Role of light, carbon dioxide and nitrogen in regulation of buoyancy, growth and bloom formation of Anabaena flos-aquae

The availability of light, CO₂ and NH₄-N interacted to controlbuoyancy and growth of the gas vacuolate blue-green alga, Anabaenaflos-aquae. At high light intensities algal growth rates werehigh; however, the alga was non-buoyant regardless of the availabilityof CO₂ or NH₄-N. The mechanism for buoyancy loss involved increasedcell turgor pressures at higher light intensities which resultedin collapse of gas vacuoles. At lower light intensities algalgrowth rates and cell turgor pressures were reduced and buoyancywas controlled by the availability of CO₂ and inorganic nitrogen.Carbon dioxide limitation increased buoyancy, while reducedinorganic nitrogen availability reduced buoyancy. Mechanismsfor buoyancy regulation at low light intensities involved changesin cellular C/N ratios which appeared to affect the rate ofsynthesis and accumulation of protein-rich gas vacuoles. Algalspecific growth rates were combined with buoyancy data to forma single index (µ_bloom) to the rate of surface bloom formationof A.flos-aquae as a function of the availability of light,CO₂ and NH₄-N. The bloom formation index was enhanced with decreasedavailability of light and CO₂, and increased availability ofNH₄-N.     

The Interrelations of Cell Turgor Pressure, Gas-vacuolation, and Buoyancy in a Blue-green Alga

The increase in pressure required to collapse gas vacuoles onsuspending the cells of the blue-green alga Anabaena flos-aquaein hypertonic sucrose solutions shows the turgor pressure tovary over the range of 265 to 459 KN m^–2 under differentculture conditions. The cell turgor increased at a rate of upto KN m^–2 h^–1 on transferring the alga from lowto high light intensity. This rise appears to be a result ofthe accumulation of photosynthate, as it is dependent on thepresence of carbon dioxide in the gas phase and is inhibitedby DCMU. Experiments using ¹⁴CO² indicate that the increasedrate of photosynthesis during the high light exposure is easilysufficient to account for the observed turgor rise. The rise in turgor can bring about collapse of sufficient ofthe alga's gas vacuoles to destroy its buoyancy. Higher turgorpressures, and consequently a lower degree of gas vacuolationand buoyancy, were maintained when the alga was kept at highlight intensitives for a week and more. The significance ofthis behaviour is discussed in relation to stratification ofplanktonic blue-green algae in natural habitats.     

Nitrogen-fixation associated with the marine blue-green alga,Trichodesmium, as measured by the acetylene-reduction technique

Summary The marine blue-green alga, Trichodesmium, was collected from the Gulf Stream, near Miami, and occurred in two distinct colonial forms both of which reduced acetylene to ethylene. Trichodesmium was more abundant during the summer but its acetylene-reducing potential showed no obvious seasonal variation. Illuminated Trichodesmium reduced acetylene to ethylene equally well either anaerobically or aerobically (20% oxygen). Acetylene-reduction in the dark, however, was oxygen-dependent and was usually 25% or less of the activity recorded in the light. 0.3–1.0 nmoles of ethylene were produced per minute per mg of protein, by illuminated cultures, and these values compare favorably with those recorded for other nitrogen-fixing blue-green algae. However, the possibility that bacteria contributed to the acetylene-reducing activity associated with Trichodesmium was not completely eliminated.Contribution No. 1578 from the University of Miami, Rosenstiel School of Marine and Atmospheric Science, 10 Rickenbacker Causeway, Miami, Florida 33149, U.S.A.     

Isolation of gas vacuole-less mutants of the blue-green alga Anabaenopsis raciborskii

From a bloom forming blue-green alga, Anabaenopsis raciborskii, spontaneous mutants, which had lost the ability to form gas vacuoles have been isolated; the mutant frequency was 4.8×10^-3. The filaments of gas vacuole-less mutants settled at the bottom of flasks in liquid culture media unlike the parent alga. The growth and nitrogen fixation were comparatively poor in the mutants.     

An investigation into the possible light-shielding role of gas vacuoles in a planktonic blue-green alga

When the gas vacuoles of Anabaena flos-aquae Bréb. ex Born. et Flah. are collapsed, the optical properties of the alga change. While this may suggest a light-shielding role, photosynthetic measurements indicate that intact gas vacuoles reduce the light falling on the thylakoids by only 4%, or less. Intact gas vacuoles offer no protection against the lethal effects of ultraviolet light. When the alga is grown at high light intensity the gas vacuoles are fewer in number but are oriented peripherally in the cells. However, this does not markedly affect their light shielding efficiency. Spectrophotometric measurements carried out by others indicate a light shielding role by gas vacuoles in a non-planktonic blue-green alga, Nostoc muscorum Kütz., but do not give a quantitative estimate of this effect. In Anabaena no definite evidence of light-shielding is obtained by such a method. All of the experiments described were conducted with dilute algal suspensions to investigate shielding effects in individual cells. Possible self-shading effects in dense suspensions and surface water blooms require further investigation.     

WATER-BLOOMS

1. Peculiarities in the ecology of planktonic blue-green algae are reviewed in relation to recent advances in understanding their physiological characteristics. 2. Dense water-blooms are always the result of buoyant migration of existing populations to the lake surface under calm weather conditions. The size of the population is the direct result of photoautotrophic growth, and is dependent upon light and the availability of inorganic nutrients; it is apparently enhanced by moderately high water temperatures, high pH, low oxygen tensions and possibly, the presence of organic solutes. The relative effectiveness of these factors is untested. 3. Buoyancy is imparted by gas vacuoles whose principal function is to regulate the position of the alga in the water column. Control is effected by two mechanisms: (i) ‘dilution’ of newly produced vacuoles during active cell division; (ii) changes in cell turgor-pressure acting on the gas-vacuole structure. Gas-vacuole production is greatest at low light intensities and the alga becomes more buoyant; at higher light intensities, increased turgor-pressure collapses the weaker vacuoles causing the alga to lose buoyancy. 4. Potentially, algae are able to poise themselves at an optimum point in the light gradient, usually towards the bottom of the euphotic zone, where the algae are likely to encounter the conditions most favouring their growth. 5. Different species of blue-green algae differ in the typical sizes of their colonies and, hence, in their rates of controlled movement. These differences are interpreted as hydrodynamic adaptations to the variations in turbulent water movements to which the algae are subject. 6. Populations of single-filamentous Oscillatoria agardhii and O. rubescens come to occupy the stable metalimnia of stratified lakes, provided that they are located within the euphotic zone. 7. The large stream-lined colonial forms occur mainly in polymictic lakes and in the unstable epilimnia of stratified lakes where light penetration is restricted to the superficial layers. These algae are adapted to sink or float rapidly to the optimum depth when turbulence subsides. Because of their potentially high rates of movement, it is the large colonial forms that commonly form blooms. 8. Bloom formation can occur when most of the algae possess excess buoyancy. Excess buoyancy is acquired when the photosynthetic rate is insufficient to develop the necessary turgor-pressure to cause collapse of the vacuoles. Photosynthesis may be sufficiently impaired under four circumstances: (i) during turbulent circulation of the population over a depth that significantly exceeds the euphotic depth; (ii) in the absence of light (e.g. at night): (iii) at limiting concentrations of carbon dioxide: and (iv) when the algal population is senescent. 9. Because bloom-formation depends upon the coincidence of persistent algal overbuoyancy with calm weather, its occurrence is incidental, and serves no vital function in the biology of blue-green algae. 10. Some possible causes for the occurrence of blue-green algal blooms in a relatively restricted range of water bodies are discussed. Large bloom-forming populations are probably restricted to moderately rich, mildly alkaline, thermally unstable lakes in all regions, except those which are permanently cold. Extremes of poverty or richness of nutrients, short water-retention times and low pH seem to be factors which select against planktonic blue-green algae.     

Counting of gas vacuoles by electron microscopy in lysates and purified fractions ofmicrocystis aeruginosa

Summary A microdroplet spray method is described to determine quantitatively the number of gas vacuoles in purified fractions and in total lysates of the blue-green alga,Microcystis aeruginosa.It is found that 6.45×10¹² gas vacuoles of 370 nm length make up one mg of protein. This supports the idea that the entire organelle is made up by a single layer of protein only. The gas vacuole number per cell varies with the stage of growth between 3,700 and 11,000.     

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.     

Planktothrix populations in subalpine lakes: selection for strains with strong gas vesicles as a function of lake depth,morphometry and circulation

1. The genus Planktothrix (Cyanobacteria) usually produces concentrated populations of filaments in the summer metalimnion of thermally stratifying lakes. This has been associated with the action of gas vesicles, cellular structures providing positive buoyancy. At the end of the summer, filaments are carried by convective mixing deeper into the water column where some gas vesicles collapse as a result of high hydrostatic pressure. They then lose their buoyancy, sink and are lost from the euphotic zone. 2. The resistance of gas vesicles to hydrostatic pressures is critical for the survival of Planktothrix in deep lakes. However, comparative observations on populations from lakes of a range of depths and hydrodynamic regimes are still needed to examine the relationships between the adaptive trait (i.e. the ‘critical’ pressure at which each gas vesicle collapses) with the environmental factor (i.e. the maximum hydrostatic pressure). 3. To explore the adaptation of Planktothrix populations to the depth of winter circulation in different systems, we collected 276 strains of P. cf. rubescens from eight lakes (z_max = 24–410 m) in Northern Italy during summer 2009 and we analysed the multicopy gene gvpC coding for a protein that crucially influences the critical pressure. 4. The strains analysed clustered into two main groups having gas vesicles with a mean critical pressure of 1.1 and 0.9 MPa, respectively. The proportion of the stronger strains was generally positively related to lake depth, although the overall pattern was complicated by individual lake morphology and hydrology. The relative frequency of stronger filaments was (i) greatest in deep basins with concave slopes and (ii) least in one deep, but permanently stratified lake. 5. The simultaneous presence of ‘weaker’ and ‘stronger’ filaments could allow for a rapid adaptive response to changes in hydrostatic pressures, related to changes in the amplitude of vertical circulation characterising deep lakes.     

Regulation of blue-green algal buoyancy and bloom formation by light,inorganic nitrogen,C02, and trophic level interactions

In highly eutrophic ponds, buoyancy of the gas-vacuolate blue-green alga Anabaenopsis Elenkinii (Miller) was regulated by complex interactions between chemical and physical parameters, as well as by biological interactions between various trophic levels. Algal buoyancy and surface bloom formation were enhanced markedly by decreased light intensity, and to a lesser extent by decreased CO₂ availability and increased availability of inorganic nitrogen. In the absence of dense populations of large-bodied Cladocera, early season blooms of diatoms and green algae reduced light availability in the ponds thus creating conditions favorable for increased buoyancy and bloom formation by A. Elenkinii. The appearance of blue-green algal blooms could be prevented by a reduced density of planktivorous fish, which allowed development of dense cladoceran populations. The cladocerans limited the growth of precursory blooms of diatoms and green algae, and given the resulting clear-water conditions, buoyancy of A. Elenkinii was reduced, and blue-green algal blooms never appeared.     

Isolation and chemical characterization of gas-vacuole membranes fromMicrocystis aeruginosa Kuetz. emend. Elenkin

Summary A method involving penicillin treatment was developed to osmotically lyse the cells of the blue-green alga,Microcystis aeruginosa Kuetz. emend. Elenkin, and release the pressure-sensitive gas vacuoles intact. The gas vacuoles were purified by liquid-polymer partitioning or by macromolecular sieving and centrifugation. The degree of purification of the gas vacuoles was followed by observation in the electron microscope and by the use of C¹⁴-labeled vacuolated and nonvacuolated strains ofM. aeruginosa. The gas-vacuole membrane is composed of only protein consisting of 10% basic, 18% acidic and 52% non-polar amino acids.Supported by U.S. Atomic Energy Commission, Contract No. AT(11-1)-1338.     

Trichodesmium thiebautii; structure of a nitrogen-fixing marine blue-green alga (Cyanophyta)

Summary Trichodesmium thiebautii was collected, as floating bundles composed of uniseriate filaments aligned in parallel, from the Kuroshio waters off Shikoku Island, Japan. The ultrastructure of this alga had basically the same general features as the related speciesT. erythraeum first described byvan Baalen andBrown (1969). InT. thiebautii long electron dense fibers and concentrically lamellated bodies were observed which were either not reported previously, or did not occur inT. erythraeum. The peripheral wall layers were generally typical ofOscillatoria-type blue-green algae, but with a distinctive finely striated outer layer. Thylakoids per cell volume were very sparse compared to most other blue-green algae. Phycobilisomes, apparently hemidiscoidal in shape, typically occurred on the stromal side of the thylakoid surface. Large gas vesicle areas occupied the main volume of the cell, including cells which seemed to be actively growing. The gas vesicle areas were distributed throughout the cell, not only in the cell periphery as inT. erythraeum. Considerable complexity was suggested by the apparent cell compartmentation, particularly because the gas vesicle areas were delimited by one to several thylakoids. Only rarely were the gas vesicle areas traversed by thylakoids. Electron dense fibers (ca. 25 nm diameter) were always observed between the gas vesicles and were usually oriented parallel with them, but they were not rigid appearing as were the gas vesicles. The gas vesicles had a smaller diameter (ca. 45 nm) than most blue-greens. Concentrically lamellated bodies (ca. 1.0 m diameter) were observed in cells of some of the bundles. Each concentric layer was ca. 1.3 nm wide. These concentrically lamellated bodies may be characteristic of older cells. Cylindrical bodies were considerably smaller (ca. 120 nm diameter) and less complex than those reported forT. erythraeum.     

Comparative Study of the Structure of Gas Vacuoles

The fine structure of gas vacuoles was examined in two blue-green algae, two green bacteria, three purple sulfur bacteria, and two halobacteria. The gas vacuole is a compound organelle, composed of a variable number of gas vesicles. These are closed, cylindrical, gas-containing structures with conical ends, about 80 to 100 nm in width and of variable length, ranging from 0.2 to over 1.0 mum. The wall of the gas vesicle is a non-unit membrane 2 to 3 nm in thickness, bearing very regular striations with a periodicity of 4 nm, oriented more or less at right angles to the long axis of the cylinder. This fine structure could be clearly resolved in isolated gas vesicles prepared from a blue-green alga and from Halobacterium halobium, and its presence in the gas vesicles of the green bacterium Pelodictyon clathratiforme was inferred from thin sections. The gas vacuole thus appears to be a homologous organelle in all of these procaryotic groups. Minor differences with respect to the length and arrangement of the gas vesicles were observed. In blue-green algae and green bacteria, the vesicles are relatively long and tend to be arrayed in parallel bundles; in purple sulfur bacteria and Halobacterium, they are shorter and more irregularly distributed in the cell.     

Chemical composition of gas vesicles isolated from Anabaena flos-aquae

Summary Chemical analysis of purified preparations of gas vesicles isolated from the filamentous blue-green alga Anabaena flos-aquae has shown that they are similar in composition to those isolated from the unicellular alga Microcystis aeruginosa by Jones and Jost (1970), being proteinaceous structures, free of lipid and carbohydrate. The gas vesicle protein from Anabaena contains the same 14 amino acids, in broadly the same proportions; in addition there is a small proportion of proline. No sulphur-containing amino acids are present. The empirical formula, suggested by the amino acid ratios, indicates a molecule of 15000 MW.     

Kinetics of the assembly of gas vacuoles in the blue-green alga Microcystis aeruginosa Kuetz. emend. Elekin.

Summary In exponentially growing cultures of the blue-green alga Microcystis aeruginosa the number of gas vacuoles per cell decreases and reaches a value of 4.5×10³ at 1.2×10⁷ cells per ml. The assembly of gas vacuoles with respect to number and length was followed after the organelles were caused to collapse by a pulse of ultrasound. The change in the number N of gas vacuoles per cell is N=224.8×t ^0.757, 0<t<24 h. After 24 h 50% of the value of non-sonicated cultures is reached. The changes in the length L of the organelles is expressed by L=87.06 ×t ^0.4084, 0<t<24 h. After 24 h 85% of the control value is reached.Abbreviations used EDTA ethylene diamine tetraacetate - BSA bovine serum albumine - P probability - N number of gas vacuoles - L length of gas vacuoles in nm - t time in hours     

The Role of Potassium in the Control of Turgor Pressure in a Gas-Vacuolate Blue-Green Alga

The contribution of K⁺ accumulation to cell turgor pressurewas investigated in the gas-vacuolate blue-green alga Anabaenaflos-aquae. The cell turgor pressure, measured by the gas vesiclemethod, drops in cells suspended in culture medium depletedof K⁺ but rapidly rises again, by 100 kPa or more, when K⁺ isresupplied. A similar though rather slower rise in turgor pressureis supported by an equivalent concentration of Rb⁺. The internalK⁺ concentration rose from 66 to 91 mM when K⁺ was suppliedat an external concentration of 0.4 mM. This rise was light-dependent.Greater increases in internal K⁺ concentration and turgor pressureoccurred when K⁺ was supplied at a higher concentration, 3.6mM. In both cases over 60% of the observed turgor pressure risecould be accounted for by accumulation of K⁺. The turgor pressurerise supported by light-stimulated K⁺ uptake can cause collapseof enough of the alga's gas vesicles to destroy its buoyancy.The effect of K⁺ availability on buoyancy regulation by planktonicblue-green algae is discussed.     

Absence of gas vesicle protein in a mutant of Anabaena flos-aquae

Filaments without gas vacuoles arose spontaneously in the gas-vacuolate alga Anabaena flos-aquae. The non-vacuolate mutant was enriched by repeated sedimentation and subsequently cloned by microsyringe transfer. No revertants have been observed. In the gas-vacuolate wild-type alga the gas vesicle protein was clearly distinguished by gel electrophoresis as one of the ten most abundant protein species present in whole cell extracts. Electrophoresis indicated that the mutant had lost the ability to synthesize the gas vesicle protein. A second mutant partially defective in production of gas vacuoles and gas vesicle protein has been isolated.Abbreviations gv gas vesicle protein - pb phycobilin - TCA trichloracetic acid     

Fine Structure of a Marine Ameba Associated with a Blue-Green Alga in the Sargasso Sea*,†

SYNOPSIS An ameba, bearing a fringe of scales on the plasmalemma surface, dwells among the filaments of the colonial, blue-green alga Trichodesmium thiebautii (Sournia), and preys upon bacteria growing within the colony. The cytoplasm is clearly differentiated into a fine fibrillar ectoplasm at the periphery of the cell and a central endoplasm containing most of the membranous organelles. The nucleus contains a spheroidal nucleolus which is centrally located, and a double membrane containing pores. The tubular mitochondria, microbodies, lysosomes, and endoplasmic reticulum are typical for protozoa. The Golgi apparatus consists of an array of elongate flattened cisternae. One surface is associated with a fine fibrillar layer and the opposite surface contains electron-dense vesicles (perhaps primary lysosomes) and scale-containing vesicles that appear to be the origin of the scales deposited on the plasma membrane. Three kinds of bacteria-containing vacuoles are presnt: (a) vacuoles surrounded by 3 membranes and containing bacteria that are either healthy or in an early stage of digestion, (b) singlemembrane vacuoles which are food vacuoles that become converted to digestive vacuoles, and (c) larger vacuoles resembling those in (b) which contain prey in an advanced stage of digestion. The presence of amebae within pelagic algal communities provides further evidence for the diversity of their habitats in the ocean.     

The buoyancy-providing role of gas vacuoles in an aerobic bacterium

The heterotrophic, freshwater bacterium Prosthecomicrobium pneumaticum Staley possesses sufficient gas vacuoles to render it buoyant at all stages of growth. Although the cells have a turgor pressure of about 300 kPa, there is no evidence that this pressure is important in causing collapse of the constituent gas vesicles. A mutant of the bacterium, which produced only 0.2% of the amount of gas vacuoles produced by the wild type, was isolated. It always sank in liquid culture. Wild type and mutant bacteria grew at the same rate in shaken culture, but in static culture the wild type, which floated to the liquid surface grew more quickly than the mutant, which sank. Other competition experiments suggested that the advantage gained in floating at the surface was simply that oxygen was more readily available there to this obligate aerobe. Similar advantages may benefit gas vacuolate forms in natural habitats.A second mutant was isolated which produced about 40% fewer gas vacuoles than the wild type in corresponding stages of growth, insufficient to provide buoyancy, and unlikely to be of selective value. The occurrence of this mutant suggests there may be duplication of the gas vacuole gene.Abbreviations T turbidity - PST pressure sensitive turbidity - kPa kilo-Pascals (100 kPa=1 bar)