Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N(2)-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N(2)-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with P(i), and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Biomass production,total protein,chlorophylls, lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes

Two green algae (Chlorella vulgaris and Scenedesmus obliquus) and four blue-green algae (Anacystis nidulans, Microcystis aeruginosa, Oscillatoria rubescens and Spirulina platensis) were grown in 81 batch cultures at different nitrogen levels. In all the algae increasing N levels led to an increase in the biomass (from 8 to 450 mg/l), in protein content (from 8 to 54 %) and in chlorophyll. At low N levels, the green algae contained a high percentage of total lipids (45 % of the biomass). More than 70 % of these were neutral lipids such as triacylglycerols (containing mainly 16:0 and 18:1 fatty acids) and trace amounts of hydrocarbons. At high N levels, the percentage of total lipids dropped to about 20 % of the dry weight. In the latter case the predominant lipids were polar lipids containing polyunsaturated C₁₆ and C₁₈ fatty acids. The blue-green algae, however, did not show any significant changes in their fatty acid and lipid compositions, when the nitrogen concentrations in the nutrient medium were varied. Thus the green but not the blue-green algae can be manipulated in mass cultures to yield a biomass with desired fatty acid and lipid compositions. The data may indicate a hitherto unrecognized distinction between prokaryotic and eukaryotic organisms.     

Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

Formation of biomass,total protein,chlorophylls, lipids and fatty acids in green and blue-green algae during one growth phase.

Batch cultures (8–32 l.) of Chlorella vulgaris and Scenedesmus obliquus and of Anacystis nidulans and Microcystis aeruginosa were grown in media containing 0.001 % KNO₃ and at several stages in growth sampled for biomass, total protein, chlorophylls, lipids and fatty acids. With increasing time and decreasing nitrogen concentrations, the biomass of all of the algae increased, whereas the total protein and chlorophyll content dropped. Green and blue-green algae, however, behaved differently in their lipid metabolism. In the green algae the total lipid and fatty acid content as well as the composition of these compounds changed considerably during one growth phase and was dependent on the nitrogen concentration in the media at any given day of growth. More specifically, during the initial stages of growth the green algae produced larger amounts of polar lipids and polyunsaturated C₁₆ and C₁₈ fatty acids. Towards the end of growth, however, these patterns changed in that the main lipids of the green algae were neutral with mainly saturated fatty acids (mostly 18:1 and 16:0). Such changes did not occur in the blue-green algae. These differences between prokaryotic and eukaryotic algae can possibly be explained by the ‘endosymbiont theory’.     

Deep-tank culture of blue-green algae in H2O and D2O

A 160-liter stainless steel algal growth tank has been constructed and has been used essentially continuously for over three years. Filamentous and unicellular blue-green algae as well as a photosynthetic bacterium have been cultured using both ordinary and heavy water (99.8 atom % ²H). By using a recycling technique, yields as high as 25 g/liter of ²H₂O have been obtained.     

The feeding biology of Hydrobia ventrosa (Montagu). I. The assimilation of different components of the food

The assimilation efficiencies for 7 species of diatoms, two of blue-green algae, and one bacterium have been measured in the deposit-feeding prosobranch Hydrobia ventrosa (Montagu). Two methods have been used, a chemical and a radiotracer one.The results of the first method were difficult to evaluate due to the fact that the time taken to clear the gut is ill-defined. The second method gave more consistent results.All diatoms are assimilated efficiently (60–71 %) as is the bacterium (about 75 %). The blue-green algae are less efficiently utilized; an Oscillatoria sp. was assimilated with an efficiency of about 50 % whereas a Chroococcus sp. was only assimilated with an efficiency of 8 %. It is concluded that resource partitioning based on differential utilization of micro-organisms cannot play an important rôle in the co-existence of Hydrobia ventrosa with other deposit-feeders.     

Aliphatic Hydrocarbons and Fatty Acids of Some Marine and Freshwater Microorganisms

Gas chromatography and combined gas chromatography-mass spectrometry have been used to study the fatty acids and hydrocarbons of a bacterium from the Pacific Ocean, Vibrio marinus, a freshwater blue-green alga, Anacystis nidulans, and algal mat communities from the Gulf of Mexico. Both types of microorganisms (bacteria and algae) showed relatively simple hydrocarbon and fatty acid patterns, the hydrocarbons predominating in the region of C-17 and the fatty acids in the range of C-14 to C-18. The patterns of V. marinus were more comparable to those of the algal populations than to patterns reported for other bacteria. An incomplete correlation between fatty acids and hydrocarbons in both types of organisms was observed, making it difficult to accept the concept that the biosynthesis of hydrocarbons follows a simple fatty acid decarboxylation process.     

Growth stimulation of Microcystis aeruginosa by a bacterium from hyper-eutrophic water (Taihu Lake, China)

Cyanobacteria are the causative organisms of the algal blooms that occur in Taihu Lake. Dissolved organic nitrogen (DON) comprises a significant composition of nitrogen (N) pool in the water and may increase the nutrient source of microalgae. In the present study, we investigated the relationship between Microcystis aeruginosa, Pseudomonas sp. A3CT isolated from Taihu, and DON compounds. Co-incubation (3 days) of the bacterium with six DON compounds (four free amino acids and two combined amino acids) was collected as six decomposed DON solutions. The decomposed DON solutions of six compounds were used to test the stimulatory effect of nutrient regeneration by the bacterium. The growth of M. aeruginosa was significantly enhanced by the six decomposed DON solutions. M. aeruginosa grew much better under the six decomposed DON solutions than the corresponding undigested DON forms. Especially, the decomposed l-lysine solution, not only avoided the inhibiting effect of lysine on M. aeruginosa, but significantly promoted the cyanobacterial growth. Further chemical tests indicated that A3CT transformed DON into NH₄ ⁺, which was utilized by M. aeruginosa. These results demonstrate that the bacterium plays an important role in decomposing unavailable DON forms into available NH₄ ⁺, which suggests that the bacterium contributes to the fast growth of M. aeruginosa. Moreover, this phenomenon, in conjunction with previous studies, indicates that the responsible and effective way of harmful blooms is reducing the N and P inputs (including DON and DOP).     

Algal nitrogen fixation on the lava field of Heimaey,Iceland

Summary Nitrogen fixation (C₂H₂ reduction) by blue-green algae occurring on the juvenile lava field of Heimaey, Iceland was examined both in the laboratory (potential at 20° C and 39° C) and in the field, three and a half years after the volcanic eruption.Already at this early stage of colonization representatives of unicellular and filamentous heterocystous and non-heterocystous blue-green algae were commonly observed. The predominating algae were Nostoc sp. (20° C) and Schizothrix sp. — Microcoleus chthonoplastes, (39° C), the former often in association with the protonemata-rhizoids of moss plants.The potential for nitrogen fixation was recorded at an average rate of 109.2 (20° C) and 138.1 (39° C) ng N g^-1 h^-1 in soil collected from localities randomly distributed over the lava field.Tests for nitrogen fixation performed in situ revealed significant fixation activities in all the eleven localities subject to examination. The activities ranged from 2.8 to 63.4 (mean 21.5) ng N g^-1 h^-1 and 1.9 to 17.7 (mean 7.9) ng N cm^-2 h^-1.All the nitrogen fixation data noted imply that blue-green algae contribute a substantial part of the nitrogen input to the lava. Further, it was found that material incubated under micro-aerophilic conditions exhibited considerably enhanced nitrogenase activity.The role of nitrogen-fixing blue-green algae in general and Nostoc muscorum in particular in being suitable as pioneering organisms preparing the bare lava for ingress of other plants is also discussed.     

Structure and composition of hydrocarbons and fatty acids from a marine blue-green alga,Synechococcus sp.

The major hydrocarbons of Synechococcus sp., a marine blue-green alga isolated from the Gulf of Mexico, were identified as 1-nonadecene and 1,14-nonadecadiene. The content of 1-nonadecene increased with culture age from about 0.5 mg/g dry weight in young cells to about 2.3 mg/g dry weight in old cells, while the content of 1,14-nonadecadiene remained constant with culture age at about 1 mg/g dry weight. Both [1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated to equal extents into the hydrocarbons. [1-¹⁴C]Stearate was incorporated into the hydrocarbons, but [³H]arachidate was not. The fatty acids of Synechococcus sp. were typical of blue-green algae, consisting of C_16:0, C_16:1, C_16:1, C_16:0, C_18:0, C_18:1, C_18:2 and C_18:3 fatty acids were detected. The hexadecenoic acid was shown to be 9-C_16:1 while the octadecenoic acid was a mixture of 93% 11-C_18:1 and 7% 9-C_18:1. The fatty acid content increased during the first 4 days of growth and then decreased slightly.     

Grazing by Talorchestia longicornis on an algal mat in a new England salt marsh

Grazing experiments using ¹⁴C and an analysis of fecal pellets and gut contents established that the gammaridean amphipod, Talorchestia longicornis Say, ingests blue-green algae on algal mats in a Massachusetts salt marsh. This grazing had a measurable effect on the lower algal mat, where the density of T. longicornis was high. Exclusion of amphipods resulted in increases in chlorophyll a content, carbon incorporation, and nitrogen fixation. This effect was not seen on the upper mat where T. longicornis was less abundant. The assimilation efficiency of T. longicornis feeding on a diet consisting mainly of blue-green algae was surprisingly high (67 %) considering that blue-green algae are usually considered as a poor quality food for herbivores. The population of T. longicornis seems to be annual, with growth of the overwintered juveniles in spring and early summer.     

Measurement of photorespiration in algae

The rates of true and apparent photosynthesis of two unicellular green algae, one diatom and four blue-green algae were measured in buffer at pH 8.0 at subsaturating concentrations of dissolved inorganic carbon (13-27 micromolar). Initial rates of depletion from the medium of inorganic carbon and ¹⁴C activity caused by the algae in a closed system were measured by gas chromatography and by liquid scintillation counting, respectively. The rate of photorespiration was calculated as the difference between the rates of apparent and true photosynthesis. The three eucaryotic algae and two blue-green algae had photorespiratory rates of 10 to 28% that of true photosynthesis at air levels of O₂. Reduction of the O₂ level to 2% caused a 52 to 91% reduction in photorespiratory rate. Two other blue-green algae displayed low photorespiratory rates, 2.4 to 6.2% that of true photosynthesis at air levels of O₂, and reduction of the O₂ concentration had no effect on these rates.     

Effects of free amino acids on the isolated symbiotic algae of the coral Plesiastrea versipora (Lamarck): absence of a host release factor response

Symbiotic algae incubated in host tissue homogenate of the coral Plesiastrea versipora for 2 h in the light released at least four and a half times as much photosynthetically fixed carbon (range 13.8±3.1 to 158±9.5 nmol C/10⁶ algae) as algae incubated in seawater (range 1.4±0.3 to 10.8±0.6 nmol C/10⁶ algae) indicating the presence of ‘host release factor’. When algae were incubated in a low molecular weight fraction of homogenate containing partially purified ‘host release factor’ they also released more carbon (range 62.2±3.7 to 279±11.4 nmol C/10⁶ algae) than algae incubated in seawater. This low molecular weight fraction contained free amino acids. We tested the hypothesis that the free amino acids in this fraction were responsible for ‘host release factor’ activity. Algae incubated in a mixture of free amino acids equivalent to those found in this fraction, released more fixed carbon (range 2.4±0.3 to 25.2±0.2 nmol C/10⁶ algae) than algae incubated in seawater but in each experiment, release was much lower than when algae were incubated in host tissue homogenate. These data indicate that the stimulation of release of photosynthetically fixed carbon from the symbiotic algae of Plesiastrea versipora incubated in partially purified host release factor is not primarily due to the presence of free amino acids. We are continuing further studies to determine the exact nature of the active compound.     

Measurement of rates of grazing of the ostracod Cyprinotus carolinensis on blue-green algae

Measurements were made of the rates of grazing of the ostracod Cyprinotus carolinensis fed ¹⁴C-labelled filamentous blue-green algae (cyanobacteria). The grazing rate was a linear function of food concentration at densities below 1.1 µg dry weight of algae · ml^-1 and independent of concentration at densities above 11.5 µg algae · ml^-1. Starvation affected grazing rates significantly, but light vs. dark feeding, animal density, and the volume of feeding container did not. Grazing on Nostoc sp. was a linear function of ostracod size. Respiration of C. carolinensis and the blue-green algae was not a significant factor in the tests of grazing.     

Photochemical energy conversion using immobilized blue-green algae

A blue-green alga, Anabaena N-7363, was immobilized in 2% κ-carrageenan gel. The hydrogen productivity of the immobilized algae was 2.4 times higher than that of free algae, with a maximum rate of hydrogen production of 3.24 mmol h⁻¹ g⁻¹ dry gel, in a nitrogen free medium under illumination (6000 lux). The immobilized blue-green algae (39 kg wet gel) was employed for continuous production of hydrogen under illumination (6000 lux), producing 0.5–1.1 ml min⁻¹ for more than 8 days. The hydrogen produced was supplied to a phosphoric acid fuel cell, which generated an approximate 50 mW power output and a current of 300 mA over a period of 4 h.     

BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS : I. Protein Amino Acids

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with ¹⁴CO₂ for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of ¹⁴CO₂ into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with ¹⁴CO₂ in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.     

DETECTION OF LIMITING OR SURPLUS NITROGEN IN ALGAE AND AQUATIC WEEDS1

The rate of NH₄+-N absorption by algae and aquatic weeds in the dark has been shown to be 4-5 times greater for plants which are N-limited as compared to plants with sufficient available N. Eight species of green algae, 2 blue-green algae, 2 diatoms, and 3 aquatic weeds were used to demonstrate the usefulness of the test in determining if available N was in surplus or limited supply in a particular environment. The test was shown not to differentiate between blue-green algae capable of fixing N (4 species) from media with NO₃-N or without combined nitrogen. The factors influencing the results of NH4⁺-N absorption tests have been investigated. In order to differentiate between plants with sufficient available N and those which are N limited, the rate of NH₄⁺-N absorption (0.1 mg N) over 1-hr incubation in the dark by 10-20 mg of algae or aquatic weed tissues is measured. The relatively simple analysis for NH₄⁺-N in the samples makes it very easy to follow the changing N nutrition of plants in cultures with a limited N supply or in the presence of possible N sources.     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

Retrieval of phycocyanin concentration from remote-sensing reflectance using a semi-analytic model in eutrophic lakes

With the rapid development of the economy in recent years, massive algal (blue-green algae in particular) blooms have often observed in Chinese eutrophic lakes. The concentration of the cyanobacterial pigment phycocyanin (PC), an accessory pigment unique to freshwater blue-green algae, is often used as a quantitative indicator of blue-green algae in eutrophic inland waters. The purpose of this study was to evaluate the semi-analytic PC retrieval algorithm proposed by Simis et al. and to explore the potential to improve this PC algorithm so that it is more suitable for eutrophic lakes, such as Taihu Lake. In this paper, we recalculated the correction coefficients γ and δ to calculate the absorptions of chlorophyll-a at 665 nm and the absorptions of phycocyanin at 620 nm in terms of in situ measurements and observed that the values of these coefficients differed from the values used by Simis et al. and Randolph et al. The two coefficients are site dependent due to the different bio-optical properties of lakes. We also observed that the specific PC absorption at 620 nm a_pc*(620) decreases exponentially with an increase in PC concentrations. Therefore, a non-linear power–function of a_pc*(620), instead of a constant value of a_pc*(620) as used by Simis et al., was proposed for our improved PC retrieval algorithm in Taihu Lake, yielding a squared correlation coefficient (R²) of 0.55 and a root mean square error (RMSE) of 58.89 μg/L. Compared with the original PC retrieval algorithm by Simis et al., the improved retrieval algorithm has generally superior performance. In evaluating the limitation of the PC retrieval algorithms, we observed that the ratio of the total suspended solids to phycocyanin can be used as a primary measure for retrieval performance. Validation in Dianchi Lake and an error analysis proved that the improved PC algorithm has a better universality and is more suitable for eutrophic lakes with higher PC concentrations.     

Isolation of individual amino acids from various microbiological sources using reversed-phase high-performance liquid chromatography

A new method for the preparative isolation of individual amino acids on a milligram scale based on reversed-phase high-performance liquid chromatography (RP-HPLC) after pre-column derivatization with carbobenzoxychloride (ZCl) has been developed. The chromatographic procedure was tested by the investigation of jack bean urease hydrolysate. The method has been applied to the preparative separation of Z-amino acids (from 10 up to 16) obtained from protein hydrolysates of various sources (green microalgae, blue-green algae, halophilic and methylotrophic microorganisms) and was proved to be reliable by the separation of deuterated amino acids (enrichment 97–99%) from Methylobacillus flagellatum (due to the bioconversion of CD₃OD and D₂O). Independent of the biological source of the protein, the amino acids were isolated with high recovery (from 68% up to 89%) and chromatographic purity (from 96% up to 99%). The method was also applied for the isolation of phenylalanine and leucine excreted by amino-acid overproducing microorganisms.