Effects of arsenate on the growth and microcystin production of Microcystis aeruginosa isolated from Taiwan as influenced by extracellular phosphate

Arsenic pollution and eutrophication are both prominent issues in the aquaculture ponds of Taiwan. It is important to study the effects of arsenic on algal growth and toxin production in order to assess the ecological risk of arsenic pollution, or at least to understand naturally occurring ponds. The sensitivity of algae to arsenate has often been linked to the structural similarities between arsenate and phosphate. Thus, in this study we examined the effects of arsenate (10⁻⁸ to 10⁻⁴ M) on Microcystis aeruginosa TY-1 isolated from Taiwan, under two phosphate regimes. The present study showed that M. aeruginosa TY-1 was arsenate tolerant up to 10⁻⁴ M, and that this tolerance was not affected by extracellular phosphate. However, it seems that extracellular phosphate contributed to microcystin production and leakage by M. aeruginosa in response to arsenate. Under normal phosphate conditions, total toxin yields after arsenate treatment followed a typical inverted U-shape hormesis, with a peak value of 2.25 ± 0.06 mg L⁻¹ in the presence of 10⁻⁷ M arsenate, whereas 10⁻⁸ to 10⁻⁶ M arsenate increased leakage of ∼75% microcystin. Under phosphate starvation, total toxin yields were not affected by arsenate, while 10⁻⁶ and 10⁻⁵ M arsenate stimulated microcystin leakage. It is suggested that arsenate may play a role in the process of microcystin biosynthesis and excretion. Given the arsenic concentrations in aquaculture ponds in Taiwan, arsenate favors survival of toxic M. aeruginosa in such ponds, and arsenate-stimulated microcystin production and leakage may have an impact on the food chain.     

Cellular microcystin content in N-limited Microcystis aeruginosa can be predicted from growth rate

Cell quotas of microcystin (Q(MCYST); femtomoles of MCYST per cell), protein, and chlorophyll a (Chl a), cell dry weight, and cell volume were measured over a range of growth rates in N-limited chemostat cultures of the toxic cyanobacterium Microcystis aeruginosa MASH 01-A19. There was a positive linear relationship between Q(MCYST) and specific growth rate (mu), from which we propose a generalized model that enables Q(MCYST) at any nutrient-limited growth rate to be predicted based on a single batch culture experiment. The model predicts Q(MCYST) from mu, mu(max) (maximum specific growth rate), Q(MCYSTmax) (maximum cell quota), and Q(MCYSTmin) (minimum cell quota). Under the conditions examined in this study, we predict a Q(MCYSTmax) of 0.129 fmol cell(-1) at mu(max) and a Q(MCYSTmin) of 0.050 fmol cell(-1) at mu = 0. Net MCYST production rate (R(MCYST)) asymptotes to zero at mu = 0 and reaches a maximum of 0.155 fmol cell(-1) day(-1) at mu(max). MCYST/dry weight ratio (milligrams per gram [dry weight]) increased linearly with mu, whereas the MCYST/protein ratio reached a maximum at intermediate mu. In contrast, the MCYST/Chl a ratio remained constant. Cell volume correlated negatively with mu, leading to an increase in intracellular MCYST concentration at high mu. Taken together, our results show that fast-growing cells of N-limited M. aeruginosa are smaller, are of lower mass, and have a higher intracellular MCYST quota and concentration than slow-growing cells. The data also highlight the importance of determining cell MCYST quotas, as potentially confusing interpretations can arise from determining MCYST content as a ratio to other cell components.     

Release of heptapeptide toxin (microcystin) during the decomposition process of Microcystis aeruginosa.

The decomposition process of toxic blue-green alga (cyanobacteria), Microcystis aeruginosa, under dark and aerobic condition was investigated in relation to the change of the amounts of heptapeptide toxins (microcystins YR and LR) by two experiments: one with Microcystis cells and the other with two purified microcystins. In the experiment with Microcystis cells, an increase of heterotrophic bacteria observed from the beginning of the experiment, was followed by decomposition of the algal cells and the subsequent release of microcystins into the filtrate fraction. The amounts of the toxins initially present in the cells were quantitatively detected in the filtrate fraction on the 35th day. The decomposition of microcystin YR began on the 42nd day. The decomposition rate of the two toxins was different. The decomposition rate of purified microcystins YR and LR, compared in distilled water and culture medium, respectively, indicated clearly that microcystin YR was more labile to decomposition than microcystin LR in the culture medium. At the end of the experiment (45th day) microcystin YR decreased to 58.6%, while 86.2% of microcystin LR remained.     

Small water clusters stimulate microcystin biosynthesis in cyanobacterial Microcystis aeruginosa

Recent research has indicated that different scales of water clusters can cause different biological effects from normal water clusters. In this study, we used the cyanobacterium Microcystis aeruginosa FACHB-905 as a model organism to investigate the effect of small water clusters (SWCs) on the growth and toxin production of toxic cyanobacteria. The results showed that SWCs were able to stimulate the growth of M. aeruginosa, which resulted in increased cell numbers and higher specific growth rates after a 20-day treatment. Moreover, the SWCs treatment up-regulated microcystin (MC) synthesis and exudation in 6 days in M. aeruginosa. Subsequently, the intracellular MC content decreased after the 16th day. SWCs had positive effects on the photochemical system as well as the uptake of nitrogen and phosphorus for the majority of the period. Moreover, the cell photosynthetic pigment concentrations were transitorily stimulated by SWCs. It is assumed that SWCs stimulated cell growth by promoting photosynthesis as well as nitrogen and phosphorus uptake, whereas the enhanced MC production is related to pigment concentrations (Chl a and carotenoid). This study reveals that SWCs is a novel environmental factor that stimulates growth and enhances MC production in M. aeruginosa.     

Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.     

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO₂ and high CO₂ conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO₂ conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and \textP_\textm^\textchla {\text{P}}_{\text{m}}^{{\text{chl}}a} , \textR_\textd^\textchla {\text{R}}_{\text{d}}^{{\text{chl}}a} , α^chla than under low CO₂ conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.     

Iron-stimulated toxin production in Microcystis aeruginosa.

Nitrate- and phosphate-limited conditions had no effect on toxin production by Microcystis aeruginosa. In contrast, iron-limited conditions influenced toxin production by M. aeruginosa, and iron uptake was light dependent. A model for production of toxin by M. aeruginosa is proposed.     

Effects of Sb(V) on Growth and Chlorophyll Fluorescence of Microcystis aeruginosa (FACHB-905)

In this study, effects of antimony Sb(V) on growth, pigments content, oxygen evolution, and photosystem II (PSII) activity of Microcystis aeruginosa were investigated. JIP-test, Q _A ^? reoxidation kinetic test and S-state test were used in this study to study the energy distribution and electron transport in PSII. Treatment with Sb(V) at various concentrations ranging from 5 to 100?mg/l had long-term effects on growth, pigments content, and oxygen evolution of M. aeruginosa. Low concentration of Sb(V) had no significant inhibition of the biomass production and PSII activity but inhibited the pigment synthesis. Growth, pigments content, oxygen evolution, and PSII activity were seriously inhibited when treated by high concentration of Sb(V) (100?mg/l). The target sites of Sb(V) toxic effect on the PSII of M. aeruginosa were mainly on the donor side and the apparatus in the light-dependent reaction. The quantum yield for photochemistry, density of reaction centers and photosynthesis performance index decreased, whereas the dissipated energy increased. PSII activity of M. aeruginosa was promoted when exposure to 50?mg/l Sb(V) by increasing the density of active reaction centers and electron transport after Q _A ^? .     

Fur from Microcystis aeruginosa binds in vitro promoter regions of the microcystin biosynthesis gene cluster

Promoter regions of the mcy operon from Microcystis aeruginosa PCC7806, which is responsible for microcystin synthesis in this organism, exhibit sequences that are similar to the sequences recognized by Fur (ferric uptake regulator). This DNA-binding protein is a sensor of iron availability and oxidative stress. In the presence of Fe(2+), a dimer of Fur binds the iron-boxes in their target genes, repressing their expression. When iron is absent the expression of those gene products is allowed. Here, we show that Fur from M. aeruginosa binds in vitro promoter regions of several mcy genes, which suggests that Fur might regulate, among other factors, microcystin synthesis. The binding affinity is increased by the presence of metal and DTT, suggesting a response to iron availability and redox status of the cell.     

Effects of light on the microcystin content of Microcystis strain PCC 7806

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 micro mol of photons m(-2) s(-1) on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell(-1) and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 micro mol of photons m(-2) s(-1), the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 micro mol of photons m(-2) s(-1) than at 10 micro mol of photons m(-2) s(-1) and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.     

水生花卉对铜绿微囊藻、斜生栅藻和小球藻生长的影响

选择黄菖蒲(Iris pseudacorus)、溪荪(I.sanguinea)、梭鱼草(Pontederia cordata)、白花水龙(Jussiaea repens)、水罂粟(Hydrocleys nymphoides)和大藻(Pistia stratiotes)6种具有较高观赏价值的水生花卉,通过将植物种植水与藻类共同培养的方式研究了不同种植时间的种植水对铜绿微囊藻(Microcystis aeruginosa)、斜生栅藻(Scenedesmus obliqnus)和小球藻(Chlorella vulgaris)生长的影响.结果表明:6种水生花卉种植水对3种藻类的化感作用具有选择性.通过6d的处理,种植水对铜绿微囊藻生长的抑制率为31.22％ ～ 96.53％,除白花水龙外,其余5种花卉的种植水对铜绿微囊藻生长的抑制率均超过70％,表现出很好的抑藻效果;种植水对斜生栅藻生长的抑制率为23.15％～77.25％;而种植水对小球藻有抑制也有促进,抑制率为-26.07％ ～75.70％,大藻、梭鱼草和溪荪抑制小球藻的生长,黄菖蒲、白花水龙表现为低促高抑,水罂粟表现为促进作用.随着种植时间的延长,种植水对3种藻类的抑制作用增强.6种水生花卉种植水对铜绿微囊藻生长的抑制作用由大到小依次为水罂粟＞黄菖蒲＞梭鱼草＞大藻＞溪荪＞白花水龙;对斜生栅藻生长的抑制作用由大到小依次为梭鱼草＞溪荪＞黄菖蒲＞水罂粟＞白花水龙＞大藻;对小球藻生长的抑制作用由大到小依次为大藻＞梭鱼草＞溪荪＞黄菖蒲、白花水龙＞水罂粟.试验表明,6种水生花卉在控制城市景观水体中的藻类水华有一定的推广价值.     

Microcystin production by Microcystis aeruginosa in a phosphorus-limited chemostat

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C(18) cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C(18) reverse-phase column. The specific growth rate (mu) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing mu. The MC-LR and MC-RR contents on a dry weight basis were highest at mu of 0.1/day at 339 and 774 microg g(-1), respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower mu, whereas the MC-producing rate was linearly proportional to mu. The C fixation rate at an ambient irradiance (160 microeinsteins m(-2) s(-1)) increased with mu. The ratios of the MC-producing rate to the C fixation rate were higher at a lower mu. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.     

Effects of Environmental Factors on Toxicity of a Cyanobacterium (Microcystis aeruginosa) under Culture Conditions

Effects of light intensity, temperature, and nutrients on the toxicity of Microcystis aeruginosa were investigated, using a toxic strain which kills mice. A marked change in toxicity was observed in the light intensity experiment, and slight changes were observed to be caused by temperature and phosphorus deficiency.     

Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions

Effects of light intensity, temperature, and nutrients on the toxicity of Microcystis aeruginosa were investigated, using a toxic strain which kills mice. A marked change in toxicity was observed in the light intensity experiment, and slight changes were observed to be caused by temperature and phosphorus deficiency.     

铜绿微囊藻(Microcystis aeruginosa)对惠氏微囊藻(Microcystis wesenbergii)的化感作用

通过混合培养和添加过滤液两种方式观察铜绿微囊藻和惠氏微囊藻的生长曲线,探讨两种微囊藻之间的化感作用。结果表明:在混合培养条件下,两者能够形成相互抑制作用;当两者起始藻密度高于0.5×106cells.mL-1、混合比为1:1时,惠氏微囊藻的生长因化感作用而受到显著抑制(P<0.05),同时惠氏微囊藻也会对铜绿微囊藻产生一定的胁迫作用;处于对数生长期的铜绿微囊藻过滤液能抑制惠氏微囊藻的生长,且惠氏微囊藻起始藻密度低于0.5×106cells.mL-1,连续滴加该过滤液后,其生长受到极显著抑制(P<0.01)。     

Identification and analysis of whole microcystin synthetase genes from two Korean strains of the cyanobacterium Microcystis aeruginosa

Microcystins are cyanobacterial hepatotoxins, and are produced by nonribosomal enzyme complexes, mcy gene cluster. In this study, we report on whole mcy gene clusters from two Korean strains of M. aeruginosa that were blooming in Lake Paldang (FCY-26) and Geum river (FCY-28). Their specific gene locus, amino acid information, and sub-cluster orientation were also characterized in both strains. Both gene clusters are of 55 kb, and also each length, number and the arrangement are identical. Their sequence analysis revealed a cluster of 10 genes (mcyA, B, C, D, E, F, G, H, I, and J) involved in the biosynthesis of microcystin, and mcyABC and mcyDEFGHIJ formed two polycistronic operon structures that are transcribed bidirectionally from a central promoter region between mcyA and mcyD. The analysis of SNPs provided different nucleotide composition and amino acid variations in two Korean strains of M. aeruginosa. This approach is useful to develop genetic indicators identifying toxic cyanobacteria and their cyanotoxins, and helpful for a better understanding of the diversities of mcy gene clusters, the biosynthesis of microcystin, and the mediation of environmental parameters causing algal blooming and HABs.