Reduced forms of nitrogen are a driver of non-nitrogen-fixing harmful cyanobacterial blooms and toxicity in Lake Erie

Western Lake Erie (WLE) experiences anthropogenic eutrophication and annual, toxic cyanobacterial blooms of non-nitrogen (N) fixing Microcystis. Numerous studies have shown that bloom biomass is correlated with an increased proportion of soluble reactive phosphorus loading from the Maumee River. Long term monitoring shows that the proportion of the annual Maumee River N load of non-nitrate N, or total Kjeldahl nitrogen (TKN), has also increased significantly (Spearman's ρ = 0.68, p = 0.001) over the last few decades and is also significantly correlated to cyanobacterial bloom biomass (Spearman's ρ = 0.64, p = 0.003). The ratio of chemically reduced N to oxidized N (TKN:NO3) concentrations was also compared to extracted chlorophyll and phycocyanin concentrations from all weekly sampling stations within WLE from 2009 to 2015. Both chlorophyll (Spearman's ρ = 0.657, p < 0.0001) and phycocyanin (Spearman's ρ = 0.714, p < 0.0001) were significantly correlated with TKN:NO3. This correlation between the increasing fraction of chemically reduced N from the Maumee River and increasing bloom biomass demonstrates the urgent need to control N loading, in addition to current P load reductions, to WLE and similar systems impacted by non-N-fixing, toxin-producing cyanobacteria.     

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.     

Monitoring changing toxigenicity of a cyanobacterial bloom by molecular methods

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the beta and alpha subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.     

16S rDNA analyses of the cyanobacterial microbiota through the water-column in a boreal lake with a metalimnic Planktothrix population

The Planktothrix population in Lake Steinsfjord has attracted particular attention, due to the potential development of toxic blooms. This population is special in the sense that mass developments of Planktothrix occur in the metalimnion. We investigated the distribution of Planktothrix, as well as other cyanobacteria, through the water-column during a Planktothrix mass development at 10-16 m depth. The analyses were done by chlorophyll measurements, microscopy, and by a recently developed 16S rDNA array-based method.These analyses showed that Planktothrix dominated the cyanobacterial community at 11 m, while cyanobacteria belonging to the order Nostocales were predominant at 4 m. The combination of analytical methods presented in this work provides a powerful tool to analyze cyanobacterial communities. We have developed a concept that enables both relative (16S rDNA array analyses) and absolute quantification (chlorophyll a measurements) of cyanobacteria through water-columns. Such approaches will be important in better understanding cyanobacterial microbiota and bloom dynamics.     

From Bacteria to Fish: Ecological Consequences of Seasonal Hypoxia in a Great Lakes Estuary

The occurrence of bottom-water hypoxia is increasing in bodies of water around the world. Hypoxia is of concern due to the way it negatively impacts lakes and estuaries at the whole ecosystem level. During 2015, we examined the influence of hypoxia on the Muskegon Lake ecosystem by collecting surface- and bottom-water nutrient samples, bacterial abundance counts, benthic fish community information, and performing profiles of chlorophyll and phycocyanin as proxies for phytoplankton and cyanobacterial growth, respectively. Several significant changes occurred in the bottom waters of the Muskegon Lake ecosystem as a result of hypoxia. Lake-wide concentrations of soluble reactive phosphorus (SRP) and total phosphorus increased with decreasing dissolved oxygen (DO). Bacterial abundance was significantly lower when DO was less than 2.2 mg L^?1. Whereas there were no drastic changes in surface chlorophyll a concentration through the season, phycocyanin increased threefold during and following a series of major wind-mixing events. Phycocyanin remained elevated for over 1.5 months despite several strong wind events, suggesting that high SRP concentrations in the bottom waters may have mixed into the surface waters, sustaining the bloom. The fish assemblage in the hypolimnion also changed in association with hypoxia. Overall fish abundance, number of species, and maximum length all decreased in catch as a function of bottom DO concentrations. The link between hypoxia and wind events appears to serve as a positive feedback loop by continuing internal loading and cyanobacterial blooms in the lake, while simultaneously eroding habitat quality for benthic fish.     

Photosynthetic action spectra and adaptation to spectral light distribution in a benthic cyanobacterial mat

We studied adaptation to spectral light distribution in undisturbed benthic communities of cyanobacterial mats growing in hypersaline ponds at Guerrero Negro, Baja California, Mexico. Microscale measurements of oxygen photosynthesis and action spectra were performed with microelectrodes; spectral radiance was measured with fiber-optic microprobes. The spatial resolution of all measurements was 0.1 mm, and the spectral resolution was 10 to 15 nm. Light attenuation spectra showed absorption predominantly by chlorophyll a (Chl a) (430 and 670 nm), phycocyanin (620 nm), and carotenoids (440 to 500 nm). Blue light (450 nm) was attenuated 10-fold more strongly than red light (600 nm). The action spectra of the surface film of diatoms accordingly showed activity over the whole spectrum, with maxima for Chl a and carotenoids. The underlying dense Microcoleus population showed almost exclusively activity dependent upon light harvesting by phycobilins at 550 to 660 nm. Maximum activity was at 580 and 650 nm, indicating absorption by phycoerythrin and phycocyanin as well as by allophycocyanin. Very little Chl a-dependent activity could be detected in the cyanobacterial action spectrum, even with additional 600-nm light to excite photosystem II. The depth distribution of photosynthesis showed detectable activity down to a depth of 0.8 to 2.5 mm, where the downwelling radiant flux at 600 nm was reduced to 0.2 to 0.6% of the surface flux.     

Photosynthetic action spectra and adaptation to spectral light distribution in a benthic cyanobacterial mat.

We studied adaptation to spectral light distribution in undisturbed benthic communities of cyanobacterial mats growing in hypersaline ponds at Guerrero Negro, Baja California, Mexico. Microscale measurements of oxygen photosynthesis and action spectra were performed with microelectrodes; spectral radiance was measured with fiber-optic microprobes. The spatial resolution of all measurements was 0.1 mm, and the spectral resolution was 10 to 15 nm. Light attenuation spectra showed absorption predominantly by chlorophyll a (Chl a) (430 and 670 nm), phycocyanin (620 nm), and carotenoids (440 to 500 nm). Blue light (450 nm) was attenuated 10-fold more strongly than red light (600 nm). The action spectra of the surface film of diatoms accordingly showed activity over the whole spectrum, with maxima for Chl a and carotenoids. The underlying dense Microcoleus population showed almost exclusively activity dependent upon light harvesting by phycobilins at 550 to 660 nm. Maximum activity was at 580 and 650 nm, indicating absorption by phycoerythrin and phycocyanin as well as by allophycocyanin. Very little Chl a-dependent activity could be detected in the cyanobacterial action spectrum, even with additional 600-nm light to excite photosystem II. The depth distribution of photosynthesis showed detectable activity down to a depth of 0.8 to 2.5 mm, where the downwelling radiant flux at 600 nm was reduced to 0.2 to 0.6% of the surface flux.     

Using Landsat and in situ data to map turbidity as a proxy of cyanobacteria in a hypereutrophic Mediterranean reservoir

Satellite remote estimates of phycocyanin (PC) have become valuable for monitoring the quality of inland waters affected by harmful cyanobacterial blooms. In this study, we developed an algorithm for mapping turbidity as a proxy of PC content through Landsat 8 Operational Land Imager (OLI) data and in situ measurements. The chosen study site is Karaoun Reservoir, in Lebanon, a hypereutrophic freshwater body where turbidity is mostly driven by cyanobacteria. Satellite images were corrected for atmospheric effects with the 6S (Second Simulation of the Satellite Signal in the Solar Spectrum) code which proved to be more accurate than the DOS (Dark Object Subtraction) approach with R = 0.98 and R = 0.5, respectively. A strong relationship was found between turbidity and PC measurements (R = 0.92, R² = 0.86), as well as between turbidity and the ratio of band 5 to band 4 of the OLI (R = 0.88, R² = 0.77). Results reveal a promising performance of the algorithm for predicting PC concentrations with high correlations determined through simple linear regression analysis for both the calibration (R = 0.92, R² = 0.85) and validation (R = 0.88, R² = 0.78) periods. An application of the approach to a set of historical Landsat images revealed a time series of cyanobacterial bloom occurrence with high variation in surface area at the study site. The algorithm is considered to be suitable for retrieving cyanobacteria in highly eutrophic waters dominated by cyanobacteria where turbidity is mostly a function of the latter. This approach will improve monitoring cyanobacterial blooms on a spatial and timely basis.     

Classification trees as a tool for predicting cyanobacterial blooms

Nutrient enrichment of aquatic ecosystems caused dramatic increase in the frequency, magnitude and duration of cyanobacterial blooms. Such blooms may cause fish kills, have adverse health effects on humans and contribute to the loss of biodiversity in aquatic ecosystems. Some 50 eutrophic to hypereutrophic ponds from the Brussels Capital Region (Belgium) were studied between 2003 and 2009. A number of the ponds studied are prone to persistent cyanobacterial blooms. Because of the related health concerns and adverse effects on ecological quality of the affected ponds, a tool for assessment of the risk of cyanobacterial bloom occurrence was needed. The data acquired showed that cyanobacteria have threshold relationships with most of the environmental factors that control them. This is negatively reflected on the predictive capacity of conventional statistical methods based on linear relationships. Therefore, classification trees designed for the treatment of complex data and non-linear relationships were used to assess the risk of cyanobacterial bloom occurrence. The main factors determining cyanobacterial bloom development appeared to be phytoplankton biomass, pH and, to a lesser degree, nitrogen availability. These results suggest that to outcompete eukaryotic phytoplankters cyanobacteria need the presence of environmental constraints: carbon limitation, light limitation and nitrogen limitation, for which they developed a number of adaptations. In the absence of constraints, eukaryotic phytoplankters appear to be more competitive. Therefore, prior build up of phytoplankton biomass seems to be essential for cyanobacterial dominance. Classification trees proved to be an efficient tool for the bloom risk assessment and allowed the main factors controlling bloom development to be identified as well as the risk of bloom occurrence corresponding to the conditions determined by these factors to be quantified. The results produced by the classification trees are consistent with those obtained earlier by probabilistic approach to bloom risk assessment. They can facilitate planning management interventions and setting restoration priorities.     

Acclimation Processes in the Light-Harvesting System of the Cyanobacterium Anacystis nidulans following a Light Shift from White to Red Light

Cyanobacteria acclimate to changes in light by adjusting the amounts of different cellular compounds, for example the light-harvesting macromolecular complex. Described are the acclimatization responses in the light-harvesting system of the cyanobacterium Anacystis nidulans following a shift from high intensity, white light to low intensity, red light.

The phycocyanin and chlorophyll content and the relative amount of the two linker peptides (33 and 30 kilodaltons) in the phycobilisome were studied. Both the phycocyanin and chlorophyll content per cell increased after the shift, although the phycocyanin increased relatively more. The increase in phycocyanin was biphasic in nature, a fast initial phase and a slower second phase, while the chlorophyll increase was completed in one phase. The phycocyanin and chlorophyll responses to red light were immediate and were completed within 30 and 80 hours for chlorophyll and phycocyanin, respectively. An immediate response was also seen for the two phycobilisome linker peptides. The amount of both of them increased after the shift, although the 33 kilodalton linker peptide increased faster than the 30 kilodalton linker peptide. The increase of the content of the two linker peptides stopped when the phycocyanin increase shifted from the first to the second phase. We believe that the first phase of phycocyanin increase was due mainly to an increase in the phycobilisome size while the second phase was caused only by an increase in the amount of phycobilisomes. The termination of chlorophyll accumulation, which indicates that no further reaction center chlorophyll antennae were formed, occurred parallel to the onset of the second phase of phycocyanin accumulation.

     

Determination of cyanobacterial diversity during algal blooms in Daechung Reservoir, Korea, on the basis of cpcBA intergenic spacer region analysis

The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.     

低功率高频超声抑制蓝藻生长的研究

为防治蓝藻水华,从超声的生物效应出发提出了新的抑藻思路。低功率高频（1.7MHz)超声高效节能地破坏藻胆体和叶绿素等蓝藻天线复合物的关键组分,或抑制其生物合成,导致光合作用受阻,从而抑制蓝藻生长。在纯顶螺旋藻对照实验中,5min超声辐照为最佳处理时间,可显著降低蓝藻浓度,并使其生长速度大大减缓。实验发现藻蓝蛋白受到的超声破坏作用尤其强烈,即高频超声对蓝藻细胞不同成分的破坏具有选择性,据此提出了高频超声量子效应的解释。     

有害藻华预警预测技术研究进展

近年来有害藻华频繁发生且危害严重,对有害藻华预警预测技术的研究可为有害藻华的预警预报、生态学防治及防灾减灾提供借鉴.本文从有害藻华的运动预测预警、指标临界值预警、数据驱动模型和生态数学模型4个方面介绍了国内外有害藻华的预警技术研究进展,分析了各类预警技术的优劣,并提出了基于细胞特征预测蓝藻生长速率以及基于藻类群落特征预警蓝藻水华的新思路.     

Fluctuation of algal alkaline phosphatase activity and the possible mechanisms of hydrolysis of dissolved organic phosphorus in Lake Barato

For freshwater cyanobacteria, the autofluorescence of phycocyanin is quite high while the in vivo fluorescence (IVF) yield of chlorophyll-a is relatively low, apparently because of low chlorophyll concentrations associated with photosystem II. In eucaryotic phytoplankton, even those with phycobili-protein accessory pigments (e.g. some cryptophytes), the opposite is true. Thus, an IVF ratio which relates phycocyanin to chlorophyll-a signals could be a good index of relative cyanobacterial abundance in the field. Spectrofluorometric scans of whole cells from laboratory cultures indicated that the ratio Em₆₆₀ @ Ex₆₃₀/Em₆₈₀ @ Ex₄₃₀ could be a very sensitive cyanobacterial indicator. Tandem flowthrough fluorometers were then fitted with the appropriate interference filters and their discriminatory power was evaluated with mixtures of cyanobacterial and eucaryotic phytoplankton. Although subject to many of the constraints of other IVF assays, tandem fluorometry should be particularly appropriate for real-time mapping of the relative spatial and temporal distributions of broad phytoplankton taxa in continuous vertical of horizontal profiles in lakes.     

Rainfall, phycocyanin, and N:P ratios related to cyanobacterial blooms in a Korean large reservoir

Nutrient concentrations and other environmental factors were measured in the Daechung Reservoir for 25 weeks from spring until autumn in 1999. The high irradiance after heavy rainfall provided optimal meteorological conditions for bloom formation during summer, therefore, rain would also appear to forecast imminent bloom. The bloom formation was largely governed by cyanobacteria, in particular, Microcystis spp. and Anabaenaspp. Phycocyanin showed higher correlation with cyanobacteria (r = 0.744, P < 0.001) compared to chlorophyll-a(r = 0.599, P < 0.01). Therefore, phycocyanin was more accurate and useful than chlorophyll-a in quantitatively measuring cyanobacterial blooms. The atomic N:P ratio of the particulate form also showed a high correlation with cyanobacteria (r = 0.541, P < 0.01), increasing from 4.3 to 14.6 during bloom formation, while that of the dissolved form decreased from 25.5 to 8.7. These results indicated that the algae assimilated N significantly without comparable P uptake during the blooming season, which was in sharp contrast to the excessive storage of P during the spring.     

Ten-year survey of cyanobacterial blooms in Ohio’s waterbodies using satellite remote sensing

Cyanobacterial blooms are on the rise globally and are capable of adversely impacting human, animal, and ecosystem health. Blooms dominated by cyanobacteria species capable of toxin-production are commonly observed in eutrophic freshwater. The presence of cyanobacterial blooms in selected Ohio lakes, such as Lake Erie and Grand Lake St. Marys, has been well studied, but much less is known about the geographic distribution of these blooms across all of Ohio’s waterbodies. We examined the geographic distribution of cyanobacterial blooms in Ohio’s waterbodies from 2002 to 2011, using a nested semi-empirical algorithm and remotely sensed data from the Medium Resolution Imaging Spectrometer (MERIS) onboard the European Space Agency’s Envisat. We identified: 62 lakes, reservoirs, and ponds; 7 rivers; 6 marshes and wetlands; and 3 quarries with detectable cyanobacteria pigment (phycocyanin) concentrations. Of the 78 waterbodies identified in our study, roughly half (54%; n = 42) have any reported in situ microcystins monitoring results from state monitoring programs. Further, 90% of the waterbodies identified reached phycocyanin pigment concentrations representative of levels potentially hazardous to public health. This gap in lakes potentially impacted by cyanobacterial blooms and those that are currently monitored presents an important area of concern for public health, as well as ecosystem health, where unknown human and animal exposures to cyanotoxins may occur in many of Ohio’s waterbodies. Our approach may be replicated in other regions around the globe with potential cyanobacterial bloom presence, in order to assess the intensity, geographic distribution, and temporal pattern of blooms in lakes not currently monitored for the presence of cyanobacterial blooms.     

Allelopathic inhibitory effect of Myriophyllum aquaticum (Vell.) Verdc. on Microcystis aeruginosa and its physiological mechanism

Eutrophication and algal blooms are the most serious environmental problems in the world, and biological tools, especially the allelopathic inhibitory effects of aquatic macrophytes on phytoplankton growth have been receiving world-wide attention. In our experiments, the allelopathic inhibitory effect of Myriophyllum aquaticum culture water on Microcystis aeruginosa and its eco-physiological mechanism were investigated by initial addition assays and continuous addition assays. The results showed that the growth of M. aeruginosa was markedly inhibited by M. aquaticum culture water. Compared with initial addition assays, M. aquaticum exhibited stronger inhibitory potential on M. aeruginosa by continuous addition assays, indicating that allelopathic compounds might be excreted continuously by M. aquaticum, and the inhibitory effects would be cumulative. We also found that the relative content of chlorophyll a (Chl a), phycocyanin (PC) and allophycocyanin (APC) of M. aeruginosa decreased to 52.7%, 15.3% and 7.6% respectively after being treated by M. aquaticum culture water for 5 days, and phycobiliprotein (especially APC) decreased more than Chl a. These results suggest that the phycobiliprotein would be the target of allelopathic inhibition of M. aquaticum on M. aeruginosa, and a new macrophyte to control cyanobacterial blooms would be found.     

Effects of iron on growth and reflectance spectrum of the bloom‐forming cyanobacterium Microcystis viridis

Iron is an important factor in algal blooms because it is involved in cyanobacterial pigment biosynthesis and therefore has the ability to influence the pigment status of algal cells. This role in pigment biosynthesis offers the opportunity for rapid monitoring of iron availability to cyanobacteria through spectral reflectance characterization. In the present study, the freshwater cyanobacterium Microcystis viridis was cultured with different levels of iron. Cell density, cellular content of iron and photosynthetic pigments, and spectral reflectivity of M. viridis were determined daily during the course of the culture experiment. The results showed that at the lowest iron concentration (0.01 μM) the growth of M. viridis was seriously limited, and the maximal cell density was only approximately 6.4% of the density observed with an iron concentration of 18 μM. Iron availability dramatically affected chlorophyll a, carotenoid and phycocyanin content, with the greatest impact on chlorophyll a. The iron‐induced changes in content and ratios of pigments were detectable through spectral reflectance. Eleven spectral indices previously developed for the estimation of concentrations and/or ratios of pigments and a newly proposed chlorophyll a/phycocyanin index were found to be suitable for generating sensitive regression models between cellular iron content and spectral parameters. The comprehensive application of key sensitive spectral indices and regression equations should help to support monitoring and diagnosis of iron availability to cyanobacteria via remote sensing.     

Relation between pigment content and photosynthetic characteristics in a blue-green algae

1. The blue-green alga Anacystis nidulans was cultured under steady state conditions at 25 and 39°C. and under several different light intensities to give five different types of cells. 2. Cells were submitted to pigment analysis based upon acetone extracts and aqueous extracts obtained by sonic disintegration. The different cell types show a threefold range of chlorophyll content and a fourfold range of phycocyanin content with only minor changes in the chlorophyll/phycocyanin ratio. Cells of highest pigment content were estimated to contain 2.8 per cent chlorophyll a and 24 per cent phycocyanin, the latter on a total chromoproteid basis. 3. Light intensity curves of photosynthesis were obtained for each of the cell types at 25 and at 39°C. The slopes of the light-limited regions of the curves are approximately linear functions of chlorophyll and phycocyanin contents. Maximum light-saturated rates of photosynthesis at 25 and 39° show no simple relation to pigment content.