Article

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments

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Abstract

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W x 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3 x 10(4) cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 microm in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.

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... The cell concentration of algae is generally used to estimate algae biomass and can be measured by several methods such as microscopic counting (Fujimoto and Sudo 1997;Lee et al. 2010;Kim et al. 2010), spectrophotometry (Rohrlack and Hyenstrand 2007;Gremberghe et al. 2009), Casy method (Wiedner et al. 2003;Schober et al. 2007;Rohrlack and Hyenstrand 2007), and flow-cytometer method (Zhou et al. 2012). Among the above mentioned methods, the microscopic counting method is the most essential and frequently used method. ...
... Casy method cannot be used to analyze natural Microcystis colonies due to its measurement limit of 150 lm. The flow-cytometer method is so expensive that it was only used in some scientific studies (Zhou et al. 2012;Zucker and Fisher 2013). The upper measurement limit of 500-1,000 lm of the flow-cytometer method is also a weakness in analysis of large Microcystis colonies (bigger than 1,000 lm frequently, Yu et al. 2007). ...
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The analysis of the cell concentration, volume concentration, and colony size of Microcystis is widely used to provide early warnings of the occurrence of blooms and to facilitate the development of predictive tools to mitigate their impact. This study developed a new approach for the analysis of the cell concentration, volume concentration, and colony size of Microcystis by applying a laser particle analyzer. Four types of Microcystis samples (55 samples in total) were analyzed by a laser particle analyzer and a microscope. By the application of the laser particle analyzer (1) when n = 1.40 and k = 0.1 (n is the intrinsic refractive index, whereas k is absorption of light by the particle), the results of the laser particle analyzer showed good agreement with the microscopic results for the obscuration indicator, volume concentration, and size distribution of Microcystis; (2) the Microcystis cell concentration can be calculated based on its linear relationship with obscuration; and (3) the volume concentration and size distribution of Microcystis particles (including single cells and colonies) can be obtained. The analytical processes involved in this new approach are simpler and faster compared to that by microscopic counting method. From the results, it was identified that the relationship between cell concentration and volume concentration depended on the colony size of Microcystis because the intercellular space was high when the colony size was high. Calculation of cell concentration and volume concentration may occur when the colony size information is sufficient.
... However, Microcystis forms colonies under natural conditions, which must be disintegrated before enumeration. Typical techniques for disintegrating Microcystis colonies are sonication (Asai et al. 2000;Briand et al. 2009;Kurmayer et al. 2003;Zhou et al. 2012), alkaline hydrolysis (Janse et al. 2004;Kardinaal et al. 2007;Reynolds and Jaworski 1978), and heating/boiling (Chaffin et al. 2011;Joung et al. 2006;Lemaire et al. 2012;Zohary and Madeira 1987). ...
... Estimating the colony size distribution of Microcystis by traditional microscopy is time consuming. A modified flow cytometer, the CytoSense, can automatically measure the size of Microcystis colonies (Zhou et al. 2012) by calculating their diameters from the intensity of forward-scattered light, but the calculation assumes that all particles are spherical and have identical optical properties (Bryan et al. 2012). ...
Article
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Colony formation plays an important role in the life history of Microcystis. However, analyzing the colony size distribution with a microscope is time consuming, and colony formation also hinders the direct monitoring of cell density. In this study, a quantitative protocol for rapidly analyzing the cell density and colony size distribution of pelagic and benthic Microcystis was developed. Microcystis colonies were disintegrated by alkaline hydrolysis with 0.01-0.05 mol L−1 sodium hydroxide at 85 °C for 6-8 min and automatically measured by the Flow Camera And Microscope (FlowCAM). Benthic Microcystis colonies were isolated from different lake sediments using 40 % Percoll solution. Alkaline hydrolysis was validated as a rapid and universally effective method to disintegrate different morphospecies of Microcystis colonies. The FlowCAM exhibited excellent accuracy, reproducibility, and efficiency in determining the cell density and size distribution of Microcystis. The developed protocol was successfully applied to field samples from Lake Caohai (China) and can accurately monitor the population dynamics and colony size distribution of bloom-forming Microcystis.
... Before used in the experiments, all equipment and medium are sterilized at 121 o C for 0.5 h. All cultures are cultivated in an illumination incubator Safe PGX under the conditions of 25 1 ± o C, 2 000 lux, and light/ dark ( / ) L D 12 h/12 h cycles [37,38] . The incubator is from Ningbo Haishu Apparatus Company of China (http://www.hktdc.com/en-buyer). ...
... After 2 d when an equilibrium is established [40] , the M. aeruginosa reaches its exponential growth phase and are inoculated into the cylinders at a density of 8×10 5 cells m/L [36] . The phytotron conditions are 25 1 ± o C, 2 000 lux, and light/dark ( / ) L D 12 h/12 h cycles [37,38] . To simulate possible natural disturbances in the Zhusan Bay of the Taihu Lake, the blade stirrer operates at different rotational speeds to represent typical situations. ...
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The eutrophication of shallow lakes is sensitive to dynamic currents (i.e., disturbances) because of their shallow depths and high contents of nutrients in bed sediments. The relation between the sediment resuspension and the algae bloom is not well understood in the field scale because the interwoven influencing factors cannot be examined individually. By combining the laboratory experiment and the field observation, this paper proposes a sediment-water-algae concept to simulate the effects of hydrodynamic disturbances on the algae growth in the Taihu Lake located in east China. The sediments are sampled from the Taihu Lake while the Microcystis aeruginosa (M. aeruginosa) algae is cultured in the laboratory and then transplanted into the experiment cylinders. The temperature and the light intensity in the experiment are adjusted to be similar with the prevalent in situ conditions. The results indicate that the M. aeruginosa populations under the disturbance condition of the rotational speed ≤300 rad/min in the experiment (corresponding to the bottom velocity flow ≤0.059 m/s, the shear stress ≤0.069 N/m2, or the wind speed ≤4 m/s in the field) are higher than those under the disturbance condition of the rotational speed is 400 rad/min (corresponding to the bottom flow velocity 0.079 m/s, the shear stress 0.124 N/m2). It is suggested that a low to moderate disturbance prompts the release of the nitrogen as well as the phosphate nutrients from the bed sediments, amplifying the eutrophication of the Taihu Lake.
... Microcystis colonies were disaggregated prior to analysis: 20 ml algal samples were divided into two 10 ml samples, one sample was filtered through 10 lm mesh and the cells in the filtrate were recorded as the single-cell sample. The second 10 ml sample was sonicated (300 W 25 kHz) for 2 min (Zhou et al., 2012) to disaggregate colonies into single cells and recorded as total cells. The number of cells in Microcystis colonies Each sediment sample was mixed thoroughly. ...
... Moisture content was analyzed as the difference between wet-weight and dry-weight. Afterwards, 5 mg dry-weight sediment samples were resuspended in 1 ml of tap water (Zhou et al., 2012). The number of single cells and total cells in the sediment was measured by flow-cytometer as described above. ...
Article
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Colony formation by Microcystis is considered to play an important role in bloom development. Population dynamics of benthic and pelagic Microcystis colonies and single cells were investigated in Lake Taihu during recruitment from the sediment. Our results showed that the number of Microcystis single cells in both the water column and sediment was much higher than that of colonies during winter and early spring. In late May, cell density of single cells in the water column was greater than 1 × 10⁵ cells ml⁻¹. This result also suggested that only a portion of the Microcystis single cells formed colonies during recruitment. Microcystis single cells formed colonies on the surface of the sediment where the temperature and light intensity were lower than in the water column. The temporal variation in morphology and colony size of Microcystis in both the water column and sediment demonstrated that a possible sequence of Microcystis colony formation is that colony-forming process begins with loose amorphous colonies formed from single cells in the sediment, then changing to firm amorphous colonies. Afterwards, firm amorphous colonies rise into the water column and some form typical M. ichthyoblabe colony morphology.
... Studying the distribution of these two bacterial groups could provide more insights into interpreting the relationships between microorganisms and the marine environment. Hence, for such purposes, a single-cell enumeration method using flow cytometry that records physical and chemical characteristics is frequently used for most aquatic microbiome studies such as sea, sewage, lake, and drinking water [37,[84][85][86]. Specifically, green fluorescence intensity from HNA is 3-5 times greater than LNA and serves as the main separation criteria for these two cell populations [37,84,87]. ...
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High throughput sequencing has emerged as one of the most important techniques for characterizing microbial dynamics and revealing bacteria and host interactions. However, data interpretation using this technique is mainly based on relative abundance and ignores total bacteria load. In certain cases, absolute abundance is more important than compositional relative data, and interpretation of microbiota data based solely on relative abundance can be misleading. The available approaches for absolute quantification are highly diverse and challenging, especially for quantification in differing biological situations, such as distinguishing between live and dead cells, quantification of specific taxa, enumeration of low biomass samples, large sample size feasibility, and the detection of various other cellular features. In this review, we first illustrate the importance of integrating absolute abundance into microbiome data interpretation. Second, we briefly discuss the most widely used cell-based and molecular-based bacterial load quantification methods, including fluorescence spectroscopy, flow cytometry, 16S qPCR, 16S qRT-PCR, ddPCR, and reference spike-in. Last, we present a specific decision-making scheme for absolute quantification methods based on different biological questions and some of the latest quantitative methods and procedure modifications.
... Joung et al. (2006) (Chung, 1993), 영국담수조류도감 (John et al., 2002(John et al., , 2011, 일본담수조류 도감 (Hirose, 1977) and Avery, 1996). 이에 따라 Olson의 사상형 남조류 길 이 측정법 (Olson, 1950)이 개발되어 자연 상태 (Gibson, 1975;Booker and Walsby, 1981;Oliver and Walsby, 1984)와 배양 상태 (Konopka et al., 1978;Konopka, 1982 (Joung et al., 2006;NIER, 2009;Zhou et al., 2012). Microcystis 속 ...
... Secondly, measuring optical properties in field or laboratory samples easily allow filtering out the contributions from all non-autotrophic particles through the intensity of the red fluorescence signal, which is mainly proportional to the concentration of pigments in photosynthetic tissues (Dubelaar and Jonker 2000). Moreover, it is now technologically feasible to measure phytoplankton optical properties from automated monitoring stations (Thyssen et al. 2014;Zhou et al. 2012). The temporal resolution from such flow cytometric time series can provide important information about the influence of biotic and abiotic factors on nitrogen quota dynamics. ...
Article
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The intracellular concentration of internal nitrogen (the “cell nitrogen quota”) is crucial to explain the rate at which phytoplankton populations grow. Hence, understanding changes in cell nitrogen quota is informative on aquatic primary productivity, phytoplankton ecology, eutrophication, and algal blooms. However, current methods to directly monitor per-cell nitrogen quota remain inaccurate, expensive, and time consuming. This study tested the hypothesis that nitrogen limitation triggers systematic optical changes in single cells, which can be rapidly and accurately monitored with a standard flow cytometer. The freshwater microalgae Desmodesmus armatus, Mesotaenium sp., Scenedesmus obliquus, and Tetraëdron sp. were reared in nitrogen-limited batch culture conditions across two treatments of initial population densities and monitored for cell nitrogen quota, medium nitrogen, and optical flow cytometric properties of red fluorescence and forward and side light scatters. Changes in nitrogen quota could be described with high accuracy (R 2 = 0.9) from observations of flow cytometric variables and medium nitrogen, and the relationship did not change across different species or initial population sizes. Red fluorescence was the most important variable explaining 77 % of the total variability in total cell nitrogen and up to 87 % when combined with side light scatter, the second most important variable. Our results indicate that optical flow cytometric variables are a convenient and reliable method to estimate nitrogen quota in microalgal cells.
... The results also indicated that the spatial and temporal diversity of microcystin producing cyanobacteria are highly associated with the environmental parameters in sediment samples [7], and the cyanobacteria with active mcyA and mcyB genotypes were the dominant species in planktonic population, which has been proved in many cases [22,26,55,56]. Compared to quantitative analysis by flow cytometer [57], quantitative PCR identified and quantified Microcystis sp. at molecular biological level. Instead of the 16S or 23S rRNA analysis, the phylogenetic relationships of mcyA genes ( Figure 4) indicated three dominant toxic Microcystis sp. after recruitment from Shanzi Reservoir sediments. ...
Article
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This study investigated the impacts of four environmental factors on the recruitment of cyanobacteria from the bottom sediments in the eutrophic Shanzi Reservoir. Temperature and light were identified as the key determinants for the recruitment of Microcystis and Oscillatoria. Cyanobacteria became dominant at higher temperature (20°C) and light intensity (2,000 lx), and Microcystis and Oscillatoria were the major species. Detailed recruitment simulation undertaken with respective gradient of temperature and light suggested that both Microcystis and Oscillatoria are temperature sensitive, and their critical temperature point was 10°C. However, distinct light impacts were observed only on Microcystis. The recruitment of Oscillatoria was light independent, whereas Microcystis had positive relationship with light intensity. Physical disturbance promoted Microcystis recruitment and also affected the structure of recruited cyanobacterial community the water-sediment interaction, based on quantitative polymerase chain reaction (qPCR) and phylogenetic analysis.
... RedThyssen et al. 2014; Zhou et al. 2012). The temporal resolution from such flow cytometric time series can provide important information about the influence of biotic and abiotic factors on nitrogen quota dynamics. ...
... Previous studies have shown that FCM is a well suited method for measuring the number of phytoplankton cells, providing comparable numbers to microscopy (Stehouwer et al. 2013). It is important to be aware, however, that single cell suspensions are essential for accurate enumeration of cells with FCM, as cell aggregates give rise to a single event only and thus cause problems for cell enumeration of colony forming algae and bacteria (Veldhuis et al. 2005;Christaki et al. 2011;Zhou et al. 2012). ...
Article
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Ballast water may, when discharged, cause the spread of nonindigenous and potentially invasive species. International ballast water treatment regulations have accelerated the development of new methods to detect, enumerate and assess the status of organisms in the water to be discharged. Flow cytometry (FCM) is a powerful technique with a broad range of applications with the possibility for multi-parametric analysis and the potential of combining it with other techniques being two strong advantages. This review will discuss whether FCM is suitable for ballast water analysis according to international ballast water regulations, and sum up the advantages and disadvantages. It will also give an overview of available labeling techniques. Finally, a discussion on the knowledge gaps and future potential for FCM within ballast water analysis is presented.
... As shown by Olson et al. 1985, marine phytoplankton distributions can be successfully measured using shipboard FCM. More recently, an FCM protocol for quantitative and rapid analysis of Microcystis cells and colonies in lake sediment was developed with a quantification limit of 3.3×10 4 cells g -1 dry weight (dw) (Zhou et al. 2012). ...
Article
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Flow cytometry (FCM) is routinely used in medical and veterinary diagnostics although it is also widely applied in environmental studies, including phytoplankton investigations. Cyanobacteria are widespread photosynthetic microorganisms that attract attention due to their ecology and potential toxicity. Therefore, novel research tools are being applied in their investigation. This paper characterizes FCM as a technique that enables photopigments (chlorophylls and phycocyanin) expressed by cyanobacteria to be excited and their emission to be subsequently detected. This feature not only allows cells to be counted in a rapid manner but also enables a wide range of potential applications in ecological and biochemical studies. The main advantages of FCM, such as rapid, automatic and precise measurements requiring small sample volumes, are also discussed in this paper along with challenges including analyses of filamentous cyanobacteria and signal overlapping. It is expected that FCM will continue to be used in some fields of cyanobacterial studies.
... The microscope counting of Lugol-preserved samples as either entities or single cells clearly demonstrates the discrepancy encountered between the way cells can be observed. Flow cytometers are not capable of making a distinction between single cells within a chain and will always count multi-cell colonies or aggregates as one entity (Veldhuis et al. 2005;Christaki et al. 2011;Zhou et al. 2012). Smaller cells will generally not be affected by disintegration processes due to the shear forces of the fluid flow, suggesting instead that the differences between the cytometer counts for the small size fraction are governed largely by flow cytometry gain settings that determine the sensitivity for the smallest cells as well as the signal threshold settings. ...
Article
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The ability to quantify vital aquatic organisms in the 2–50 µm size range was compared between five different flow cytometers and several different microscopes. Counts of calibration beads, algal monocultures of different sizes as well as organisms in a Wadden Sea sample were compared. Flow cytometers and microscopes delivered different bead concentrations. These differences between the instruments became larger for algal monocultures and were even higher for the Wadden Sea sample. It was observed that the concentration differences were significant between flow cytometer and microscope counts, and that this difference increased with the size of the objects counted. Microscope counts were more accurate for larger (50 µm) objects because cytometers struggled with bigger particles that clogged the instruments. Contrary to microscopy, the flow cytometers were capable of accurately enumerating cultured cells in the 2–10 µm size range and cells in the lower size range of the 10–50 µm size class. Flow cytometers were also well-suited to assess low abundance samples due to their ability to process larger volumes than microscopes. The results were used to indicate which tools are suitable for ballast water monitoring: flow cytometry is a suitable technology for an indicative and real time analysis of ballast water samples whilst only microscopy would be robust enough for detailed taxonomical analyses.
... Using Fig. 1 as a timeline guide of recent studies conducted in this area, there are a few general conditions that are applied in previous studies to study algicidal or inhibitory effects of bacterial isolates on targeted cyanobacteria. On a laboratory scale, the studies are conducted primarily through & Plaque assays on solid agar medium (Rashidan and Bird 2001;Gumbo et al. 2010) & Liquid medium growth assays (Nakamura et al. 2003;Choi et al. 2005;Gumbo et al. 2010) & Flow cytometric assessments of cell viability and death through live and dead stains, microscopy and cell surface monitoring (Cellamare et al. 2010;Zhou et al. 2012;Gumbo et al. 2014) The methods listed above are among the most commonly used, with chlorophyll measurements being the primary measurement of cell death. With Microcystis specifically, this has proven to be difficult as it is not an accurate estimation of cell abundance or death. ...
Article
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Biological control of cyanobacteria is a well-researched area with a central focus on laboratory-scale studies. Numerous reports have been made on algicidal isolates, with bacteria as a major component of the antagonists. The research in this review draws a brief summary of what is currently known in the area of freshwater cyanobacteria being inhibited by bacterial isolates. Proteobacteria, Bacteroidetes and Firmicutes are among the most commonly reported phyla of bacteria associated with or employed in this research area. However, there are limited reports of upscaling these control measures beyond the laboratory scale. Lytic control agents are the most commonly reported in the literature with subsequent cyanotoxin release. From a water quality perspective, this is not feasible. Based on the available literature, temperature, pH and nutrient changes have been explored in this short review as possible contributors to less optimal bacterial performance. Moreover, the investigation into optimising some of these parameters may lead to increased bacterial performance and, therefore, viability for upscaling this biological control. Through the compilation of current research, this review offers insight to live predator-prey cell interactions between cyanobacteria and algicidal bacteria.
... Algal growth was determined by counting cells using a Sedgewick-Rafter Chamber after fixing 1 mL of culture with 1% Lugol's Iodine solution. For colonial species S. uvella, ultrasonic method was used to disperse algal cells before counting (Zhou et al., 2012;Lee et al., 2012). Preliminary tests were conducted to get the ultrasonic intensity and time conditions as follows: sonicating the samples at 300 W for 10 s using Branson Sonifier 250 (America) with tapered microtip (1/800). ...
Article
Fishy odor has become one of the most often encountered aesthetic water quality problems in drinking water. While fresh water algae living in colder water can produce offensive fishy odors, their environmental behaviors remain poorly understood. In this study, two chrysophyte species (Synura uvella and Ochromonas sp.), which are often associated with fishy odor events, were selected to investigate the effect of temperature (8, 16, and 24 C) and light intensity (10, 41, and 185 mmol photons m À2 s À1) on algal growth and odorant production. Five polyunsaturated aldehyde derivatives, including 2,4-heptadienal, 2-octenal, 2,4-octadienal, 2,4-decadienal, and 2,4,7-dectridienal, were identified as fishy/cod liver oil/fatty/ rancid descriptors in the cultures of the two algae based on gas chromatography-olfactometry-mass spectrometry and comprehensive two-dimensional gas chromatography mass spectrometry. While biomass yield increased with the increase in temperature for both species, significantly higher odorant yields (production of odorants per cell) were obtained at 8 C. The total odorant production and cell yield of the odorants decreased with the increase in light intensity from 10 to 185 mmol photons m À2 s À1. The biodegradation half-lives for the released odorants were 6e10 h at 8 C and 2e4 h at 24 C, whereas the volatilization half-lives were 36e97 d at 8 C and 6e17 d at 24 C, suggesting that temperature-dependent biodegradation was an important factor controlling the fate of fishy compounds in aquatic environments. The results of this study will help clarify why most fishy odor events occur in cooler seasons, and provide knowledge related to cold water persistence for the management of fishy odor problems associated with algae.
... To be able to accurately count natural samples which regularly contain colony forming species, the image analysis procedure needs to be refined and could be solved by using more advanced approaches such as other automatized quantification and species identification programs (PlanktoVision, ImageJ) (Schulze et al. 2011(Schulze et al. , 2013. Similar issues are also encountered when using flow cytometry (Christaki et al. 2011;Zhou et al. 2012;Peperzak et al. 2018) and FlowCAM (Reavie et al. 2010;Camoying and Yñiguez 2016;Romero-Martínez et al. 2017). ...
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To limit the spreading of aquatic invasive species, regulations require ships’ ballast water to be treated before discharge. To validate ballast water treatment system (BWTS) performance, treated water is analyzed for living organisms in different size classes. Quantitative assessment of the size class 10–50 μm (mainly phytoplankton) is carried out using the vital stain method, which requires labor-intensive manual microscope counts of fluorescent (i.e., living) cells. The method is slow, demands specialized personnel, and is challenged by subjectivity and mobile organisms. Using a high-content screening platform (HCS-Platform) and image analysis, we developed an automated, objective and faster quantification method. The automated method neutralized subjectivity by using fixed cell recognition parameters for image analysis. The implementation of membrane filters gently manipulated the organisms into a 2D plane that reduced mobility. Quantifications were performed at different concentrations using monocultures of slow-moving Rhodomonas salina, highly mobile Tetraselmis suecica and natural algae. Results were compared to the standard manual counting procedure. Automated counts of monocultures were comparable to manual counts at low and medium concentration levels. Manual counts of T. suecica at high concentration levels were significantly lower compared to automated counts stressing the challenge to count mobile cells in 3D. Natural algal counts were similar for both counting approaches, but accuracy was challenged by colony forming species and high number of algal species ~ 10 μm. Automated counts were significantly faster than manual counts. In conclusion, the HCS-Platform showed promising results as an alternative quantitative phytoplankton assessment method for BWTS validation.
... For instance, phenotypic identification through microscopy can be time-consuming, especially with high volumes of samples for monitoring purposes. Molecular identifications such as polymerase chain reaction (PCR) are more accurate in terms of specificity and sensitivity; however, they are not cost effective and cannot detect single cells [33]. Moreover, biochemical and immunoassays can be utilized, but require a field expert, expensive equipment, and may result in false positives [34]. ...
Article
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Eutrophication is a process that occurs due to the excessive accumulation of nutrients, primarily nitrogen and phosphorus, from natural and anthropogenic sources. This phenomenon causes cyanobacterial overgrowth, which over time leads to cyanobacterial harmful algal blooms (CHABs) that affect public drinking water sources and water sites with recreational usage. The rapid detection of bloom-forming cyanobacteria in freshwater bodies is critical in order to implement prevention strategies. Cyanobacteria contain phycobiliproteins such as phycoerythrin and allophycocyanin as part of the phycobilisome that allows autofluorescence. In this study, samples from 36 freshwater bodies in 14 New Jersey counties were collected and analyzed using flow cytometry with forward-scatter phycoerythrin and allophycocyanin parameters. Pure cultures of Synechococcus sp. IU 625, Cylindrospermum spp. and Microcystis aeruginosa were used as references. The results revealed that 17 out of the 36 analyzed sites contained all three references and related species. Seven sites showed Microcystis and Cylindrospermum-like species, while four sites indicated Microcystis and Cylindrospermum-like species. Six water bodies showed Cylindrospermum-like species, and two sites showed Microcystis-like species. Polymerase chain reaction (PCR)-based assays further confirmed the flow cytometric results. The findings from this study suggest that flow cytometry could potentially serve as a rapid method for freshwater cyanobacteria detection and screening.
... Products of this type have been arriving on the market over the last 5 years and are used to monitor drinking water treatment processes and reservoirs, where an increase in cell counts can be indicative for failure of the treatment or contamination of a reservoir. They are also used to detect harmful algal blooms [36], for quality control in aquaculture and to monitor ballast water to prevent dispersion of nonindigenous organisms [37]. ...
Chapter
Sensorsdeployedinsmartwatersystemsneedtomeetanumberofcriteria, first and foremost robustness of performance, autonomous operation and low maintenance.Solid-state,opticalsensorsareattheforefrontinthedevelopmentofthenext generationofsensorsforsmartwatersystems.Arangeofopticalsensortechnologies is currently in use for (near) real-time water quality analysis and between them can cover most of the relevant quality parameters. Technologies used in the water industry include UV/Vis absorbance, fluorescence and NIR absorbance spectroscopy. Spectroscopic methods with potential for broader application include Raman spectroscopy and laser-induced breakdown spectroscopy. All approaches share the following properties: fully solid-state hardware, no reagents or other consumables required for their operation, and automatic interpretation of the sensor data performed by increasingly advanced chemometric solutions. This chapter provides a brief overview of the fundamentals behind these technologies and reviews their use in water quality monitoring applications.
... This larger size fraction could be quantified by traditional microscopy or image analysis, as these cells or colonies could be easily identified and enumerated. Nevertheless, it would be also advisable to apply suitable methods to count individual cells from colonies, for example, via sonication (38,39) and by that avoid potential problems related to large cell sizes. However, by using approaches to separate single cells, it needs to be kept in mind that the ecological relevant phenotype would be modified and therefore critical for morphological based classification shown in this work. ...
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Ponds are an insufficiently studied research object but represent a biodiversity hotspot and have a high value for ecosystem services like recreation, water retention, or angling. Especially urban ponds create a direct contact for citizens experiencing nature. But on the other side, these systems also suffer from several pressures caused by humans, for example , high nutrient and salt influxes or high temperatures. Phytoplankton organisms are a crucial part of ponds ecosystem and an understanding of community composition is crucial especially when eutrophication and high temperatures lead to dominance of unpleasant toxic cyanobacteria. With traditional microscopic methods for phytoplankton analysis, monitoring is not feasible with high spatial resolution and frequency. Therefore, a new approach of imaging flow cytometry to classify phytoplankton species in either tax-onomic or morphologically based functional groups (MBFGs) is suggested. In this study, both classifications could be successfully applied to a natural phytoplankton community in an urban pond in Leipzig with minor modifications. Both classifications in combination provide a good mechanistic understanding of phytoplankton community dynamics. In addition, a great advantage of the measurements is the archivability of microscopic images allowing a comprehensive respective data analysis. Two examples of detailed trait and image analysis are demonstrated to investigate single-cell traits for cyanobacteria and chlorophytes/euglenophytes and to follow the fate of a cyanobacterial bloom affected by a fungal infection.
... For example, recently, an FCM protocol for quantitative and rapid analysis of Microcystis cells and colonies in lake sediment was developed [118]. Because of its many advantages, including rapidity, sensitivity, multiparametric analysis, and cell sorting capacity, FCM has been increasingly applied to environmental studies with aquatic microorganisms. ...
Chapter
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Self-fluorescence is a powerful tool for investigation of living photosynthetic microorganisms. Since the physiological state of such microorganisms is closely related to the operation and activity of photosynthetic system; thus, any variations in spectroscopic properties of their self-fluorescence indicate the changes in their physiological state. Cyanobacteria (or blue green alga) are one of the most widespread photosynthetic organisms in nature, and the ecological aspect in their investigation is quite valuable. On the other hand, thousands of strains belonging to different cyanobacterial species are cultivated in biolaboratories all over the world for different biotechnological applications such as biofuel cells, food production, pharmaceuticals, and fertilizers. Thus, the novel noninvasive methods of their investigation are quite important for on-line monitoring of cyanobacterial cultures. In this chapter, several fluorescence techniques are presented for investigation of living cyanobacterial cells and cultures.
... In situ flow cytometry has revolutionized our understanding of marine picocyanobacteria population dynamics (Hunter-Cevera et al., 2020) though this approach has not yet been attempted in freshwater microbiology. The colony or chain/filament forming habit of many freshwater cyanobacteria potentially complicates the use of cytometry although some cytometry systems have more potential than others in coping with colonies (Zhou et al., 2012). In terms of the biomarkers that have been applied to cyanobacteria to attempt a categorization of the cause of cell death (annexin, caspase activity, and TUNEL labeling; see Hu and Rzymski, 2019) difficulties are raised due to the findings of variable staining between taxa and the difficulties of interpretation due to the lack of satisfactory positive controls (Kozik et al., 2019). ...
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Cyanobacteria are ancient and versatile members of almost all aquatic food webs. In freshwater ecosystems some cyanobacteria form “bloom” populations containing potent toxins and such blooms are therefore a key focus of study. Bloom populations can be ephemeral, with rapid population declines possible, though the factors causing such declines are generally poorly understood. Cell death could be a significant factor linked to population decline. Broadly, three forms of cell death are currently recognized – accidental, regulated and programmed – and efforts are underway to identify these and standardize the use of cell death terminology, guided by work on better-studied cells. For cyanobacteria, the study of such differing forms of cell death has received little attention, and classifying cell death across the group, and within complex natural populations, is therefore hard and experimentally difficult. The population dynamics of photosynthetic microbes have, in the past, been principally explained through reference to abiotic (“bottom-up”) factors. However, it has become clearer that in general, only a partial linkage exists between abiotic conditions and cyanobacteria population fluctuations in many situations. Instead, a range of biotic interactions both within and between cyanobacteria, and their competitors, pathogens and consumers, can be seen as the major drivers of the observed population fluctuations. Whilst some evolutionary processes may theoretically account for the existence of an intrinsic form of cell death in cyanobacteria, a range of biotic interactions are also likely to frequently cause the ecological incidence of cell death. New theoretical models and single-cell techniques are being developed to illuminate this area. The importance of such work is underlined by both (a) predictions of increasing cyanobacteria dominance due to anthropogenic factors and (b) the realization that influential ecosystem modeling work includes mortality terms with scant foundation, even though such terms can have a very large impact on model predictions. These ideas are explored and a prioritization of research needs is proposed.
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Effective assessment of the health risk of cyanobacterial blooms requires an early warning system, which enables rapid detection of species of concern and determination of whether their cell concentrations exceed advisory guidelines. Advanced digital flow cytometry using FlowCam® (Fluid Imaging Technologies) in combination with light microscopy is a solid prospect for tracking cyanobacterial communities in a timely manner. However, implementation of such a method poses several challenges for the user. We first address sample preparation, instrumentation, taxonomic enumeration, and trouble-shooting to facilitate high throughput of analyses of water samples for total cyanobacterial cell counts and their species composition. Preservation and initial screening of samples using light microscopy to estimate community size structure are endorsed to insure their archival quality and avoid clogging of the flow cell. We show that the highest magnification (×20 objective) is needed to achieve representative total and species-specific cell enumerations. We also report that total cyanobacterial cell counts for samples analyzed using FlowCam vs. inverted light microscopy show significant positive correlation, as do those for preserved vs. live samples. Quantification of community composition using FlowCam vs. light microscopy also shows strong concordance. Although our FlowCam method performs well in the context of the World Health Organization advisory threshold of a total cyanobacterial count of 100,000 cells mL⁻¹, it remains a work in progress in terms of reliably automated species-level identifications. © 2018 The Authors Limnology and Oceanography: Methods published by Wiley Periodicals, Inc. on behalf of Association for the Sciences of Limnology and Oceanography.
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Accurate soil moisture indicator is critically important for hydrological applications such as water resource management and hydrological modelling. Modern satellite remote sensing has shown a huge potential for providing soil moisture measurements at a large scale. However its effective utilisation in the aforementioned areas still needs comprehensive research. This chapter focuses on exploring the advances and potential issues in the current application of satellite soil moisture observations in hydrological modelling. It has been proposed that hydrological application of soil moisture data requires the data relevant to hydrology. In order to meet the requirement, the following two research tasks are suggested: the first is to carry out comprehensive assessments of satellite soil moisture observations for hydrological modelling, not merely based on evaluations against point-based in situ measurements; the second is that a soil moisture product (e.g. soil moisture deficit) directly applicable to hydrological modelling should be developed. Only fully accomplishing these two steps will push forward the utilisation of satellite soil moisture in hydrological modelling to a greater extent.
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The evaluation about the relative distribution of cyanobacterial biomass between the sediment and water column would be indispensable to understand if benthic cyanobacteria are important to cyanobacterial biomass in the water column. A separation method for the rapid quantification of benthic cyanobacteria in Chaohu Lake was developed by density-gradient centrifugation. A 2⁴ full factorial design and response surface methodology was employed to optimize the extraction protocol. Under the optimal operating parameters including 29% Percoll solution, 30 min centrifugation time, 7200 r/min centrifugation speed, and a 1:10 ratio between the volume of sediment and Percoll solution, the recovery rate of cyanobacteria in sediment was 96.73%. Temporal and spatial variations in cyanobacterial biomass in water and sediment were investigated monthly throughout a whole year. In general, cyanobacterial biomass per square centimeter in the water column showed high spatial-temporal changes, tending to increase in February and reaching a peak in April at some sites due to the growth of Dolichospermum. The second peak arrived in July and September and was caused by the rapid growth of Microcystis. Concurrently, cyanobacteria biomass per unit area in sediment showed a clear temporal change pattern, increasing from October and reaching a peak level in February at all the sampling sites. The average ratio of cyanobacterial biomass in water to that in sediment was lowest in January at 1.48 and increased to the highest level in July at 318.61. Although Microcystis and Dolichospermum were dominant species in the water column, only Microcystis was observed in the Percoll solution extraction from sediment. Microscopic observation revealed that a very small fraction of Microcystis cells could survive in sediment, and most of the cells decomposed when the water temperature increased after June. Therefore, the contribution of the recruitment of cyanobacteria could be negligible in Lake Chaohu.
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A brief overview is given of current applications of flow cytometry (FCM) in marine phytoplankton research. This paper presents a selection of highlights and various technical and analytical problems we encountered during the past 10 years. In particular, the conversion of the relative values obtained in terms of size and fluorescence applying FCM to quantitative estimates of cell size, pigment concentration, genome size etc., is addressed. The introduction of DNA -cell-cycle analysis made easily assessable by flow cytometry has been of great impor tance, allowing in situ measurement of species specific growth rates. Key questions in ecology such as factors determining the wax and wane of phytoplankton bloom can now be better answered in terms of species specific growth and mortality. Finally, flow cytometry provides detailed information of the physiological status of the individual algal cells. New staining methods enable us to distinguish between viable and non-viable cells and so will help us to elucidate the importance of 'automortality' in aquatic ecosystems.
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A 4 year series of field, light-microscope and ultrastructural observations is presented to illustrate biological aspects of the annual cycle of natural Microcystis populations enclosed in Lund tubes. Nine morphological stati, all referable to M. aeruginosa f. aeruginosa, feature at various stages of the cycle. Summer bloom-forming populations originate from vegetative colonial stock that overwinters on the bottom sediment each year, but there is no mass transfer of these colonies to the water column: intensive growth from individual cells in the old colonies leads to the formation of new infective colonies, being stimulated when the bottom water approaches anoxia and light penetrates to the bottom sediments. Growth is slow but the developing populations sustain only minor losses through grazing and settling out, eventually becoming dominant over other species. Allelopathy possibly contributes to this effect. In postmaximal populations, several mechanisms can contribute to net buoyancy loss and a (usually) rapid recruitment of vegetative colonies to the sediments is observed. Hypotheses are advanced to account for the observed behaviour, and some of these have been tested in the laboratory. The apparent physiological flexibility of Microcystis seems well suited to growth and survival in the microenvironments encountered in eutrophic lakes.
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Phytoplankton observation is the product of a number of trade-offs related to sampling processes, required level of diversity and size spectrum analysis capabilities of the techniques involved. Instruments combining the morphological and high-frequency analysis for phytoplankton cells are now available. This paper presents an application of the automated high-resolution flow cytometer Cytosub as a tool for analysing phytoplanktonic cells in their natural environment. High resolution data from a temporal study in the Bay of Marseille (analysis every 30 min over 1 month) and a spatial study in the Southern Indian Ocean (analysis every 5 min at 10 knots over 5 days) are presented to illustrate the capabilities and limitations of the instrument. Automated high-frequency flow cytometry revealed the spatial and temporal variability of phytoplankton in the size range 1−∼50 μm that could not be resolved otherwise. Due to some limitations (instrumental memory, volume analysed per sample), recorded counts could be statistically too low. By combining high-frequency consecutive samples, it is possible to decrease the counting error, following Poisson’s law, and to retain the main features of phytoplankton variability. With this technique, the analysis of phytoplankton variability combines adequate sampling frequency and effective monitoring of community changes.
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Flow cytometry is an alternative to traditional methods of studying the distribution and abundance of phytoplankton that allows rapid, objective analysis of light scattering and fluorescence of individual cells in a natural water sample. We report here the first flow cytometric analysis of phytoplankton communities carried out at sea. Light scatter and autofluorescence of individual cells were measured in water samples from the Gulf Stream. These flow cytometric ‘signatures’ allowed us to discriminate between different groups of phytoplankton, enumerate them, and detect changes in average pigment fluorescenee resulting from changes in population structure. The results indicate that flow cytometric techniques can be used on board ship with minimal modification of available equipment. They also show promise for automated analyses of plankton and particles in the sea.
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Cyanobacteria (blue-green algae) commonly occur in the phytoplankton of lakes and reservoirs and sometimes develop as blooms which can cause deoxygenation, toxin production and nuisance odours1. The factors which govern the occurrence and seasonal development of such blooms in surface waters are imprecisely understood and little is known about the origins of the bloom-forming populations within particular water bodies (see, however, refs 2,3). Using 15N as tracer, we show here that the appearance of Microcystis in the phytoplankton of an experimental enclosure in the summer of 1977 correlated directly with the presence of particulate 15N which could only have been sediment-derived and which originated mainly from Microcystis cells deposited on the sediment surface the previous year. The most plausible explanation is that Microcystis overwinters on the sediment surface and by so doing provides an inoculum of colonies from which the epilimnetic population develops the following summer4-8. Possible mechanisms which allow the mass recruitment of Microcystis to the plankton are considered elsewhere2,3.
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The cell counting of colonial Microcystis spp. is a rather difficult and error-prone proposition, as this genus forms irregularly-shaped and irregularly-sized colonies, which are packed with cells. Thus, in order to facilitate a cell count, four methods of dividing the colonies into single cells were compared, including vortexing, sonication, TiO2 treatment, and boiling. As a result, the boiling method was determined to generate the greatest number of single cells from a colony, and all colonies were found to have divided completely after only 6 min of treatment. Furthermore, no significant cell destruction, which might alter the actual cell density, was detected in conjunction with the boiling method (P = 0.158). In order to compute the cell number more simply, the relationship between the colony size and the cell number was determined, via the boiling method. The colony volume, rather than the area or diameter was correlated more closely with the cell number (r2 = 0.727), thereby suggesting that the cell numbers of colonial Microcystis sp. can also be estimated effectively from their volumes.
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While the performance of biological sensors in real time monitoring networks is limited to bulk values like chlorophyll fluorescence, in practice the implementation of automated phytoplankton taxonomy remains a remote option. Aiming to reduce this gap we developed a flow cytometer called CytoBuoy for autonomous in situ operation, for instance in a moored buoy with wireless data transfer. Although not comparable to microscopy, flow cytometers detect and count par ticles allowing a limited level of particle characterization based on the light scatter and fluorescence properties of the individual particles. CytoBuoy analyses a large size range of particles, typical for marine coastal zones and fresh waters. The 'field' design implies a tradeoff between the accuracy and versatility of laboratory flow cytometers and the qualities needed for trouble free autonomous operation in situ. The optics and electronics however were designed for maximal reflection of the par ticle morphology in the measured signals. Whereas standard cytometers reduce these to single peak or area 'listmode' numbers, the signal courses are preserved fully by CytoBuoy and transferred to the computer as raw data, which allows more extended morphological analysis. Extended field tests will have to show how the system holds in various environments and weather conditions.
Article
Preface 1. What is phytoplankton? 2. Mechanisms of suspension 3. Spatial and temporal distribution of phytoplankton 4. Photosynthetic activity of phytoplankton 5. Nutrients 6. Growth and survival 7. Loss process 8. Periodicity and change in phytoplankton composition Glossary and symbols References Index to lakes and rivers Index to genera/species General index.
Article
Seasonal variations of colony numbers of Microcystis aeruginosa(Kütz.) Kütz. and M. wesenbergii(Komárek) Komárek in N. V. Kondrat. in sediments of Lake Biwa were investigated over a period of 1 year. At two stations located in the shallow South Basin of Lake Biwa (ca. 4 m water depth), the colony number of Microcystisfluctuated seasonally. The number had a tendency to gradually decrease from winter to early summer, while it increased through mid-summer and autumn. Since the Microcystispopulation in sediment was rather small, intensive growth and accumulation in the water column should be important for the formation of Microcystisblooms in Lake Biwa. Microcystiscolonies in the sediment samples after June were observed to be floating in a counting chamber under a microscope. The observation suggests that the recruitment of Microcystis colonies into the water column mostly occurs in early summer. The number of Microcystiscolonies in the deep North Basin of Lake Biwa (70 – 90 m water depth) was larger than in the South Basin. Because the seasonal variation of colony numbers was not observed in the North Basin, and Microcystiscells do not have gas vesicles, these colonies will not return into the water column. The colonies isolated from the sediment of the North Basin were able to grow in cultured conditions, in the same way as those from the sediment of the South Basin. Therefore, Microcystiscolonies may survive for a long time under stable conditions of low temperature (ca. 8 °C) and darkness, in the sediment of the deep North Basin, accumulating gradually each year.
Article
The standing crop and photosynthetic activity of Microcystis aeruginosa Kütz. in both the plankton and sediment were investigated from November 1979 to May 1982 in Lake Kasumigaura, Japan. The number of planktonic colonies of this species decreased from early autumn to early spring, but increased in the sediment during late summer and autumn. The overwintering colonies in the sediment were -100-1000 times greater per unit area than those in lake water. No photoinhibition of photosynthesis could be observed in overwintering Microcystis. The values of the initial slopes of photosynthesis-light (P-I) curves were similar to those of the summer population, although the maximum photosynthetic rate (P max) measured at 20°C was lower than that of the summer planktonic population. In winter the values of initial slope of the P-I curve, and the ratio of phycobilin to chlorophyll a sorted from sediment were higher than in colonies from the plankton.
Colonies of Microcystis aeruginosa have dominated the phytoplankton in Lund tube C, a limnetic enclosure in Blelham Tarn, English Lake District, during the summer and autumn in recent years. Following holomixis in autumn the previously buoyant colonies sedimented from the water column onto the bottom mud. In all samples gas vesicles, which provided the colonies with buoyancy, were present in sufficient volume to negate the combined ballast provided by protein, carbohydrate, lipid and phosphate, the major cell components. The gas vesicles, which accounted for about 10% of the cell protein, were too strong to be collapsed or regulated by cell turgor pressure. Consequently, the loss of buoyancy could not be explained by an increase in cell ballast or by disappearance of gas vesicles. Colonies collected in sediment traps were found to be buoyant after they had been agitated and diluted with lake water, which removed a colloidal precipitate from them. Similarly, 66% of the sinking fraction of a net tow sample was found to be buoyant after it had been treated in the same way. Previously buoyant colonies could be made to sink on mixing with the colloidal precipitate. This demonstrated the ability of the precipitate to trap colonies and to cause their sedimentation. The colloid comprised approximately equal amounts of organic and inorganic matter and was rich in iron. Colloids of this type form when the dissolved iron in the anoxic water of the hypolimnion becomes oxidized on mixing with the aerated water of the epilimnion.
Article
Benthic stages of the annual life cycle of the meroplanktonic cyanobacterium Microcystis spp. in relation to microcystin (MCYST) dynamics in sediments of a shallow lake (Quitzdorf Reservoir, Germany) were investigated. Based on changes in the absolute abundance of benthic Microcystis, the annual life cycle was subdivided into four phenological stages: reinvasion, pelagic growth, sedimentation, and overwintering. Habitat-coupling processes, such as reinvasion of the pelagic zone in spring as well as autumnal sedimentation, were particularly triggered by changes in water temperature. During reinvasion substantial losses of Microcystis were detected. Only a minor part of benthic Microcystis (about 3%) formed the inoculum for pelagic growth. Between 65% and 85% of the benthic Microcystis stock disappeared during the reinvasion phase. Because these colonies were neither detected within the sediments nor in the pelagic inoculum, it was concluded that they were subjected to decay. The occurrence of extracellular MCYSTs in the pelagic zone during this period, which cannot solely originate from the pelagic Microcystis population, supports this conclusion. Dynamics of benthic Microcystis and MCYSTs were characterized by almost identical successions with a decrease during reinvasion, an increase during sedimentation, and remarkable invariability throughout pelagic growth and overwintering. It can be deduced that MCYSTs are preserved within benthic resting stages of Microcystis because they could play a role during overwintering or reinvasion.
Article
Recruitment of Microcystis from sediments to the water column was investigated in shallow (1–2 m) and deep (6–7 m) areas of Lake Limmaren, central Sweden. Recruitment traps attached to the bottom were sampled on a weekly basis throughout the summer season ( June–September). A comparison between the two sites showed that the recruitment from the shallow bay was significantly higher over the entire season for all three Microcystis species present in the lake. Maximum rates of recruitment were found in August, when 2.3 × 105 colonies m−2·day−† left the sediments of the shallow area. Calculated over the entire summer, Microcystis colonies corresponding to 50% of the initial abundance in the surface sediments were recruited in the shallow bay, whereas recruitment from the deep area was only 8% of the sediment colonies. From these results we conclude that shallow areas, which to a large extent have been overlooked in studies of recruitment of phytoplankton, may be crucial to the dynamics of these organisms by playing an important role as inoculation sites for pelagic populations.
Article
The phytoplankton community structure of a hypertrophic lake was quantitatively determined with the aid of flow cytometry. The flow cytometry signals were calibrated to obtain cell-specific information, such as the chl a content and the biovolume per cell. The reliability of this method was tested with laboratory cultures. The results of the phytoplankton structure in a hypertrophic lake with respect to chl distribution in the different algal groups obtained by flow cytometry were compared with the results from HPLC pigment fingerprinting. Both methods yield the percentage contribution of the different algal groups to total chl a. The chl a specific absorption coefficient of the phytoplankton (a*Phy) was determined via visible (VIS) spectroscopy of samples taken from a hypertrophic lake (Auensee) in 2003. The results indicated that a*Phy of the total cell suspension is dependent on the phytoplankton structure as well as on environmental factors. The linear relationship between a*Phy at 675 nm and the product of the chl a content per cell and the biovolume offered the possibility to normalize phytoplankton absorption spectra to acquire the taxon-specific a*Phy. The estimated a*Phy (675 nm) values were used to normalize single cell absorption spectra at this wavelength to obtain the a*Phy between 400 and 750 nm for representatives of the major algal groups. Our measurements show that the absorption coefficient for the whole phytoplankton community varies within the season. Finally, we used the a*Phy and the chl a distribution to calculate the light absorption of each algal group in the hypertrophic lake.
Chapter
IntroductionDescription of Sampling Methods Description of the Subsampling ProceduresDescription of the Counting TechniquesConsiderations Regarding the Use of the MicroscopeAlgae as BioindicatorsConclusions
Article
In some lakes, large amounts of the potentially toxic cyanobacterium Microcystis overwinter in the sediment. This overwintering population might inoculate the water column in spring and promote the development of dense surface blooms of Microcystis during summer. In the Dutch Lake Volkerak, we found photochemically active Microcystis colonies in the sediment throughout the year. The most vital colonies originated from shallow sediments within the euphotic zone. We investigated whether recruitment of Microcystis colonies from the sediment to the water column was an active process, through production of gas vesicles or respiration of carbohydrate ballast. We calculated net buoyancy, as an indication of relative density, using the amounts and densities of the major cell constituents (carbohydrates, proteins, and gas vesicles). Carbohydrate content of benthic Microcystis cells was very low throughout the year. Buoyancy changes of benthic Microcystis were mostly a result of changes in gas vesicle volume. Before the summer bloom, net buoyancy and the amount of buoyant colonies in the sediment did not change. Therefore, recruitment of Microcystis from the sediment does not seem to be an active process regulated by internal buoyancy changes. Instead, our observations indicate that attachment of sediment particles to colonies plays an important part in the buoyancy state of benthic colonies. Therefore, we suggest that recruitment of Microcystis is more likely a passive process resulting from resuspension by wind-induced mixing or bioturbation. Consequently, shallow areas of the lake probably play a more important role in recruitment of benthic Microcystis than deep areas.
Article
Variations in microbial biomass and activity in the sediments of hypereutrophic Lake Vallentunasjn were followed during a period of 5 years. The data were compared to the calculated release of phosphorus from the sediments during the same period. A strong co-variation was found between biomass of Microcystis, heterotrophic bacterial activity in the sediments and internal phosphorus loading. These parameters exhibited mainly a declining trend during the investigation period. A pronounced stability of the sediment chemistry, including the fractional composition of the sediment phosphorus, during the studied period indicates that microbial activity affected the phosphorus release from the sediments. Calculations of the percentage of sediment bacteria that was associated to the mucilage of Microcystis colonies imply, together with the specific bacterial production, that Microcystis in the sediment stimulates bacterial production. In the highly phosphorus-saturated sediments of Lake Vallentunasjn this would ultimately lead to an increased release of phosphorus from the sediment. Lake Vallentunasjn does not follow the common pattern of recovery after reduction of external phosphorus loading. The large biomasses and long survival of Microcystis in the sediment are probably important reasons for the delayed recovery of the lake.
Article
The seasonal variation of microbial biomass and activity in the surface sediments (0–10 cm) of the shallow, eutrophic Lake Vallentunasjn was followed during one year. OverwinteringMicrocystis colonies dominated the microbial community during all seasons, constituting 60–90% of the total microbial biomass. Expressed on an areal basis, the benthic biomass was, throughout the year, larger than or similar to the planktonic biomass during the peak of the summer bloom, indicating an ability of the colonies to survive in the sediments for extended periods. Abundance of other, non-photosynthetic bacteria varied in the range 3.0–15.5 1010 cells g–1 d. w. over the year with minimum values in summer and maximum values in autumn in connection with the sedimentation of theMicrocystis bloom. A substantial part of the non-photosynthetic bacteria, up to circa 40%, was associated with the mucilage of healthyMicrocystis colonies. Bacterial production (3H-thymidine incorporation) appeared to be strongly temperature dependent and less influenced by the seasonal sedimentation pattern. Our data indicate an increasing proportion of non-growing cells in autumn and winter. Biomass-bound phosphorus constituted a significant portion, circa 10%, of the phosphorus content in Lake Vallentunasjn sediments. This pool has normally been overlooked in studies on phosphorus dynamics in lake sediments. Different mechanisms whereby organic phosphorus can be released from the sediments are discussed.
Article
The benthic recruitment of Microcystis was simulated in vitro in order to characterize the colonies of Microcystis recruited and to study the impact of intracellular and extracellular microcystins (MCs), and the influence of colony size on the recruitment process. We observed recruitment dynamics consisting of a lag phase followed by a peak and then a return to low recruitment rates, mainly controlled by passive resuspension throughout the experiment, and by physiological processes during the recruitment peak. Ninety-seven percent of the Microcystis colonies recruited were <160 μm in maximum length, and their cells contained much greater amounts of MCs (0.26 ± 0.14 pg eq microcystin leucine-arginine variant [MC-LR] · cell−1) than those in benthic colonies (0.021 ± 0.004 pg eq MC-LR · cell−1). The MC content of recruited Microcystis varied significantly over time and was not related to changes in the proportion of potentially toxic genotypes, determined using real-time PCR. On the other hand, the changes in MC content in the potentially toxic Microcystis recruited were closely and negatively correlated with recruitment dynamics; the lowest MC contents corresponded to high recruitment rates, and the highest MC contents corresponded to low recruitment rates. Thus, depending on temperature and light conditions, these variations are thought to result from the selection of various subpopulations from among the smallest and the most toxic of the initial benthic population. Adding purified MC-LR to experimental treatments led to a decreased recruitment of Microcystis and more specifically of mcyB genotypes.
Article
An experiment for studying the effects of sediment dredging on denitrification in sediments was carried out through a one-year incubation of undredged (control) and dredged cores in laboratory. Dredging the upper 30 cm of sediment can significantly affect physico-chemical characteristics of sediments. Less degradation of organic matter in the dredged sediments was found during the experiment. Denitrification rates in the sediments were estimated by the acetylene blockage technique, and ranged from 21.6 to 102.7 nmol N2/(g dry weight (dw) x hr) for the undredged sediment and from 6.9 to 26.9 nmol N2/(g dw x hr) for dredged sediments. The denitrification rates in the undredged sediments were markedly higher (p < 0.05) than those in the dredged sediments throughout the incubation, with the exception of February 2006. The importance of various environmental factors on denitrification was assessed, which indicated that denitrification was regulated by temperature. Nitrate was probably the key factor limiting denitrification in both undredged and dredged sediments. Organic carbon played some role in determining the denitrification rates in the dredged sediments, but not in the undredged sediments. Sediment dredging influenced the mineralization of organic matter and denitrification in the sediment; and therefore changed the pattern of inherent cycling of nitrogen.
Article
Although Microcystis-based toxins have been intensively studied, previous studies using laboratory cultures of Microcystis strains are difficult to explain the phenomenon that microcystin concentrations and toxin variants in natural blooms differ widely and frequently within a short-term period. The present study was designed to unravel the mechanisms for the frequent variations of intracellular toxins related to the differences in cyanobacterial colonies during bloom seasons in Lake Taihu, China. Monitoring of Microcystis colonies during warm seasons indicated that the variations in microcystins in both concentrations and toxin species were associated with the frequent alteration of Microcystis colonies in Lake Taihu. High concentration of microcystins in the blooms was always associated with two Microcystis colonies, Microcystis flos-aquae and Microcystis aeruginosa, whereas when Microcystis wesenbergii was the dominant colonial type, the toxin production of the blooms was low. Additionally, environmental factors such as temperature and nutrition were also shown to have an effect on the toxin production of the blooms, and may also potentially influence the Microcystis species present. The results of the present study provides insight into a new consideration for quick water quality monitoring, assessment and risk alert in cyanobacterium- and toxin-contaminated freshwaters, which will be beneficial not only for water agencies but also for public health.
Article
Optical properties of eight algae species were measured on a flow cytometer. Forward and perpendicular light scatter measurements provide information on the size and shape of algae cells. The intensity of chlorophyll fluorescence varies greatly among the studied algae species and can be used to distinguish them. Measurements of chlorophyll fluorescence after excitation with different wavelengths provide a fluorescence excitation spectrum for each species over the available wavelength range. These spectra reflect the different photosynthetic pigment contents of the species. Staining algae cells with the DNA stains, Hoechst 33342 and DAPI, provides two additional optical parameters to distinguish algae populations: blue nuclear fluorescence and yellow granular fluorescence. The combination of these optical measurements enables the distinction of each algae species into a small cluster in a hyperspace of parameters. The automation of phytoplankton analysis on the flow cytometer may lead to the rapid and objective assessment of water quality.
Article
The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.
Article
The spatio-temporal distribution of benthic colonies of Microcystis aeruginosa in Grangent Reservoir (France) in 2000 was not homogeneous and appeared to be controlled by many external factors: lake depth, station morphometry, substratum and hydraulic regime (lacustrine or fluvial). A most important concentration of benthic colonies was found at deep sites with fine sediment or at sites where the sediment was rich in organic matter. In spite of a stable water level and a minimum flow during summer, the number of benthic colonies showed great variation in the lacustrine downstream part of the reservoir. These variations may be explained by the dynamics of planktonic cyanobacteria.
Article
In the present paper, sorption, persistence, and leaching behavior of three microcystin variants in Chinese agriculture soils were examined. Based on this study, the values of capacity factor and slope for three MCs variants in three soils ranged from 0.69 to 6.00, and 1.01 to 1.54, respectively. The adsorption of MCs in the soils decreased in the following order: RR > Dha7 LR > LR. Furthermore, for each MC variant in the three soils, the adsorption rate in the soils decreased in the following order: soil A > soil C > soil B. The calculated half-time ranged between 7.9 and 17.8 days for MC-RR, 6.0-17.1 days for MC-LR, and 7.1-10.2 days for MC-Dha7 LR. Results from leaching experiments demonstrated that recoveries of toxins in leachates ranged from 0-16.7% for RR, 73.2-88.9% for LR, and 8.9-73.1% for Dha7 LR. The GUS value ranged from 1.48 to 2.06 for RR, 1.82-2.88 for LR, and 1.76-2.09 for Dha7 LR. Results demonstrated the use of cyanobacterial collections as plant fertilizer is likely to be unsafe in soils.
Article
For the purpose of understanding the environmental fate of microcystins (MCs) and the potential health risks caused by toxic cyanobacterial blooms in Lake Taihu, a systematic investigation was carried out from February 2005 to January 2006. The distribution of MCs in the water column, and toxin bioaccumulations in aquatic organisms were surveyed. The results suggested that Lake Taihu is heavily polluted during summer months by toxic cyanobacterial blooms (with a maximum biovolume of 6.7 x 10(8)cells/L) and MCs. The maximum concentration of cell-bound toxins was 1.81 mg/g (DW) and the dissolved MCs reached a maximum level of 6.69 microg/L. Dissolved MCs were always found in the entire water column at all sampling sites throughout the year. Our results emphasized the need for tracking MCs not only in the entire water column but also at the interface between water and sediment. Seasonal changes of MC concentrations in four species of hydrophytes (Eichhornic crassipes, Potamogeton maackianus, Alternanthera philoxeroides and Myriophyllum spicatum) ranged from 129 to 1317, 147 to 1534, 169 to 3945 and 124 to 956 ng/g (DW), respectively. Toxin accumulations in four aquatic species (Carassius auratus auratu, Macrobrachium nipponensis, Bellamya aeruginosa and Cristaria plicata) were also analyzed. Maximum toxin concentrations in the edible organs and non-edible visceral organs ranged from 378 to 730 and 754 to 3629 ng/g (DW), respectively. Based on field studies in Lake Taihu, risk assessments were carried out, taking into account the WHO guidelines and the tolerable daily intake (TDI) for MCs. Our findings suggest that the third largest lake in China poses serious health threats when serving as a source of drinking water and for recreational use. In addition, it is likely to be unsafe to consume aquatic species harvested in Lake Taihu due to the high-concentrations of accumulated MCs.
Article
Blooms of cyanobacteria, or blue-greens, are known to produce chemicals, such as microcystins, which can be toxic to aquatic and terrestrial organisms. Although previous studies have examined the fate of microcystins in freshwater lakes, primary elimination pathways and factors affecting degradation and loss have not been fully explained. The goal of the present study was to explore sources of algal toxins and investigate the distribution and biodegradation of microcystins in water and sediment through laboratory and field analyses. Water and sediment samples were collected monthly from several locations in Lake Taihu from February 2005 to January 2006. Samples were analyzed for the presence of microcystin. Water and sediment were also used in laboratory studies to determine microcystin degradation rates by spiking environmental samples with known concentrations of the chemical and observing concentration changes over time. Some water samples were found to efficiently degrade microcystins. Microcystin concentrations dropped faster in water collected immediately above lake sediment (overlying water). Degradation in sediments was higher than in water. Based on spatial distribution analyses of microcystin in Lake Taihu, higher concentrations (relative to water concentrations) of the chemical were found in lake sediments. These data suggest that sediments play a critical role in microcystin degradation in aquatic systems. The relatively low levels of microcystins found in the environment are most likely due to bacterial biodegradation. Sediments play a crucial role as a source (to the water column) of bio-degrading bacteria and as a carbon-rich environment for bacteria to proliferate and metabolize microcystin and other biogenic toxins produced by cyanobacteria. These, and other, data provide important information that may be applied to management strategies for improvement of water quality in lakes, reservoirs and other water bodies.
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