Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa

A method for the Cryopreservation of Microcystis aeruginosa f. aeruginosa is described. For the five strains tested, dimethyl sulfoxide (DMSO) (3% v/v) was the only effective cryoprotectant for freezing to, and thawing from -196°C and allowed the successful recovery (>50%) of all the strains. The viability of frozen material was independent of the period of storage in liquid nitrogen. The strain NIES-44 (National Institute for Environmental Studies) had a recovery level of greater than 90% at 3–10% (v/v) DMSO in both two step and rapid cooling methods. The other three strains, NIES-87, 88 and 89 had greater than 60% of viability after freeze/thawing in presence of both 3% and 5% DMSO concentrations. On the other hand, the strain NIES-90 showed approximately 50% of viability in only 3% DMSO solution after two step cooling to and thawing from -196°C. This strain was damaged by greater than 4% DMSO and by rapid cooling to -196°C. It was found that cold shock injury and the cytotoxicity of DMSO were different at a strain level.     

Photoinactivation of photosystem II during photoinhibition in the cyanobacterium Microcystis aeruginosa

Sites of photoinhibition and photo-oxidative damage to the photosynthetic electrontransport system of the unicellular cyanobacterium Microcystis aeruginosa were identified by studies of the kinetics of chlorophyll fluorescence induction by whole cells at room temperature and from partial photosynthetic electron-transport reactions in vitro in thylakoid preparations. Chlorophyll fluorescence intensity decreased following photoinhibitory light treatment. This was attributed to decreases both in the activity of photosystem II and in electron flow through the primary electron acceptor, Q. This inhibition was only partially reversed over a 50-min dark recovery period. Partial photosynthetic electron-transport experiments in vitro demonstrated that photosystem I was not affected by the photoinhibitory treatment. Light damage was associated exclusively with the light reactions, of photosystem II, at a site close to the reaction centre, between the site where diphenylcarbazide can donate electrons and the site where silicomolybdate can accept electrons. This damage presumably reduced production of ATP by noncyclic photophosphorylation and production of NADPH by photosystem I, decreasing the availability of these co-factors for reducing CO₂ in the dark reactions of photosynthesis. The importance of these findings is discussed.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenylcarbazide - PSI photosystem I - PSH photosystem II     

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.     

Intraspecific variation in growth and morphology of the bloom-forming cyanobacterium Microcystis aeruginosa

In the laboratory, we documented large variation in the morphology, toxicity, and maximum population growth rates for 32 Microcystis aeruginosa strains isolated from 12 lakes. Growth rates and mean colony sizes varied significantly across strains and were positively correlated. However, growth rates were unrelated to toxin production.     

Inhibition of Chlorella growth by the lipids of cyanobacterium Microcystis aeruginosa

The total lipids of the cyanobacterium Microcystis aeruginosa have been isolated and fractionated into its components. Of these lipid components, only the fatty acid-containing fraction inhibited the growth of the green alga Chlorella pyrenoidosa. The inhibitory activity appears to be due to linoleic and linolenic acids, which are both present in significants quantities. These acids may be the substances responsible for the reported toxicity of Microcystis aeruginosa to Chlorella.     

Investigation of a toxic water-bloom of Microcystis aeruginosa (Cyanophyceae) in Lake Akersvatn,Norway

During the summer and fall of 1984 and 1985, the eutrophic Lake Akersvatn in south-eastern Norway, used as reserve drinking water reservoir, was found to produce heavy water-blooms of the colonial blue-green alga Microcystis aeruginosa. Samples of the water-bloom were found to be toxic using the mouse bioassay. No toxin was found free in the water as detected by HPLC and mouse bioassay. The toxic cells (minimum lethal dose 8–15 mg/kg body weight in mice) and purified toxin (minimum lethal dose 50 μg/kg body weight in mice) showed signs of poisoning in laboratory rats and mice identical to that of other hepatotoxin-producing M. aeruginosa blooms and strains reported from other parts of the world. The toxin has chemical properties similar to the cyclic heptapeptide reported for a South African M. aeruginosa toxin. The toxin from Lake Akersvatn bloom material has a molecular weight of 994. The toxic bloom of M. aeruginosa persisted from August to November in 1984 and reappeared in July of 1985. While water from Lake Akersvatn was not used for municipal water supply during this period, the presence of toxic blue-green algae in a drinking water reservoir indicates the need to develop monitoring and detection methods for toxic cells and toxin(s).     

Characteristics of the freshwater cyanobacterium Microcystis aeruginosa grown in iron-limited continuous culture

A continuous culturing system (chemostat) made of metal-free materials was successfully developed and used to maintain Fe-limited cultures of Microcystis aeruginosa PCC7806 at nanomolar iron (Fe) concentrations (20 to 50 nM total Fe). EDTA was used to maintain Fe in solution, with bioavailable Fe controlled by absorption of light by the ferric EDTA complex and resultant reduction of Fe(III) to Fe(II). A kinetic model describing Fe transformations and biological uptake was applied to determine the biologically available form of Fe (i.e., unchelated ferrous iron) that is produced by photoreductive dissociation of the ferric EDTA complex. Prediction by chemostat theory modified to account for the light-mediated formation of bioavailable Fe rather than total Fe was in good agreement with growth characteristics of M. aeruginosa under Fe limitation. The cellular Fe quota increased with increasing dilution rates in a manner consistent with the Droop theory. Short-term Fe uptake assays using cells maintained at steady state indicated that M. aeruginosa cells vary their maximum Fe uptake rate (ρ(max)) depending on the degree of Fe stress. The rate of Fe uptake was lower for cells grown under conditions of lower Fe availability (i.e., lower dilution rate), suggesting that cells in the continuous cultures adjusted to Fe limitation by decreasing ρ(max) while maintaining a constant affinity for Fe.     

Photoinactivation in vivo of superoxide dismutase and catalase in the cyanobacterium Microcystis aeruginosa

Abstract The planktonic cyanobacterium Microcystis aeruginosa is particularly sensitive to photoinhibition by visible light, Photosystem II and ribulose 1,5-bisphosphate (RuBP) carboxylase activities being affected. Although the organism contains superoxide dismutase (SOD) and catalase, these protective enzymes are also photoinactivated during the illumination of whole cells by visible light.     

Functional analysis of PilT from the toxic cyanobacterium Microcystis aeruginosa PCC 7806

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 +/- 1.8 nmol P(i) min(-1) mg protein(-1), with a requirement for Mg(2+). Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.     

Lack of phylogeographic structure in the freshwater cyanobacterium Microcystis aeruginosa suggests global dispersal

Background

Free-living microorganisms have long been assumed to have ubiquitous distributions with little biogeographic signature because they typically exhibit high dispersal potential and large population sizes. However, molecular data provide contrasting results and it is far from clear to what extent dispersal limitation determines geographic structuring of microbial populations. We aimed to determine biogeographical patterns of the bloom-forming freshwater cyanobacterium Microcystis aeruginosa. Being widely distributed on a global scale but patchily on a regional scale, this prokaryote is an ideal model organism to study microbial dispersal and biogeography.

Methodology/Principal Findings

The phylogeography of M. aeruginosa was studied based on a dataset of 311 rDNA internal transcribed spacer (ITS) sequences sampled from six continents. Richness of ITS sequences was high (239 ITS types were detected). Genetic divergence among ITS types averaged 4% (maximum pairwise divergence was 13%). Preliminary analyses revealed nearly completely unresolved phylogenetic relationships and a lack of genetic structure among all sequences due to extensive homoplasy at multiple hypervariable sites. After correcting for this, still no clear phylogeographic structure was detected, and no pattern of isolation by distance was found on a global scale. Concomitantly, genetic differentiation among continents was marginal, whereas variation within continents was high and was mostly shared with all other continents. Similarly, no genetic structure across climate zones was detected.

Conclusions/Significance

The high overall diversity and wide global distribution of common ITS types in combination with the lack of phylogeographic structure suggest that intercontinental dispersal of M. aeruginosa ITS types is not rare, and that this species might have a truly cosmopolitan distribution.     

Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa

The effects of a cyclic peptide toxin, isolated from the cyanobacterium Microcystis aeruginosa, on cell morphology and ion transport in human erythrocytes, isolated rat hepatocytes and mouse fibroblasts (3T3) were studied. Neither in erythrocytes nor in fibroblasts did the toxin cause morphological alterations. In hepatocytes the toxin induced marked morphological alterations at a concentration of about 50 nM. In erythrocytes and fibroblasts no effects on ion transport were observed. In hepatocytes the toxin induced a significant increase in both phosphate and potassium efflux at concentrations far below the concentration causing morphological alterations (0.1 and 1 nM, respectively). It is suggested that the cytotoxicity of the toxin is not due to a non-specific interaction with the plasma membrane and that the effects of the toxin in hepatocytes are probably due to an interaction of the toxin with cytoskeletal elements.     

Selective inhibitory potential of silver nanoparticles on the harmful cyanobacterium Microcystis aeruginosa

Silver nanoparticles (SNPs) at 1 mg/l inhibited the growth of the toxic cyanobacterium, Microcystis aeruginosa, by 87%. Similar results were obtained in field experiments. M. aeruginosa was more sensitive to SNPs than were green algae. SNPs may be a useful selective biocidal agent for the control of M. aeruginosa.     

Isolation and characterization of a cyanophage infecting the toxic cyanobacterium Microcystis aeruginosa

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.     

Growth inhibition of bloom-forming cyanobacterium Microcystis aeruginosa by rice straw extract

AIMS: To inhibit the growth of the bloom-forming cyanobacterium Microcystis aeruginosa using a rice straw extract. METHODS AND RESULTS: The cell numbers of the algal strain M. aeruginosa UTEX 2388 significantly decreased after treatment with different concentrations (0.01, 0.1, 1 and 10 mg l(-1)) of a rice straw extract for an 8-day cultivation period. Among seven tested allelochemicals from rice straw, salicylic acid at 0.1 mg l(1) exhibited the highest allelopathic activity (26%) on day 8. A synergistic effect on algal growth inhibition was found when adding two or three phenolic compounds from the rice straw. CONCLUSIONS: The growth of M. aeruginosa was inhibited by rice straw extract concentrations ranging from 0.01 to 10 mg l(1). This activity was due to the synergistic effects of various phenolic compounds in the rice straw. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of rice straw as an effective material for the growth inhibition of M. aeruginosa implies it may have the potential to be used as an environment-friendly biomaterial for controlling the algal bloom of M. aeruginosa in eutrophic water.     

Occurrence of mycosporine-like amino acids (MAAs) in the bloom-forming cyanobacterium Microcystis aeruginosa

Here we report the finding of two mycosporine-like amino acids(shinorine and Porphyra-334) in both a culture of the cyanobacteriumMicrocystis aeruginosa isolated from Lake Taihu (China) anda natural phytoplankton sample collected from this lake whichincluded Microcystis spp. Our results are the first to clearlydocument the occurrence of these UV-sunscreen compounds in afreshwater bloom-forming cyanobacterium.     

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.     

Responses of the toxic cyanobacterium Microcystis aeruginosa to iron and humic substances.

Iron is an essential element to marine biota. Different types of dissolved organic matter (DOM), such as humic substances have impacts on the marine coastal waters iron chemistry. The aim of the study was to examine how the presence of humic substances (both aquatic and sedimentary) may affect iron bioavailability to the bloom-forming cyanobacterium Microcystis aeruginosa Kutzing incubated on standard and modified mineral BG-11 media. The final iron concentrations in the growth media ranged from 0.1 to 100microM. The results demonstrate that both the growth rate and the concentration of chlorophyll a in cultures of M. aeruginosa are limited by insufficient (<10microM) Fe concentrations. The addition of aquatic humic substances in the presence of iron in concentrations <0.1microM increased the optical density 25-fold, and the production of chlorophyll a 15-fold as compared with the cultures exposed to iron only at the same concentration. Sedimentary humic acids in the presence of iron at a concentration of 10microM reduced the growth and production of chlorophyll a by 50% as compared to the cultures exposed to iron only at the same concentration. Possible mechanisms of humic substances - metal ion - alga interactions are discussed. It is suggested that aquatic humic substances could be of great importance in the formation of cyanobacteria blooms.     

Activity of some Nile River aquatic macrophyte extracts against the cyanobacterium Microcystis aeruginosa

The anti-algal activity of five macrophyte extracts on the cyanobacterium Microcystis aeruginosa in Egypt was investigated in 2013. Extract activity varied according to plant type, extracting solvent and its concentration. The highest inhibitory activity was achieved with ethanol extract at a concentration of 80 mg l^?1, followed by chloroformic extracts, at 60 mg l^?1. Methanolic extracts of Eichhornia crassipes and Polygonum tomentosum inhibited growth of Microcystis aeruginosa at all concentrations. Acetonic extracts inhibited algal growth at 60 mg l^?1, except for the extract of Ceratophyllum subdemersum, which showed stimulation of M. aeruginosa growth. Eichhornia crassipes ethanolic extract exerted the most powerful inhibition by more than five-fold, 570.17%, followed by those of P. tomentosum, Saccharum spontaneum, Ceratophyllum demersum and C. subdemersum, 559.48, 553.99, 544.11 and 366.51%, respectively. Phytochemical screening for the tested plant extracts revealed the presence of biologically active substances of different concentrations, with P. tomentosum having the highest polyphenols, 1.95% of dry weight.