The diversity and distribution of toxigenic Microcystis spp. in present day and archived pelagic and sediment samples from Lake Erie

The reoccurrence of significant cyanobacterial blooms in Lake Erie during the last 13 years has raised questions concerning the long-term persistence of microcystin-producing cyanobacteria and the presence of natural sediment reservoirs for potentially toxic cyanobacteria in this large lake system. To address these questions, we analyzed phytoplankton and sediment samples which were collected and preserved in the 1970s as well as samples collected in 2004 from locations within Lake Erie. The identification of microcystin-producing cyanobacteria in Lake Erie was examined via PCR amplification of the mcyA gene fragment. Based on the high % sequence similarity, the mcyA sequences from all 1970s phytoplankton and sediment samples were determined to belong to Microcystis spp., in spite of reports suggesting that Lake Erie was dominated by filamentous cyanobacteria in the 1970s. In sediment samples from 2004, signature genes for Microcystis were distributed and preserved not only in the surface sediments but also up to 10–12 cm in depth. Based on cell quantities determined by a quantitative polymerase chain reaction (qPCR) method, 0.18% of eubacteria in the sediments were Microcystis cells, of which 4.8% were potential microcystin producers. In combination with experiments showing that Microcystis cells can be cultured from Lake Erie surface sediments, this paper demonstrates the potential for these sediments to act as a reservoir for pelagic Microcystis populations and that the composition of the population of microcystin-producing cyanobacteria in Lake Erie has not changed remarkably since the 1970s.     

Densification of cyanobacteria from a lake leading to production of β‐cyclocitral and related volatile organic compounds and species change

The purpose of the present study was to demonstrate that the lysis with the blue color formation was caused by densification of the cyanobacteria, and related events of the species change in the cyanobacteria were induced by the resulting volatile organic compounds (VOCs), particularly β‐cyclocitral. In order to obtain a high cell density of cyanobacteria in the laboratory, a concentration technique (graduated cylinder method) using the buoyancy of the gas vesicles was successfully used. The collected scum contained mainly Dolichospermum spp. and Microcystis, and the dispersed cyanobacteria were concentrated in the surface layer after several hours and the concentration ratio became approximately 10. The concentrated cyanobacteria were gradually lysed, while some of the cyanobacteria sank to the bottom, which finally died and disappeared. This method has the additional advantage that it is possible to visualize the entire lysis process. During the concentration process, β‐cyclocitral and its oxidation products together with β‐ionone were significantly detected. Because β‐cyclocitral was easily oxidized to the corresponding carboxylic acid, the pH of the water in the graduated cylinder decreased to approximately 6. Under favorable conditions, lysis with the blue color from phycocyanin could be observed due to the acid stress. Overall, the results of the present study were consistent with the hypothesis that VOCs were produced when the cyanobacteria are highly dense, and that the lysis with the blue color formation occurs due to the higher density.     

Mesozooplankton and microzooplankton grazing during cyanobacterial blooms in the western basin of Lake Erie

Lake Erie is the most socioeconomically important and productive of the Laurentian (North American) Great Lakes. Since the mid-1990s cyanobacterial blooms dominated primarily by Microcystis have emerged to become annual, late summer events in the western basin of Lake Erie yet the effects of these blooms on food web dynamics and zooplankton grazing are unclear. From 2005 to 2007, grazing rates of cultured (Daphnia pulex) and natural assemblages of mesozooplankton and microzooplankton on five autotrophic populations were quantified during cyanobacterial blooms in western Lake Erie. While all groups of zooplankton grazed on all prey groups investigated, the grazing rates of natural and cultured mesozooplankton were inversely correlated with abundances of potentially toxic cyanobacteria (Microcystis, Anabaena, and Cylindrospermopsis; p < 0.05) while those of the in situ microzooplankton community were not. Microzooplankton grazed more rapidly and consistently on all groups of phytoplankton, including cyanobacteria, compared to both groups of mesozooplankton. Cyanobacteria displayed more rapid intrinsic cellular growth rates than other phytoplankton groups under enhanced nutrient concentrations suggesting that future nutrient loading to Lake Erie could exacerbate cyanobacterial blooms. In sum, while grazing rates of mesozooplankton are slowed by cyanobacterial blooms in the western basin of Lake Erie, microzooplankton are likely to play an important role in the top-down control of these blooms; this control could be weakened by any future increases in nutrient loads to Lake Erie.     

Targeted deep sequencing reveals high diversity and variable dominance of bloom-forming cyanobacteria in eutrophic lakes

Cyanobacterial blooms in eutrophic lakes are severe environmental problems worldwide. To characterize the spatiotemporal heterogeneity of cyanobacterial blooms, a high-throughput method is necessary for the specific detection of cyanobacteria. In this study, the cyanobacterial composition of three eutrophic waters in China (Taihu Lake, Dongqian Lake, and Dongzhen Reservoir) was determined by pyrosequencing the cpcBA intergenic spacer (cpcBA-IGS) of cyanobacteria. A total of 2585 OTUs were obtained from the normalized cpcBA-IGS sequence dataset at a distance of 0.05. The 238 most abundant OTUs contained 92% of the total sequences and were classified into six cyanobacterial groups. The water samples of Taihu Lake were dominated by Microcystis, mixed Nostocales species, Synechococcus, and unclassified cyanobacteria. Besides, all the samples from Taihu Lake were clustered together in the dendrogram based on shared abundant OTUs. The cyanobacterial diversity in Dongqian Lake was dramatically decreased after sediment dredging and Synechococcus became exclusively dominant in this lake. The genus Synechococcus was also dominant in the surface water of Dongzhen Reservoir, while phylogenetically diverse cyanobacteria coexisted at a depth of 10 m in this reservoir. In summary, targeted deep sequencing based on cpcBA-IGS revealed a large diversity of bloom-forming cyanobacteria in eutrophic lakes and spatiotemporal changes in the composition of cyanobacterial communities. The genus Microcystis was the most abundant bloom-forming cyanobacteria in eutrophic lakes, while Synechococcus could be exclusively dominant under appropriate environmental conditions.     

Phylogenies of Microcystin-Producing Cyanobacteria in the Lower Laurentian Great Lakes Suggest Extensive Genetic Connectivity

Lake St. Clair is the smallest lake in the Laurentian Great Lakes system. MODIS satellite imagery suggests that high algal biomass events have occurred annually along the southern shore during late summer. In this study, we evaluated these events and tested the hypothesis that summer bloom material derived from Lake St. Clair may enter Lake Erie via the Detroit River and represent an overlooked source of potentially toxic Microcystis biomass to the western basin of Lake Erie. We conducted a seasonally and spatially resolved study carried out in the summer of 2013. Our goals were to: 1) track the development of the 2013 summer south-east shore bloom 2) conduct a spatial survey to characterize the extent of toxicity, taxonomic diversity of the total phytoplankton population and the phylogenetic diversity of potential MC-producing cyanobacteria (Microcystis, Planktothrix and Anabaena) during a high biomass event, and 3) compare the strains of potential MC-producers in Lake St. Clair with strains from Lake Erie and Lake Ontario. Our results demonstrated a clear predominance of cyanobacteria during a late August bloom event, primarily dominated by Microcystis, which we traced along the Lake St. Clair coastline downstream to the Detroit River''s outflow at Lake Erie. Microcystin levels exceeded the Province of Ontario Drinking Water Quality Standard (1.5 µg L⁻¹) for safe drinking water at most sites, reaching up to five times this level in some areas. Microcystis was the predominant microcystin producer, and all toxic Microcystis strains found in Lake St. Clair were genetically similar to toxic Microcystis strains found in lakes Erie and Ontario. These findings suggest extensive genetic connectivity among the three systems.     

Lake Erie Microcystis: Relationship between microcystin production, dynamics of genotypes and environmental parameters in a large lake

Cyanobacteria of genus Microcystis sp. have been commonly found in Lake Erie waters during recent summer seasons. In an effort to elucidate relationships between microcystin production, genotypic composition of Microcystis community and environmental parameters in a large lake ecosystem, we collected DNA samples and environmental data during a three-year (2003–2005) survey within Lake Erie and used the data to perform a series of correlation analyses. Cyanobacteria and Microcystis genotypes were quantified using quantitative real-time PCR (qPCR). Our data show that Microcystis in Lake Erie forms up to 42% of all cyanobacteria, and that Microcystis exists as a mixed population of potentially toxic and (primarily) non-toxic genotypes. In the entire lake, the total abundance of Microcystis as well as the abundance of microcystin-producing Microcystis is strongly correlated with the abundance of cyanobacteria suggesting that Microcystis is a significant component of the cyanobacterial community in Lake Erie during summer seasons. The proportion of total Microcystis of all cyanobacteria was strongly linked to the microcystin concentrations, while the percentage of microcystin-producing genotypes within Microcystis population showed no correlation with microcystin concentrations. Correlation analysis indicated that increasing total phosphorus concentrations correlate strongly with increasing microcystin concentrations as well as with the total abundance of Microcystis and microcystin-producing Microcystis.     

Dynamics of the Volatile Organic Substances Associated with Cyanobacteria and Algae in a Eutrophic Shallow Lake

The volatile substances present in a eutrophic shallow lake were determined qualitatively and quantitatively during the growing season, allowing a first-time analysis of the dynamics of these compounds in a water body. The major compounds found were β-cyclocitral, α-cyclocitral, β-ionone, 1,3,3-trimethylcyclohexene, 2,2,6-trimethylcyclohexanone, 2,6,6-trimethylcyclohex-2-en-1-one, eucalyptol, geosmin, two argosmin isomers, pent-1-en-3-ol, pent-1-en-3-one, heptadec-1-ene, heptadec-cis 5-ene, heptadeca-1, cis 8-diene, decanal, and hexanal. Most of the substances have not been detected before in natural waters. The occurrence of geosmin, the argosmin isomers, heptadec-cis 5-ene, and an unknown sesquiterpene could clearly be correlated with the appearance of Aphanizomenon gracile, as could the occurrence of β-cyclocitral with the appearance of Microcystis wesenbergii. The data presented indicate that a rapid metabolism or the elimination of these compounds occurs in lake water.     

Molecular Characterization of Potential Microcystin-Producing Cyanobacteria in Lake Ontario Embayments and Nearshore Waters

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 μg MC-LR_eq liter⁻¹. MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns.     

Biotransformation of Hg(II) by Cyanobacteria

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl₂ in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl₂. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.     

Analysis of environmental drivers influencing interspecific variations and associations among bloom-forming cyanobacteria in large,shallow eutrophic lakes

Non-diazotrophic Microcystis and filamentous N₂-fixing Aphanizomenon and Dolichospermum (formerly Anabaena) co-occur or successively dominate freshwaters globally. Previous studies indicate that dual nitrogen (N) and phosphorus (P) reduction is needed to control cyanobacterial blooms; however, N limitation may cause replacement of non-N₂-fixing by N₂-fixing taxa. To evaluate potentially counterproductive scenarios, the effects of temperature, nutrients, and zooplankton on the spatio-temporal variations of cyanobacteria were investigated in three large, shallow eutrophic lakes in China. The results illustrate that the community composition of cyanobacteria is primarily driven by physical factors and the zooplankton community, and their interactions. Niche differentiation between Microcystis and two N₂-fixing taxa in Lake Taihu and Lake Chaohu was observed, whereas small temperature fluctuations in Lake Dianchi supported co-dominance. Through structural equation modelling, predictor variables were aggregated into ‘composites’ representing their combined effects on species-specific biomass. The model results showed that Microcystis biomass was affected by water temperature and P concentrations across the studied lakes. The biomass of two filamentous taxa, by contrast, exhibited lake-specific responses. Understanding of driving forces of the succession and competition among bloom-forming cyanobacteria will help to guide lake restoration in the context of climate warming and N:P stoichiometry imbalances.     

Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Phosphorus strategy in bloom-forming cyanobacteria (Dolichospermum and Microcystis) and its role in their succession

Dolichospermum (formerly Anabaena) and Microcystis cause harmful cyanobacterial blooms in freshwater ecosystems worldwide. Input reduction of both nitrogen (N) and phosphorus (P) are commonly recognized as basic ways of controlling blooms, but little is known about the roles of nutrients and their using strategy among cyanobacteria in triggering the succession of diazotrophic to non-diazotrophic cyanobacteria. In this study, we investigated in situ responses of cyanobactria to ambient P status during the transition from Dolichospermum flos-aquae to Microcystis spp. in Lake Taihu and Lake Chaohu. While dominant in phytoplankton community, D. flos-aquae experienced P deficiency as evidenced by qualitative detection of extracellular phosphatase via enzyme labeled fluorescence (ELF). The percentage of ELF-labelled D. flos-aquae cells was 33% when it dominated the phytoplankton community, and was 78% when it co-dominated with Microcystis spp., indicating an increase in P deficiency. Meanwhile, no ELF-labelled Microcystis cells were observed while polyphosphate body (PPB) were present, suggesting that Microcystis spp. were not P deficient. Additionally, the percentages of Microcystis cells containing PPB showed an inverted “U-shaped” relationship with concentrations on soluble reactive phosphorus (SRP). To validate the field observation, a laboratory study of the monocultures of the dominant cyanobacteria was conducted. Extracellular alkaline phosphatase activity (APA) and PPB accumulation were regulated by P availability in monocultures of D. flos-aquae. Interestingly, no cell bound extracellular phosphatase was found on Microcystis aeruginasa even in the culture without P supply. Consistently, the expressions of phosphatase encoding gene phoX showed no differences among the treatments. The way in which PPB accumulation occurred in Microcystis spp. in response to P availability in the cultures was similar to that observed in the field, demonstrating a strategy of energy conservation over P accumulation. The competitive advantage of Microcystis spp. was displayed at low P concentrations: where it could rapidly uptake and store inorganic P, which also increased the P deficiency of the coexisting phytoplankton species. Responses of P-transport gene pstS confirmed this hypothesis. The physiological and molecular mechanisms mentioned above enable Microcystis to survive and proliferate in environment with low available P supply more efficiently. In conclusion, different cyanobacterial species have distinct ways of responding to P availability, suggesting that the control of cyanobacterial blooms by targeted nutrient reduction is largely dependent upon the dominant species. P reduction is more effective in controlling diazotrophic cyanobacteria than non-diazotrophic cyanobacteria.     

Development of Models for Predicting the Predominant Taste and Odor Compounds in Taihu Lake,China

Taste and odor (T&O) problems, which have adversely affected the quality of water supplied to millions of residents, have repeatedly occurred in Taihu Lake (e.g., a serious odor accident occurred in 2007). Because these accidents are difficult for water resource managers to forecast in a timely manner, there is an urgent need to develop optimum models to predict these T&O problems. For this purpose, various biotic and abiotic environmental parameters were monitored monthly for one year at 30 sites across Taihu Lake. This is the first investigation of this huge lake to sample T&O compounds at the whole-lake level. Certain phytoplankton taxa were important variables in the models; for instance, the concentrations of the particle-bound 2-methylisoborneol (p-MIB) were correlated with the presence of Oscillatoria, whereas those of the p-β-cyclocitral and p-β-ionone were correlated with Microcystis levels. Abiotic factors such as nitrogen (TN, TDN, NO₃-N, and NO₂-N), pH, DO, COND, COD and Chl-a also contributed significantly to the T&O predictive models. The dissolved (d) T&O compounds were related to both the algal biomass and to certain abiotic environmental factors, whereas the particle-bound (p) T&O compounds were more strongly related to the algal presence. We also tested the validity of these models using an independent data set that was previously collected from Taihu Lake in 2008. In comparing the concentrations of the T&O compounds observed in 2008 with those concentrations predicted from our models, we found that most of the predicted data points fell within the 90% confidence intervals of the observed values. This result supported the validity of these models in the studied system. These models, basing on easily collected environmental data, will be of practical value to the water resource managers of Taihu Lake for evaluating the probability of T&O accidents.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Method for Isolation and Purification of Cyanobacteria

A method employing nutrient saturated glass fiber filters allowed the isolation of the same numbers of cyanobacteria from freshwater as were obtained with medium solidified with agar, while providing a 2- to 15-fold reduction in the number of accompanying heterotrophic bacteria. Imipenem, a broad-spectrum β-lactam antibiotic which inhibits peptidoglycan biosynthesis, was superior to some other β-lactam antibiotics for reducing the numbers of heterotrophic bacterial contaminants associated with freshly isolated cyanobacteria to a level which facilitated the production of axenic cyanobacterial cultures.     

Nitrogen Fixed By Cyanobacteria Is Utilized By Deposit-Feeders

Benthic communities below the photic zone depend for food on allochthonous organic matter derived from seasonal phytoplankton blooms. In the Baltic Sea, the spring diatom bloom is considered the most important input of organic matter, whereas the contribution of the summer bloom dominated by diazotrophic cyanobacteria is less understood. The possible increase in cyanobacteria blooms as a consequence of eutrophication and climate change calls for evaluation of cyanobacteria effects on benthic community functioning and productivity. Here, we examine utilization of cyanobacterial nitrogen by deposit-feeding benthic macrofauna following a cyanobacteria bloom at three stations during two consecutive years and link these changes to isotopic niche and variations in body condition (assayed as C:N ratio) of the animals. Since nitrogen-fixing cyanobacteria have δ¹⁵N close to -2‰, we expected the δ¹⁵N in the deposit-feeders to decrease after the bloom if their assimilation of cyanobacteria-derived nitrogen was substantial. We also expected the settled cyanobacteria with their associated microheterotrophic community and relatively high nitrogen content to increase the isotopic niche area, trophic diversity and dietary divergence between individuals (estimated as the nearest neighbour distance) in the benthic fauna after the bloom. The three surface-feeding species (Monoporeia affinis, Macoma balthica and Marenzelleria arctia) showed significantly lower δ¹⁵N values after the bloom, while the sub-surface feeder Pontoporeia femorata did not. The effect of the bloom on isotopic niche varied greatly between stations; populations which increased niche area after the bloom had better body condition than populations with reduced niche, regardless of species. Thus, cyanobacterial nitrogen is efficiently integrated into the benthic food webs in the Baltic, with likely consequences for their functioning, secondary production, transfer efficiency, trophic interactions, and intra- and interspecific competition.     

Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.     

Lysis of Yeast Cell Walls: Glucanases from Bacillus circulans WL-12

Endo-β-(1 → 3)- and endo-β-(1 → 6)-glucanases are produced in high concentration in the culture fluid of Bacillus circulans WL-12 when grown in a mineral medium with bakers' yeast cell walls as the sole carbon source. Much lower enzyme levels were found when laminarin, pustulan, or mannitol was the substrate. The two enzyme activities were well separated during Sephadex G-100 chromatography. The endo-β-(1 → 3)-glucanase was further purified by diethylaminoethyl-cellulose and hydroxyapatite chromatography, whereas the endo-β-(1 → 6)-glucanase could be purified further by diethylamino-ethyl-cellulose and carboxymethyl cellulose chromatography. The endo-β-(1 → 3)-glucanase was specific for the β-(1 → 3)-glucosidic bond, but it did not hydrolyze laminaribiose; laminaritriose was split very slowly. β-(1 → 4)-Bonds in oat glucan in which the glucosyl moiety is substituted in the 3-position were also cleaved. The kinetics of laminarin hydrolysis (optimum pH 5.0) were complex but appeared to follow Michaelis-Menten theory, especially at the lower substrate concentrations. Glucono-δ-lactone was a noncompetitive inhibitor and Hg²⁺ inhibited strongly. The enzyme has no metal ion requirements or essential sulfhydryl groups. The purified β-(1 → 6)-glucanase has an optimum pH of 5.5, and its properties were studied in less detail. In contrast to the crude culture fluid, the two purified β-glucanases have only a very limited hydrolytic action on cell wall of either bakers' yeast or of Schizosaccharomyces pombe. Although our previous work had assumed that the two glucanases studied here are responsible for cell wall lysis, it now appears that the culture fluid contains in addition a specific lytic enzyme which is eliminated during the extensive purification process.     

Controlling Harmful Cyanobacteria: Taxa-Specific Responses of Cyanobacteria to Grazing by Large-Bodied Daphnia in a Biomanipulation Scenario

Lake restoration practices based on reducing fish predation and promoting the dominance of large-bodied Daphnia grazers (i.e., biomanipulation) have been the focus of much debate due to inconsistent success in suppressing harmful cyanobacterial blooms. While most studies have explored effects of large-bodied Daphnia on cyanobacterial growth at the community level and/or on few dominant species, predictions of such restoration practices demand further understanding on taxa-specific responses in diverse cyanobacterial communities. In order to address these questions, we conducted three grazing experiments during summer in a eutrophic lake where the natural phytoplankton community was exposed to an increasing gradient in biomass of the large-bodied Daphnia magna. This allowed evaluating taxa-specific responses of cyanobacteria to Daphnia grazing throughout the growing season in a desired biomanipulation scenario with limited fish predation. Total cyanobacterial and phytoplankton biomasses responded negatively to Daphnia grazing both in early and late summer, regardless of different cyanobacterial densities. Large-bodied Daphnia were capable of suppressing the abundance of Aphanizomenon, Dolichospermum, Microcystis and Planktothrix bloom-forming cyanobacteria. However, the growth of the filamentous Dolichospermum crassum was positively affected by grazing during a period when this cyanobacterium dominated the community. The eutrophic lake was subjected to biomanipulation since 2005 and nineteen years of lake monitoring data (1996–2014) revealed that reducing fish predation increased the mean abundance (50%) and body-size (20%) of Daphnia, as well as suppressed the total amount of nutrients and the growth of the dominant cyanobacterial taxa, Microcystis and Planktothrix. Altogether our results suggest that lake restoration practices solely based on grazer control by large-bodied Daphnia can be effective, but may not be sufficient to control the overgrowth of all cyanobacterial diversity. Although controlling harmful cyanobacterial blooms should preferably include other measures, such as nutrient reductions, our experimental assessment of taxa-specific cyanobacterial responses to large-bodied Daphnia and long-term monitoring data highlights the potential of such biomanipulations to enhance the ecological and societal value of eutrophic water bodies.