N₂ and CO₂ fixation, are dictated by dissolved inorganic carbon (DIC) availability for A. oscillarioides photosynthetic production, ii) associated bacteria exhibit preferences for recently fixed excreted N compounds, iii) bacterial utilization of excreted N is independent of ambient light conditions, and iv) lag times between N₂ fixation and detectable bacterial assimilation of excreted fixed N compounds are ca. 1–2 h. Both ¹⁴NH₄Cl dilution and Hg(NH₃)₂ Cl₂ precipitation techniques indicate that NH₃ is a major excretion product from A. oscillarioides, particularly during DIC limited growth. Active N and C excretion and transfer to associated bacteria are features of viable A. oscillarioides filaments. Hence, transfer of these metabolites reflects complex mutualistic, and possibly symbiotic associations rather than solely signaling senescence.     

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.     

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.     

INTERACTION OF PLECTONEMA BORYANUM (CYANOPHYCEAE) AND THE LPP-CYANOPHAGES IN CONTINUOUS CULTURE1

Continuous culture techniques are used to study long-term population interactions between Plectonema boryanum Gomont, a filamentous bluegreen alga, and the LPP-viruses which infect it. After LPP-I (virulent cyanophage) infection of sensitive algae, 3 oscillations occur in cell density with concomitant oscillations in virus titer before final stabilization of both algal and viral concentrations. After LPP-ID and LPP-2 (temperate viruses) infection, oscillation in cell density occurred with burst of virus particles. Resistant algae always repopulated the chemostat; lysogeny was not established. The interaction between Plectonema that was resistant to virus infection and the 3 LPP-cyanophages resulted in rapid elimination of the viruses from the chemostat in the effluent. When lysogenic P. boryanum was tested, a law population of virus was present in the chemostat throughout the incubation period indicative of spontancous induction. Clones of lysogenic algae were isolated.     

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.     

THE MECHANISMS OF FLUORIDE TOXICITY AND FLUORIDE RESISTANCE IN SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYCEAE)1

Fluoride was supplied as dissolved NaF at concentrations ranging from 0.26 to 7.9 mM (5–150 ppm) to three freshwater microalgae: Synechococcus leopoliensis (Racib.) Komarek (Cyanophyta), Oscillatoria limnetica Lemmermann (Cyanophyta) and Chlorella pyrenoidosa Chick (Chlorophyta). Growth of C. pyrenoidosa was unaffected by fluoride, and uptake of fluoride by this organism was not detectable. Growth of the cyanophytes was temporarily inhibited by NaF. The duration of this growth lag increased markedly as the pH was lowered at constant external fluoride concentration. In S. leopoliensis, fluoride uptake and inhibition of photosynthesis by NaF increased in the same way as did the growth lag in response to pH. Growth-inhibitory NaF treatments decreased the ATP level in cells of S. leopoliensis by 75% and also abolished phosphate uptake. Cells of S. leopoliensis in which fluoride-resistance was induced by prior growth in non-growth-inhibitory levels of NaF accumulated much less fluoride than did normal (“sensitive”) cells, and also did not respond to fluride by reduction of the ATP pool. It is suggested (1) that fluoride enters sensitive cells of S. leopoliensis principally as undissociated HF; (2) that its major inhibitory effect in these cells is the reduction in cellular ATP; (3)that fluoride-resistant cells accumulate less fluoride by developing incresed permeability to the fluoride anion.     

ON SPECTRAL CONTROL OF PIGMENTATION IN ANACYSTIS NIDULANS (CYANOPHYCEAE)1,2

Growth of Anacystis nidulans (Richt.) Drouet & Daily in wavelengths of light predominantly absorbed by chlorophyll a causes a dramatic lowering in chlorophyll content and in the chlorophylllphycocyanin ratio. A limit to the effect is reached in far red (680 nm) light where the ratio chlorophylllphycobilinogen falls to <0.3. A special water bath for culture tubes was illuminated by tungsten-halogen lamps through far red (>650 nm)filters; it gave the same extreme pigment ratio together with a high specific growth rate of 2.5 day^-1. Compared with normally pigmented cells there are three features which accompany the low-chlorophyll condition of far red light. At 677 nm relative quantum efficiency increases but action decreases. Synthesis of total pigment and total cell material in far red light also decreases. These observations suggest that the low chlorophyll response to far red light reflects an incompetence rather than an adaptation.     

INTERACTIONS BETWEEN LIGHT,NH4+, AND CO2 IN BUOYANCY REGULATION OF ANABAENA FLOS-AQUAE (CYANOPHYCEAE)1

Buoyancy of the gas-vacuolate alga Anabaena flosaquae Brébisson was measured under various levels of light, NH₄⁺, and CO₂. At high irradiance (50 μE · m^?2·^?1) the alga was non-buoyant regardless of the availability of CO₂ and NH₄⁺. At low irradiance (≤10 μE · m ^?2· s^?1) buoyancy was controlled by the availability of NH₄⁺ and CO₂. When NH₄⁺ was abundant, algal buoyancy was high over a wide range of CO₂ concentrations. In the absence of NH₄⁺, algal buoyancy was reduced at high CO₂ concentrations, however as the CO₂ concentration declined below about 5 μmol · L^?1, algal buoyancy increased. These results help explain why gas vacuolate, nitrogen-fixing blue-green algae often form surface blooms in eutrophic lakes.     

GROWTH RESPONSE OF A NITROGEN FIXER (ANABAENA FLOS-AQUAE,CYANOPHYCEAE) TO LOW NITRATE1

Anabaena flos-aquae (Lyngb.) Bréb. was grown in varying concentrations of nitrate. Specific growth rates, as estimated in batch culture, were constant and approached the maximum rate at all concentrations of NO₃^?-N tested bewteen 0 and 400 μ/L. Steady-state biomass, as determined in semicontinuous culture, did not vary with NO₃^? at slower dilution rates. However at a faster dilution rate, significantly less biomass occurred in intermediate concentrations of NO₃^? than in either higher or lower concentrations. The results indicate that both growth rate and standing crop are maximized by either N₂ fixation or NO₃^? assimilation, but extracellular NO₃^? reduces the rate of N₂ fixation. Consequently, at very low NO₃^? concentrations, growth is virtually maximized by N₂ fixation alone, and at high concentrations of NO₃^?, N₂ fixation is inhibited but growth is maximized by assimilation of NO₃^?. At intermediate concentrations of NO₃^?, growth becomes a function of NO^3? assimilation augmented by N₂ fixation. In this case, full growth potential is realized only if hydraulic residence time is sufficiently long to compensate for the reduced rate of N₂ fixation. Growth rate and standing crop are not diminished in response to the large amount of energy allocated to N₂ fixation. Instead, other cellular processes are probably affected negatively during N₂ fixation.     

INTERACTION OF NITROGEN FIXATION AND PHOSPHORUS LIMITATION IN APHANIZOMENON FLOS-AQUAE (CYANOPHYCEAE)1

The effect of simultaneous nitrogen fixation and phosphorus limitation on the physiological adaptation and growth performance of Aphanizomenon flos-aquae (L.) Ralfs PCC 7905 was studied in continuous culture. In the absence of ammonia, N₂ fixation occurred and the maximum growth rate (as determined in diluted batch cultures) was lower. However, no distinction could be made between the steady-state N uptake rates (based on cellular N contents) of N₂-fixing cells and cells grown with ammonia. At the higher dilution rates, the residual P concentration increased with increasing dilution rate, more so under N₂-fixing conditions, compared to the cultures grown in the presence of ammonia. More generally, the yield of biomass per consumed P, as the biomass concentration itself, decreased with increasing dilution rate, and both were lower under N₂-fixing conditions. The restricted biomass production under N₂-fixing conditions suggests that reduction of N loading may benefit lake restoration projects. The influence of N₂-fixation on the severity of P limitation is discussed in terms of metabolic control analysis. From the increase of the residual P concentration on switching from ammonium to N₂-fixing conditions, it is deduced that under N₂-fixing and P-limited conditions, control of growth is shared by N and P metabolism.     

DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The mechanism of photosynthetic carbon dioxide fixation in the green flagellate Dunaliella tertiolecta Butcher varies during growth in batch culture. Evidence for this change comes from three sources: i) algae from the stationary phase incorporated a greater proportion of the fixed carbon into amino arids and protein than did cells from the mid-exponential phase; ii) the activity of phosphoenolpyruvate carboxylase relative to that of ribulose-1, 5-di-phosphate carboxylase increased with age in batch culture; and, iii) cells from the stationary phase appeared to utilize the bicarbonate ion as the substrate for photosynthesis, whereas those from mid-exponential phase appeared to utilize fire carbon dioxide. These data suggest that a change of photosynthetic mechanism can occur within a single species of alga, depending on its physiological state.     

SPECTROPHOTOMETRICALLY ASSAYED INHIBITORY EFFECTS OF MERCURIC COMPOUNDS ON ANABAENA FLOS-AQUAE AND ANACYSTIS NIDULANS (CYANOPHYCEAE)1

The growth-related inhibitory effects of mercuric chloride (MC), methylmercuric chloride (MMC) and phenylmercuric acetate (PMA) (each at 1, 10, 10,² 10³ ppb) were measured in Anabaena flos-aquae (Lyng.) Bréb. and Anacystis nidulans (Richt.) Drouet & Daily. Optical density changes of control cultures compared against those of experimental cultures showed that MC was the least inhibitory of the compounds. MMC. was the most inhibitory, producing statistically significant inhibition at a concentration as low as I ppb in Anabaena. PMA was more inhibitory than MC but less than MMC. Effects caused by the mercury compounds included bleaching of individual cells, cell size changes and destruction of whole cells; the degree and extent of these effects depended on the compound and its concentration in the nutrient medium. The high sensitivities of the algae tested suggested the possibility of using them as test organisms in bioassays for mercury.     

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.     

INFLUENCE OF TRICHOME LENGTH ON LINEAR TRANSLATION IN OSCILLATORIA PRINCEPS (CYANOPHYCEAE)1

Motile trichomes of Oscillatoria princeps Voucher were examined to determine the relationship between trichome length and the ability to glide through hardened or viscous media. A minimum length for movement in solid media was found to be 0.1 mm in 0.5% agar (w/v).Viscous media tests revealed that coordinated movements through methyl cellulose media were not seen in trichomes 15–40 μ long at the viscosity employed. The minimum length motility increases with the viscosity of the medium and the gliding rates observed are greater in longer trihomes. These findings are discussed in relation to trichome surface area and the hypothesis that the motility system is restricted to lateral surfaces of the trichome.     

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.     

PHYSIOLOGICAL PROFILES OF SYNECHOCOCCUS (CYANOPHYCEAE) IN IRON-LIMITING CONTINUOUS CULTURES1

We examined the physiology and biochemistry associated with the iron-limited continuous culture of the halotolerant cyanobacterium Synechococcus PCC 7002. Biomass production, photosynthetic pigment levels, photosynthetic efficiency, and the production of hydroxamate- and catechol-type siderophores are reported for cells grown over a range of available iron concentrations. The relationship between the yield of Synechococcus PCC 7002 in iron-limited chemostats and the concentration of available iron was not linear. Synechococcus PCC 7002 expressed an inducible physiological response that led to alterations either in the cellular iron quotient or, more likely, in levels of available iron due to induced iron-scavenging processes. During iron limitation these cyanobacteria produced components consistent with the activation of a high-affinity iron transport system; both hydroxamate- and catechol-type siderophores were detected. Iron-limited Synechococcus PCC 7002 also reduced CO₂ fixation rates from luxury levels to a rate that matched the cellular growth rate, presenting interesting implications for oceanic carbon flux models.