Thesis (M.S.)--Oregon State University, 2009. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Cyanobacteria are widely distributed in marine, freshwater and terrestrial habitats. Some cyanobacterial genera can convert di-nitrogen gas (N2) to bioavailable ammonium, i.e. perform nitrogen (N) fixation, and are therefore of profound significance for N cycling. N fixation by summer blooms of cyanobacteria is one of the largest sources of new N for the Baltic Sea. This thesis investigated N fixation by cyanobacteria in the Baltic Sea and explored the fate of fixed N at different spatial and temporal scales. In Paper I, we measured cell-specific N fixation by Aphanizomenon sp. at 10 ºC, early in the season. Fixation rates were high and comparable to those in late summer, indicating that Aphanizomenon sp. is an important contributor to N fixation already in its early growth season. In Paper II, we studied fixation and release of N by Aphanizomenon sp. and found that about half of the fixed N was rapidly released and transferred to other species, including autotrophic and heterotrophic bacteria, diatoms and copepods. In Paper III, we followed the development of a cyanobacterial bloom and related changes in dissolved and particulate N pools in the upper mixed surface layer. The bloom-associated total N (TN) increase was mainly due to higher particulate organic N (PON) concentrations, but also to increases in dissolved organic nitrogen (DON). About half the PON-increase could be explained by the sum of N-fixing cyanobacteria, other phytoplankton (&gt;2µm) and zooplankton, indicating that production was stimulated by the N fixation. In Paper IV, we used a growth model based on measured photosynthesis–irradiance relationships to explore the production potential of Aphanizomenon sp. The model included data on irradiance, biomass, temperature and light attenuation (1999–2013). Until the bloom peak, the modelled production matched the measured biomass, indicating low production losses. Over the whole season, the modelled production could explain a substantial part of the summer TN increase, assuming that plausible losses (such as grazing or cell lysis) are retained within the upper mixed layer. Complementing the other data, we also investigated the nutrient content (Paper I) and varying cell width (Paper IV) of Aphanizomenon sp. By a combination of approaches, this thesis has contributed new information on cyanobacterial N fixation rates, the transfer of fixed N to other organisms in the food web and shown the potential for fixed N to stimulate summer primary and secondary production in the Baltic Sea. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

The ability to regulate buoyancy (sinking and floating) using cellular gas vesicles is a unique characteristic that allows many common bloom-forming cyanobacteria to accumulate at water surfaces and dominate systems. Typical control and management strategies include nutrient manipulation and phosphorus reduction, which are effective but do not reduce the advantage of buoyancy control. Since buoyancy control is based upon a mechanism that is driven by photosynthesis along with environmental conditions that trigger vesicle formation and ion exchange, buoyancy regulation can be influenced by manipulating extracellular conditions. In this study I manipulated extracellular conditions using wetland water and additions of potassium, sodium, and calcium in small-scale lab experiments and larger scale, near-lake containers with Aphanizomenon flos-aquae from Upper Klamath Lake, Oregon. The results indicate a target mixture of 10% wetland water reduces surface accumulation, increases cellular turgor pressure (a measure of the ability of gas vesicle forming cells to control buoyancy), and leads to fewer rafts at the surface of the water column. By adding ions at the same concentration as the target wetland mixture, similar results were found. This research represents the basis of a possible strategy for mitigating surface blooms of buoyant cyanobacteria in lakes using wetland water and/or ion additions that could be used in tandem with nutrient manipulation and phosphorus reduction.

<p>Summer blooms of filamentous, diazotrophic cyanobacteria are typical of the Baltic Sea Proper, and are dominated by <i>Aphanizomenon </i>sp<i>.</i> and the toxic <i>Nodularia spumigena.</i> Although occurring every summer, the blooms vary greatly in timing and spatial distribution, making monitoring difficult and imprecise. This thesis studies how the spatial variability of Baltic cyanobacterial blooms influences estimates of abundance, vertical and horizontal distribution and N₂-fixation. Implications for sampling and monitoring of cyanobacterial blooms are also discussed.</p><p>The results of the thesis confirm the importance of diazotrophic cyanobacteria in providing N for summer production in the Baltic Proper. It also highlights the large spatial and temporal variation in these blooms and argues that improved spatial coverage and replication could make monitoring data more useful for demonstrating time trends, and for identifying the factors regulating the blooms. The vertical distribution of <i>Aphanizomenon</i> and <i>Nodularia</i> was found to be spatially variable, probably as a combination of species-specific adaptations and ambient weather conditions. Vertical migration in <i>Aphanizomenon</i> was more important towards the end of summer, and is probably regulated by a trade-off between P-availability and light and temperature.</p>

Summer blooms of filamentous, diazotrophic cyanobacteria are typical of the Baltic Sea Proper, and are dominated by Aphanizomenon sp. and the toxic Nodularia spumigena. Although occurring every summer, the blooms vary greatly in timing and spatial distribution, making monitoring difficult and imprecise. This thesis studies how the spatial variability of Baltic cyanobacterial blooms influences estimates of abundance, vertical and horizontal distribution and N2-fixation. Implications for sampling and monitoring of cyanobacterial blooms are also discussed. The results of the thesis confirm the importance of diazotrophic cyanobacteria in providing N for summer production in the Baltic Proper. It also highlights the large spatial and temporal variation in these blooms and argues that improved spatial coverage and replication could make monitoring data more useful for demonstrating time trends, and for identifying the factors regulating the blooms. The vertical distribution of Aphanizomenon and Nodularia was found to be spatially variable, probably as a combination of species-specific adaptations and ambient weather conditions. Vertical migration in Aphanizomenon was more important towards the end of summer, and is probably regulated by a trade-off between P-availability and light and temperature.

Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.

Cyanobacteria are an ancient lineage of gram-negative photosynthetic prokaryotes that play an important role in the nitrogen cycle in terrestrial and aquatic systems. Widespread cyanobacterial blooms have prompted numerous studies on the classification of this group, however defining species is problematic due to lack of clarity as to which characters best define the various taxonomic levels. The genera Anabaena, Aphanizomenon and Nostoc form one of the most controversial groups and are typically paraphyletic within phylogenetic trees and share similar morphological characters. This study’s purpose was to determine the taxonomic and phylogenetic relationships among isolates from these three genera using 16S rRNA and bacterial elongation factor P (efp) gene sequences as well as morphological analyses. These data confirmed the non-monophyly of Anabaena and Aphanizomenon and demonstrated that many of the isolates were intermixed among various clades in both gene phylogenies. In addition, the genus Nostoc was clearly not monophyletic and this finding differed from previous studies. The genetic divergence of the genus Nostoc was confirmed based on 16S rRNA gene sequence similarities (≥85.1%), and the isolates of Anabaena were genetically differentiated, contrary to previous studies (16S rRNA gene sequence similarities ≥89.4%). The morphological diversity was larger than the molecular diversity, since the statistical analysis ANOSIM showed that the isolates were morphologically well differentiated; however, the 16S rRNA gene sequence similarities showed some isolates as being related at the species level. Planktonic and benthic strains were not distinguished phylogenetically, although some well-supported clusters were noted. Cellular measurements (length and width of vegetative cells, end cells, heterocysts and akinetes) were noted to be the morphological characters that best supported the differentiation among isolates, more than qualitative characterization. Among the metric parameters, the length of akinetes resulted in better differentiation among isolates. The efp gene sequence analyses did not appear to be useful for the taxonomic differentiation at lower taxonomic levels, but gave well-supported clusters for Aphanizomenon that was supported by the morphological analyses. Both gene regions gave similar trees with the exception of the Aphanizomenon isolates which clustered together in phylogenetic trees based on the efp gene. This differed from the 16S rRNA gene in which this genus was paraphyletic with Anabaena species that were similar in morphology to Aphanizomenon. Hence, the application of multiple taxonomic criteria is required for the successful delineation of cyanobacterial species.

Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.

In the Himmerfjärden Bay a large excess of nitrogen over phosphorus in the discharge from a large sewage treatment plant (STP) has suppressed growth of diazotrophic cyanobacteria in its inner parts. Implementation of nitrogen removal in the STP in 1997 drastically reduced nitrogen load and triggered growth of diazotrophs, mainly Aphanizomenon sp. This study is part of a long-term series of experiments with the overall aim to test how algal biomass and production in a receiving area can be reduced, without stimulating nitrogen fixation and biomass growth by diazotrophic cyanobacteria. Nitrogen removal was discontinued in the STP during two years (2007-8) and resumed in 2009, and the discharge shifted from 25 to 10 m depth, above the seasonal pycnocline. Cellular 15N showed that N2 was the most important N source for diazotrophic cyanobacteria, and that uptake of combined nitrogen was insignificant. As biomass was declining and at the end of the productive season, we could detect a small, but significant, increase in cellular δ15N at the inner bay stations (H3 and H4). However, this coincided with an increased proportion of Anabaena spp. of the total diazotrophic biomass. This may indicate that Anabaena spp. has a higher uptake of combined nitrogen compared with Aphanizomenon sp. or that declining populations of Aphanizomenon sp. take up combined nitrogen. We also found no evidence of uptake of combined nitrogen during the winter months when nitrogen supply is ample and Aphanizomenon sp. is devoid of heterocysts. During the first half of summer (week 21-27) heterocyst frequencies were higher at the outer stations B1 and H2, compared to the inner bay stations (H4 and H5). The lower frequencies at the inner bay stations are likely due to the reduced growth rate suffered by the Aphanizomenon sp. due to stronger competition for phosphorus by non-diazotrophs at these stations and hence lower need for heterocysts. Towards the end of summer conditions even out along the bay, as the surplus phosphorus from the spring bloom is used up at the outer stations and no heterocyst gradient is present. Heterocyst frequency varied significantly over the summer, with minimum values in the beginning of July, coinciding with the highest water temperatures, and higher frequencies in early and late summer. We suggest this is primarily due to a more efficiently functioning nitrogenase enzyme at high temperatures with a reduced need for “expensive” heterocysts. Spring heterocyst differentiation occurred around 4-6 weeks after depletion of dissolved inorganic nitrogen (DIN) and only when water temperature was 5-9 oC and a pycnocline established. It seems that temperature and light in concert will initiate growth, leading to an internal nitrogen deficiency which starts heterocyst differentiation. / Himmerfjärden eutrophication study