Ammonia may play an important role in the succession of cyanobacterial blooms and the distribution of common algal species in shallow freshwater lakes

With the human intensification of agricultural and industrial activities, large amount of reduced nitrogen enter into the biosphere, which consequently results in the development of global eutrophication and cyanobacterial blooms. However, no research had reported the effect of ammonia toxicity on the algal succession. In this study, we investigated the ammonia toxicity to 19 algal species or strains to test the hypothesis that ammonia may regulate the succession of cyanobacterial blooms and the distribution of common algal species in freshwater lakes. The bloom‐forming cyanobacterium Microcystis aeruginosa PCC 7806 suffered from ammonia toxicity at high pH value and light intensity conditions. Low NH₄Cl concentration (0.06 mmol L^?1) resulted in the decrease of operational PSII quantum yield by 50% compared with the control exposed to 1000 μmol photons m^?2 s^?1 for 1 h at pH 9.0 ± 0.2, which can be reached in freshwater lakes. Furthermore, the tolerant abilities to NH₃ toxicity of 18 freshwater algal species or strains were as follows: hypertrophication species > eutrophication species > mesotrophication species > oligotrophication species. The different sensitivities of NH₃ toxicity in this study could well explain the distributing rule of common algal species in the freshwater lakes of different trophic states. Meanwhile, the cyanobacterial bloom (e.g. M. aeruginosa) always happened at the low concentration of ammonia in summer, and disappeared with the decrease of ammonia. This may be attributed to the toxic effect of ammonia to M. aeruginosa in spring (the average and maximum ammonia concentration were 0.08 and 0.72 mmol L^?1 in 33 Chinese lakes), and the low level of NH₃‐N in summer and fall in the lakes might be used as preferred nitrogen nutrition by M. aeruginosa, rather than with toxicity. Therefore, ammonia could be a key factor to determine the distribution of common algal species and cyanobacterial bloom in the freshwater systems.     

Copper Uptake by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Infected Burn Patients

Pseudomonas aeruginosa was isolated from infected burn patients and characterized by standard biochemical tests. The in vitro copper uptake was compared between this isolated pathogenic strain and two non-pathogenic control strains of Gram positive bacteria Bacillus thuringiensis strain Israelis as well as Gram negative bacteria Enterobacter aerogenes. Maximum copper uptake of 470 ppm/g biomass was obtained by P. aeruginosa strain, while the control strains B. thuringiensis and Enterobacter aerogenes had copper uptake of 350 and 383 ppm/g biomass, respectively. However, the lowest copper uptake (60 ppm/g biomass) was observed with another control the saprophytic strain Pseudomonas (Shewanella) putrefaciens. A further investigation regarding the effect of copper toxicity on bacterial growth, gave an MIC score of 600 ppm for P. aeruginosa strain compared to 460 and 300 ppm for the two Gram positive and Gram negative control strains, respectively. In tandem with these in vitro findings, blood analysis on burn patients infected with P. aeruginosa has indicated a selective decrease of copper (hypocupremia) and ceruloplasmin plasma levels. The iron metabolism was also affected by this copper deprivation leading to a similar decrease in plasma levels of PCV, iron, total iron binding capacity, and transferrin. All these hematological changes were significantly different (P < 0.05) from the matched group of non-infected burn patients. The observed hypocupremia in infected burn patients was attributed to demanding scavenger ability by P. aeruginosa strain for the copper of plasma.     

Phosphorus cycling between the colonial cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and attached bacteria, <Emphasis Type="Italic">Pseudomonas</Emphasis>

Phosphorus (P) transfer between Microcystis aeruginosa and the attached bacterium Pseudomonas was studied using radioactive P (³²P) and green fluorescence protein-labeled Pseudomonas. M. aeruginosa in P-starved condition took up most ³²P (70%) in water and about 50% of ³²P in ³²P-saturated bacteria in individual experiments. However, only 26% of ³²P in the ³²P-saturated M. aeruginosa was transferred to P-starved bacteria. The P-starved M. aeruginosa had an advantage to take up P over the bacteria and its growth rates and abundance were higher in combined cultures, with bacteria as the biotic P source. The rate of P transfer from bacteria to the cyanobacteria was slow. P cycles predominantly between M. aeruginosa and Pseudomonas with little variation in the water. This ability is very useful for the colony-forming M. aeruginosa, especially if phosphate concentrations in water are low during water bloom periods.     

Antagonistic effect of divalent cations Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> on the morphological development of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The automated docking program DOCK 5.3.0 was applied to screening for quorum sensing inhibitors (QSIs) of Peudomonus aeruginosa from a database containing 51 active components of Traditional Chinese Medicines with antibacterial activity. Five potential QSIs were revealed by the computer-based virtual screening. The compounds 3, 4, 5, 6, 7 inhibit biofilm formation of P. aeruginosa at a concentration of 200 μM. Compound 4 (baicalein) does not inhibit the growth of P. aeruginosa; however, it significantly inhibits biofilm formation of the bacteria at a lower concentration of 20 μM and promoted proteolysis of the signal receptor TraR protein in Escherichia coli at 4–40 mM. Baicalein and ampicillin showed synergistic activity against P. aeruginosa. These results suggested that baicalein can interfere with quorum sensing system of P. aeruginosa and will be developed as antibacterial agent with novel target.     

Effect of mixed diets (cyanobacteria and green algae) on the population growth of the cladocerans <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis> and <Emphasis Type="Italic">Moina macrocopa</Emphasis>

Microcystis aeruginosa, a cosmopolitan form, is a colonial cyanobacterium, which is also common in many freshwater bodies in Mexico. In eutrophic water bodies cyanobacteria are often the main phytoplankton that co-exist with cladocerans. We evaluated the effect of mixed diets, comprising 0, 25, 50, 75, and 100% on dry weight basis of M. aeruginosa, and the rest of one of two green algal species (Chlorella vulgaris and Scenedesmus acutus), on the population growth of the cladocerans Ceriodaphnia dubia and Moina macrocopa. Regardless of the share of M. aeruginosa in the mixed diet, C. dubia fed Chlorella had a longer initial lag phase. However, in mixed diet with S. acutus, the lag phase of C. dubia increased with increasing proportion of M. aeruginosa. When raised on 100% M. aeruginosa, the population growth of C. dubia was lowered compared with 100% S. acutus or 100% C. vulgaris. Increased proportion of M. aeruginosa in the mixed diet also resulted in decreased abundance of M. macrocopa. Irrespective of diet type, M. macrocopa had a shorter lag phase than C. dubia. Depending on the diet type, the rate of population increase (r) of C. dubia varied from 0.07 to 0.26 d⁻¹ while that of M. macrocopa was higher (0.14–0.61 d⁻¹). For both cladoceran species, the lower r values were obtained when fed Microcystis. Our study showed that the strain of M. aeruginosa was not highly toxic to cause total elimination of either C. dubia or M. macrocopa. Addition of a green algal component to the diet improved the population growth rates of both cladoceran species.     

Effects of Iron on Growth,Pigment Content,Photosystem II Efficiency,and Siderophores Production of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Microcystis wesenbergii</Emphasis>

Changes in growth, photosynthetic pigments, and photosystem II (PS II) photochemical efficiency as well as production of siderophores of Microcystis aeruginosa and Microcystis wesenbergii were determined in this experiment. Results showed growths of M. aeruginosa and M. wesenbergii, measured by means of optical density at 665 nm, were severely inhibited under an iron-limited condition, whereas they thrived under an iron-replete condition. The contents of chlorophyll-a, carotenoid, phycocyanin, and allophycocyanin under an iron-limited condition were lower than those under an iron-replete condition, and they all reached maximal contents on day 4 under the iron-limited condition. PS II photochemical efficiencies (maximal PS II quantum yield), saturating light levels (I _k ) and maximal electron transport rates (ETR_max) of M. aeruginosa and M. wesenbergii declined sharply under the iron-limited condition. The PS II photochemical efficiency and ETR_max of M. aeruginosa rose , whereas in the strain of M. wesenbergii, they declined gradually under the iron-replete condition. In addition, I _k of M. aeruginosa and M. wesenbergii under the iron-replete condition did not change obviously. Siderophore production of M. aeruginosa was higher than that of M. wesenbergii under the iron-limited condition. It was concluded that M. aeruginosa requires higher iron concentration for physiological and biochemical processes compared with M. wesenbergii, but its tolerance against too high a concentration of iron is weaker than M. wesenbergii.     

The effect of <Emphasis Type="Italic">Poterioochromonas</Emphasis> abundance on production of intra- and extracellular microcystin-LR concentration

Due to its capability for producing various microcystins, Microcystis aeruginosa is recognized as one of the most toxic, bloom-forming cyanobacteria. In this study, the fates of intra- and extracellular microcystin-LR (MC-LR) were investigated when the mixotrophic golden alga Poterioochromonas sp. (ZX1) was grazing on M. aeruginosa cells. In the control groups, the total MC-LR concentration increased with the growth of M. aeruginosa with an MC-LR content per cell of 0.5–1.5 × 10⁻⁸ μg cell⁻¹. In the treatment with ZX1, the total MC-LR decreased linearly throughout the incubation period. In particular, intracellular MC-LR disappeared with a loss of M. aeruginosa cells in the first few days. Part of the intracellular MC-LR was released to the medium under the grazing stress, resulting in an increase of extracellular MC-LR. The degradation rate of MC-LR was positively related to the initial abundance of ZX1 and negatively related to that of M. aeruginosa. The inhibition ratio of MC-LR production dropped sharply from 98 to 67% when the initial abundance of M. aeruginosa increased from 10⁶ to 10⁷ cells ml⁻¹. However, it increased from 84 to 99% when the initial ZX1 abundance increased from 10⁴ to 10⁵ cells ml⁻¹. The effective removal of both M. aeruginosa cells and MC-LR was observed under lower M. aeruginosa abundance (<10⁶ cells ml⁻¹) and higher ZX1 abundance (>1% of M. aeruginosa abundance). Light had little impact on MC-LR degradation, but MC-LR degradation decreased due to the loss of ZX1 after 10 days of darkness. This study showed that the interactions between M. aeruginosa and ZX1 were strongly influenced by their initial abundances.     

A fast algal bioassay for assessment of copper toxicity in water using <Emphasis Type="Italic">Euglena gracilis</Emphasis>

A rapid, sensitive algal bioassay was investigated to monitor copper toxicity using photosynthetic activity and motility parameters as end points in the photosynthetic flagellate Euglena gracilis. Effects on motility parameters were determined using the automatic bioassay ECOTOX. Photosynthetic efficiency was measured using chlorophyll fluorescence by means of a fast, noninvasive pulse amplitude modulated fluorometer. These parameters were assessed regarding their effectiveness as end points for short- (0–24 h) and long-term (1–5 days) toxicity tests. The model organism showed significant responses to the tested concentrations at both motility and photosynthesis examined levels. EC₅₀ values for movement parameters immediately after the cells were mixed with copper are 19.09 (300.4 μM), 20.4 (312 μM) and 23.09 (363.35 μM) mg L⁻¹ for motility, r-value and velocity, respectively. The current study proofs that Euglena is a convenient biotest organism where motility parameters show rapid sensitive responses to copper (direct exposure to copper). Also, the photosynthetic parameters appear to be appropriate for acute and chronic toxicity tests for higher and lower copper concentrations, respectively.     

Effects of Calcium Levels on Colonial Aggregation and Buoyancy of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Colonial aggregation of Microcystis plays a key role in bloom formation. Limited studies have been reported about effects of environmental factors on the aggregation of Microcystis. Calcium is an important chemical element in water system. In this study, we investigated the effects of a low- (0.015 g l⁻¹) and a high-concentration of calcium (0.100 g l⁻¹) on the aggregation and buoyancy of a colonial strain M. aeruginosa XW01. Results show that compared to the low concentration of calcium, the high-calcium condition results in bigger colonial size, higher level of buoyancy and increased production of extracellular polysaccharides (EPS) of M. aeruginosa XW01. Increased production of EPS induced by the high-calcium concentration should contribute to the colonial aggregation and buoyancy of M. aeruginosa XW01. These results suggest that an increase in calcium concentration may be beneficial for Microcystis blooms occurring in a soft water lake.     

Quantitative studies on phosphorus transference occuring between <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and its attached bacterium (<Emphasis Type="Italic">Pseudomonas</Emphasis> sp.)

Phosphorus release from Microcystis aeruginosa and attached bacterium (Pseudomonas sp.) isolated from Lake Taihu was examined using a phosphorus isotope tracer in order to investigate the phosphorus transference between the two species. Our results reveal that the amount of phosphorus released form ³²P-saturated M. aeruginosa is determined by its growth phase and most of phosphorus is assimilated by Pseudomonas finally while the amount of phosphorus released from ³²P-saturated Pseudomonas is also determined by the growth phase of M. aeruginosa and most of them are assimilated by M. aeruginosa. The results suggest that phosphorus transference occurs between M. aeruginosa and its attached Pseudomonas . This process makes microenvironment of mucilage of M. aeruginosa attached bacteria maintain relative high amounts of phosphorus. Attached bacteria may be a temporary phosphorus bank to the growth of M. aeruginosa, and assimilation of phosphorus by M. aeruginosa becomes easy when M. aeruginosa is in lag growth phase. Thus, the phosphorus exchange between M. aeruginosa and attached Pseudomonas in microenvironment may be important to microfood web and cyanobacteria bloom.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> siderophores in the sputum of patients with cystic fibrosis

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.     

Antialgal activity of a hepatotoxin-producing cyanobacterium,Microcystis aeruginosa

Antimicrobial activity of toxin produced by a freshwater bloom-forming cyanobacterium Microcystis aeruginosa has been studied. When tested against certain green algae, cyanobacteria, heterotrophic bacteria and fungi, the toxin inhibited growth of only green algae and cyanobacteria. The toxin has been partially purified employing Thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC) techniques and appears to be microcystin-LR (leucine–arginine). Both crude and purified toxins showed toxicity to mice, the clinical symptoms in test mice being similar to those produced by hepatotoxin. Purified toxin at a concentration of 50 g ml^–1 caused complete inhibition of growth followed by cell lysis in Nostoc muscorum and Anabaena BT1 after 6 days of toxin addition. Addition of toxin (25 g ml^–1) to the culture suspensions of the Nostoc and Anabaena strains caused instant and drastic loss of O₂ evolution. Furthermore a marked reduction (about 87%) in the ¹⁴CO₂ uptake was also observed at a concentration of 50 g ml^–1. Besides its inhibitory effects on photosynthetic processes, M. aeruginosa toxin (50 g ml^–1) also caused 90% loss of nitrogenase activity after 8 h of its addition. Experiments performed with ¹⁴C-labelled toxin indicate that the toxin uptake by cyanobacterial cells occurs both in light and dark. These results demonstrate that the toxin is strongly algicidal and point to the possibility that it may have an important role in establishment and maintenance of toxic blooms of M. aeruginosa in freshwater ecosystems. The relative significance of the hepatotoxic effect and the algicidal effect of the toxin is discussed with reference both to survival and dominance of M. aeruginosa in nature.     

Effect of Filtered Cultures of Flagellate Ochromonas sp. on Colony Formation in Microcystis aeruginosa

The possible effect of filtered cultures of flagellate Ochromonas sp. on colony formation in M. aeruginosa was investigated in this paper. The results show that filtered cultures of flagellates fed with M. aeruginosa could induce colony formation in M. aeruginosa. Furthermore, induction strength is clearly dependent on the concentration of flagellates and filtered cultures. However, no colonial M. aeruginosa was found in the treatments of filtered cultures of flagellates fed with Microcystis wesenbergii, filtered cultures of flagellate fed with Chlorella pyrenoidosa, and algae homogenates. This suggests that infochemicals released from flagellates fed with M. aeruginosa may be a trigger for colony formation in M. aeruginosa. The clearance rates of flagellates on algae were markedly decreased when they were cultivated with induced colonial M. aeruginosa. These indicate that colony formation in M. aeruginosa is a predator‐induced defense which could reduce predation risk from flagellates (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Plasmid distribution among unicellular and filamentous toxic cyanobacteria

Plasmid content of 5 hepatotoxin and 2 neurotoxin producing cyanobacterial strains were analyzed. Among the hepatotoxin-producing strains, Microcystis aeruginosa PCC7820, M. aeruginosa M228 and M. aeruginosa UV027 were found to carry plasmids, whereas other hepatotoxin and neurotoxin producing strains did not harbor any plasmids. Correlations were sought between toxicity and the presence of plasmids in toxic cyanobacteria as a function of age. Aged cultures of M. aeruginosa PCC7820 exhibited toxicity and harbored plasmids. In other cyanobacterial strains, plasmids were not detected. The data add to and support the current understanding that plasmids are probably not involved in toxin production in cyanobacteria.Author for Correspondence     

Strain-specific Influence of Microcystis Aeruginosa on Food Ingestion and Assimilation of some Cladocerans and Copepods

Ingestion rates where estimated for daphnids, Cyclops spp. and Bosmina (Eubosmina) coregoni thersites fed hepatotoxic and non-toxic M. aeruginosa either separate or mixed with the readily available food alga Ankistrodesmus falcatus. The ingestion rates of hepatotoxic strains of M. aeruginosa are very low compared with those of A. falcatus or non-toxic M. aeruginosa HUB 5-3 fed to Daphnia magna or D. longispina. However, a close relationship between ingestion rate of different M. aeruginosa strains and their toxicity could not be observed. Addition of the toxic strain M. aeruginosa HUB 5-2-4 reduces the ingestion rates of A. falcatus progressively due to increased food rejection by D. magna. Additionally, the assimilation efficiency of M. aeruginosa HUB 5-2-4 is two times lower compared with A. falcatus and M. aeruginosa HUB 5-3 leading to strong starvation.     

The effects of Anabaena spiroides (Cyanophyceae) exopolysaccharide on copper toxicity to Simocephalus serrulatus (Cladocera, Daphnidae)

1. This work investigated the consumption of a microalgal (Anabaena spiroides) exopolysaccharide by the cladoceran Simocephalus serrulatus (Cladocera, Daphnidae) and its effect on copper toxicity. 2. Total organic carbon concentration was used to quantify the microalgal exopolysaccharide. Both copper‐complexed exopolysaccharides and copper‐free exopolysaccharides were taken up by S. serrulatus. 3. A reduction of free copper ions was obtained in the presence of A. spiroides exopolysaccharide. Copper toxicity (EC₅₀) and zooplankton concentrations were inversely correlated to exopolysaccharide concentration in experimental media. 4. An increase of free copper ions in experimental media was obtained after exopolysaccharide consumption, suggesting that S. serrulatus excreted copper to the surrounding environment.     

Growth characteristics and growth modeling of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Planktothrix agardhii</Emphasis> under iron limitation

Although iron is a key nutrient for algal growth just as are nitrogen and phosphorus in aquatic systems, the effects of iron on algal growth are not well understood. The growth characteristics of two species of cyanobacteria, Microcystis aeruginosa and Planktothrix agardhii, in iron-limited continuous cultures were investigated. The relationships between dissolved iron concentration, cell quota of iron, and population growth rate were determined applying two equations, Monod’s and Droop’s equations. Both species produced hydroxamate-type siderophores, but neither species produced catechol-type siderophores. The cell quota of nitrogen for both M. aeruginosa and P. agardhii decreased with decreasing cell quota of iron. The cell quota of phosphorus for M. aeruginosa decreased with decreasing cell quota of iron, whereas those for P. agardhii did not decrease. Iron uptake rate was measured in ironlimited batch cultures under different degrees of iron starvation. The results of the iron uptake experiments suggest that iron uptake rates are independent of the cell quota of iron for M. aeruginosa and highly dependent on the cell quota for P. agardhii. A kinetic model under iron limitation was developed based on the growth characteristics determined in our study, and this model predicted accurately the algal population growth and iron consumption. The model simulation suggested that M. aeruginosa is a superior competitor under iron limitation. The differences in growth characteristics between the species would be important determinants of the dominance of these algal species.     

Evolutionary Changes in Growth Rate and Toxin Production in the Cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> Under a Scenario of Eutrophication and Temperature Increase

Toxic blooms of the cyanobacterium Microcystis aeruginosa affect humans and animals in inland water systems worldwide, and it has been hypothesized that the development of these blooms will increase under the future scenario of global change, considering eutrophication and temperature increase as two important consequences. The importance of genetic adaptation, chance and history on evolution of growth rate, and toxin production of M. aeruginosa was studied under these new conditions. The experiment followed the idea of “replaying life’s tape” by means of the simultaneous propagation of 15 independent isolates of three M. aeruginosa strains, which were grown under doubled nutrient concentration and temperature during c. 87 generations. Adaptation by new mutations that resulted in the enhancement of growth rate arose during propagation of derived cultures under the new environmental conditions was the main component of evolution; however, chance also contributed in a lesser extension to evolution of growth rate. Mutations were selected, displacing the wild-type ancestral genotypes. In contrast, the effect of selection on mutations affecting microcystin production was neutral. Chance and history were the pacemakers in evolution of toxin production. Although this study might be considered an oversimplification of the reality, it suggest that a future scenario of global change might lead to an increase in M. aeruginosa bloom frequency, but no predictions about the frequency of toxicity can be made.     

COPPER TOXICITY TO SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1,2

Copper toxicity to Skeletonema costatum (Grev.) Cleve has been studied in batch cultures of chemically defined culture media. The alga is relatively insensitive to cupric ion activity, demonstrating no effect on growth up to (Cu²⁺) = 10^?8.5 M. Cultures inoculated from stationary phase stocks exhibit a prolongation of the lag phase with increasing copper concentrations near and above the point of precipitation of the copper. The toxicity of copper is a function of the silicic acid concentration in the medium. This effect is observed in a range of Si(OH)₄ concentrations (10^?5 M to 10^?4 M) above known values for the saturation of silicon uptake kinetics, thus suggesting an influence of copper on silicate metabolism.