MESHWR / Department of Ecology and Resources Management / The presence of harmful algal blooms (HABs) and cyanobacteria toxins in drinking water sources are known to pose a great threat to humans. The main aim of this study was to use molecular technique to determine the origins of the cyanobacteria species at Musina raw water abstraction point by identifying and comparing the non-toxic and toxic cyanobacteria species in the Limpopo River and some of its tributaries based on the phylogenetic analyses of 16S rRNA gene. The Musina water treatment plant is located downstream of a weir and the Beit bridge on the Limpopo River and the raw water supply is abstracted from 22 boreholes of which 14 are along the Limpopo River and 8 boreholes are inside the Limpopo River channel. The bottom sediments samples were collected from these rivers: Limpopo, Crocodile, Mokolo, Mogalakwena, Nzhelele, Lephalale, Sand rivers (South Africa); Notwane (Botswana), Shashe River and Mzingwane River (Zimbabwe). The physical-chemical analysis of the bottom sediments showed the availability of nutrients, nitrates and phosphates, in excess of 0.5 mg/l for most the of rivers, alkaline pH and salinity in excess of 500 mg/l. Total genomic DNA were extracted from cyanobacteria species on the bottom sediments and Polymerase Chain Reaction (PCR) method was used to detect the genetic profile of the cyanobacteria species. Molecular identification of cyanobacteria was based on PCR amplification and sequencing of the 16S rRNA gene. The 16S rRNA gene was absent from sediments of the Mogalakwena and Lephalale rivers but present in all other selected rivers. The cyanotoxins detection was also based on PCR by amplification of microcystin/nodularin and cylindrospermopsin polyketide synthetase genes. Most of the samples showed no amplification of the toxin genes. While two samples showed the amplification of cylindrospermopsin polyketide synthetase gene (Sand River and Nzhelele River Next to Tshipise) and two samples showed amplification for microcystin/nodularin synthetase gene, Crocodile River and Mzingwane River. The first was the confirmation of similarity of samples from Crocodile River downstream of hartbeespoort Dam and Shashe River to Leptolyngbya boryana with 99% bootstrap confidence. The similarity of sample from Musina borehole to Sand River upstream to Alkalinema pantanalense with 98% bootstrap. Thus, the presence of toxic genes may imply the presence of toxic cyanobacteria species in the river sediments and may be hazardous to humans because rural communities and commercial farmers abstract water from Limpopo River catchment for human consumption, livestock and irrigation. The waters of the Limpopo River basin also provide drinking water to wildlife and a habitant for aquatic organisms/animals.

Les secteurs de la parfumerie et de la cosmétique occupent une place proéminente dans la société moderne. De nombreuses entreprises se positionnent depuis plusieurs années sur les produits cosmétiques à ingrédients naturels. Les plantes, longtemps considérées comme matière première principale pour le domaine de la cosmétique, sont aujourd’hui concurrencées par les microalgues dont la biomasse devient plus facile à obtenir grâce aux avancées en biotechnologie bleue. Ainsi, Cosmo International Ingredients se positionne à travers cette thèse pour élargir son panel de matières premières valorisables dans le domaine de la cosmétique. Dans un premier temps, l’étude phytochimique de microalgues péruviennes a permis d’isoler et identifier une famille majeure de métabolites chez les microalgues : les glycolipides. Une recherche de conditions d’extraction optimale pour cette famille a été effectuée et a permis de proposer une méthodologie verte, spécifique et peu coûteuse. Les cyanobactéries connues pour leur production de métabolites structurellement diversifiés ont été sélectionnées pour la culture suivant des critères spécifiques. Cette approche a permis d’isoler et de caractériser 5 composés à forte valeur ajoutée dont 4 peptides et un alcaloïde. Enfin, un forçage métabolique a été effectué sur Microcystis aeruginosa afin d’optimiser la production des 4 peptides cibles. Il en est ressorti que les paramètres température et intensité lumineuse jouent un rôle important dans la production peptidique / The sectors of fragrances and cosmetics play a prominent role in the modern society. During the last decades, several companies have been focusing on nature to provide innovative products. Plants have historically been considered the main raw material in the cosmetic field but, recently, microalgae have been identified as a worthy competitor due to the facility to obtain biomass. Thus, the company Cosmo International Ingredients supported this PhD. thesis to broaden their range of raw materials that can be used for the cosmetic industry. First, the phytochemical study of Peruvian microalgae allowed the isolation of a major family of metabolites: glycolipids. An environmentally-friendly, selective and low-cost method for their extraction from the biomass has been developed. Cyanobacteria known for their production of structurally diverse metabolites have been selected for culture following specific criteria; as a result 5 compounds have been isolated and fully characterized, 4 of which were peptides and one was an indole alkaloid. Finally, to optimize the production of the targeted bioactive peptides, a kinetic study was performed for 3 different temperatures and 3 different light intensities. These parameters were found to play a critical role for the peptide production

Algal blooms caused by toxigenic cyanobacterial species are an increasing economic burden globally, as high anthropogenic inputs of nitrogen and phosphorous, coupled with rising levels of atmospheric CO2, promote eutrophication and enhance bloom proliferation. Of the freshwater bloom forming species, Microcystis aeruginosa has garnered the most attention due to the production of toxic secondary metabolites known as microcystins. These cyclic peptides are potent eukaryotic protein phosphatase 1 and 2A inhibitors, and can induce hepatic damage if concentration levels reach above the World Health Organization level of 1 µg/L. Current mitigation strategies of water column disruption or by use of broad acting chemicals, are limited in their range and may cause unwanted off target effects to the surrounding biota. Antisense oligonucleotides are short single-stranded DNA polymers that hybridize with transcribed mRNA, and suppress translation of protein products through steric hindrance of ribosomes, or by RNAse H degradation of the DNA/RNA bound complex. While antisense oligonucleotide applications have proven successful in the pharmaceutical industry, their potential remains largely unexplored in environmental contexts. For this reason, we investigated the knockdown of microcystin synthetase gene cluster mcyE in M. aeruginosa. We found that ionic charge neutralization coupled with heat shock were effective chemical competence based methods for delivery, mcyE transcript abundance in cells treated with phosphodiester linked antisense oligonucleotides significantly decreased in RT-qPCR analysis, and production of intracellular microcystin significantly decreased over a 24 hour period (-1.9 fg/cell). This work demonstrates a novel proof of concept for the potential use of exogenous antisense oligonucleotides to target M. aeruginosa in harmful algal bloom occurrences.

Photoautotrophic organisms have been highlighted as carbon capture and conversion platforms for sustainable production of agricultural and chemicals in KSA. Previously two cyanobacterial strains, Geitlerinema spp. CT7801 and CT7802, were isolated from an industrial brine outfall site in the Eastern Province of the Kingdom of Saudi Arabia. Initial characterization of their growth characteristics showed growth at high temperature (38 ºC) and high salinity ( > 60 PSU), making them potentially good candidates for industrial applications. In this study, quantitative growth assays were performed using standardized methods developed for the analysis of Red Sea photosynthetic microorganisms supported by microscopic observations, optimal growth media preference assays, CO2 concentration effect, photoperiod effect, mixotrophic and heterotrophic growth tests. Data was recorded for absorbance (600 and 750 nm wave lenght), dry cell weight (DCW), colorimetric observations, and chlorophyll a content. Both CT7801 and CT7802 exhibited a clear preference for Walne's Red Sea medium. An analysis on media composition highlights B and Fe as growth enhancers, as well as a base requirement of seawater. Tests on the effect of supplied concentration of CO2 showed that air enhanced with 1 % v/v CO2 allows approximately 2-fold increase in DCW for Geitlerinema spp. CT7802. Photoperiod tests showed that continuous light is disadvantageous for phototrophic growth of Geitlerinema spp. CT7801 and CT7802. Results for mixotrophic and heterotrophic growth of Geitlerinema spp. CT7801 and CT7802 revealed their ability to metabolize glycerol. Analysis on the complete genome of CT7802 identified three key enzymes, glycerol kinase, glycerol-3-phosphate dehydrogenase and triosephosphate isomerase, which may catalyze the glycerol metabolic pathway in the strain. Utilization of glycerol, a residue of the biodiesel industry, might provide a sustainable alternative for growth of Geitlerinema sp. CT7802.

No description available.

Cyanobateria are promising cell factories due to their minimal nutrient requirements and utilization of asmospheric carbon di-oxide as its sole carbon source. In particular, polyhydroxybutyrate (PHB) is an industrially useful bio plastic that is produced naturally by some cyanobacteria. Furthermore, PHB biosynthetic pathway is a starting point for production of the biofuel, 1-butanol. There has been much genetic engineering effort toward increasing the production of PHB from cyanobacteria. These have been focused on increasing the pool of acetyl-CoA precursor, or increasing the amount of the reductant NADPH. The upstream process for increasing these reactants is complex and involves many genes. In this contect, cyanobacteria libraries will contribute to reveal genes or gene fragments that are responsible for production of PHB, alkanes and other high value compounds. In pursuit of finding these novel genes or genefragments, a transcription factor library is created in this study with 50 transcription factors. Furthermore, the process is optimized towards the creation of genomic fragment library and metagenomic fragment library with 26 diverse strains. Membersof the transcription factor library are over-expressed by a PHB - producing host Synechocystis PCC 6803 and the process towards creation of genomic and metagenomic libraries is optimized. The members of the metagenomic library can be screened for increased PHB, alkanes, lactate and other high value products and the potential members can be isolated and characterized.

Taste-and-odor (T&O) compounds are frequently produced by cyanobacterial blooms in bodies of water. Geosmin, perhaps the most common T&O compound produced by these blooms, is not effectively removed by conventional water treatment processes and frequently causes the tap water to have an off flavor. Although geosmin is not harmful when ingested, it damages the consumers' confidence in the cleanliness of their water. There are treatment options for geosmin removal, but the most common methods are often not implemented until complaints are made by consumers.There has been an increasing amount of research on the use of polymerase chain reaction (PCR)-based methods that can detect the presence of the geosmin synthase gene which is responsible for the production of geosmin. If the geosmin synthase gene is found to be present in an emerging cyanobacterial bloom, water treatment facilities can prepare in advance to treat for geosmin. In this study, we developed a qPCR (quantitative polymerase chain reaction) assay that can detect the presence of the geosmin synthase gene in several species of cyanobacteria within the Anabaena genus. We tested our assay, as well as PCR assays designed by Giglio et al. (2008) and Suurnäkki et al. (2015) on extracted Anabaena flos-aquae DNA, biosynthesized Anabaena ucrainica DNA and DNA extracted from environmental samples of Deer Creek Reservoir, Strawberry Reservoir, and Utah Lake. It is important to note that the geosmin gene was not confirmed to be present in any of the environmental samples nor in the Anabaena flos-aquae DNA and our assay did not test positive on these samples. Our qPCR assay was very successful when used with the biosynthesized Anabaena ucrainica DNA. We used the results to estimate a DNA standard curve that can be used to estimate the starting concentration of the geosmin synthase gene. Because our assay was not successfully used with any extracted DNA, further testing and calibration may be necessary to produce a DNA standard curve that is representative of DNA that is extracted. Further calibration of the DNA standard curve was not done because there were no geosmin events during the course of the research.Development of PCR-based methods of detecting geosmin-producing cyanobacteria requires genetic sequencing information of the target-organisms. Thus, further development of PCR-based methods requires that the local geosmin-producers be identified and sequenced. Our assay as well as the assay designed by Moore (2019) can assist with the identification of these species by classifying their genus.

Excess nutrients from human activity trigger toxic cyanobacterial and algal blooms, creating expansive hypoxic dead zones in lakes, damaging ecosystems, hurting local economies, undermining food and water security, and directly harming human health. To identify when and where nutrients limit phytoplankton and cyanobacterial growth, and cyanotoxin concentrations across Utah Lake, USA we conducted four in-situ bioassay studies (563 cubitainers or experimental units) that experimentally added N, P or N+P over the spring, early summer, summer, late summer, and fall in lake water from the top 20 cm of the water column. For our purpose, we defined total phytoplankton as all prokaryotic or eukaryotic organisms containing chlorophyll-a. We evaluated changes in chlorophyll-a and phycocyanin concentrations; the abundance of cyanobacterial species and total phytoplankton species or divisions; cyanotoxin concentrations of the microcystin, anatoxin-a, and cylindrospermopsin; DIN, SRP, TP, and TN concentrations; and other water chemistry parameters. We found that the nutrient limitation of cyanobacteria, and to a lesser extent phytoplankton, was influenced by season and space. Cyanobacteria were often co-limited in the spring or early summer, limited by a single nutrient in the summer, and not limited by N or P in the late summer and fall. Alternatively, phytoplankton were co-limited from the summer into the fall in the main body of the lake and either N limited or co-limited continually in Provo Bay. Microcystis, Aphanocapsa, Dolichospermum, Merismopedia, and Aphanizomenon spp., and Aulacoseira and Desmodesmus spp. and two taxonomical categories of algae (i.e., unicellular and colonial green algae) were primarily associated with cyanobacteria and phytoplankton nutrient limitations. Concentrations of the three cyanotoxins demonstrated a seasonal signal and loosely followed the growth of specific cyanobacteria but was not dependent on total cyanobacterial cell density. The DIN and SRP were biologically available in all water and nutrient treatments with nutrient concentrations declining over the incubation period, suggesting that nutrient levels were not oversaturated. Our results offer insights into specific nutrient targets, species, and, and cyanotoxins to consider in the future to manage Utah Lake.

Climate change and eutrophication, accelerated by anthropogenic activities, have impacted aquatic ecosystems worldwide. These impacts have stimulated the expansion of cyanobacterial blooms which pose severe threats to ecosystem functioning, environmental health, and the economy. However, the long-term effects of environmental change on bloom-forming cyanobacteria are not well understood as traditional paleolimnological approaches are of limited use in the reconstruction of cyanobacterial dynamics through time. Here, sediment DNA (sedDNA) was used to investigate long-term cyanobacterial trends using sediments from two experimental (fertilized L227 and acidified L223) and two reference (L224 and L442) lakes in the Experimental Lakes Area, Canada. First, to determine whether taxonomic bias might arise from the cyanobacterial sediment record, I performed a 1-year incubation experiment comparing the degradation rates of selected cyanobacterial genes under contrasting environmental conditions. Based on first-order linear decay models, Synechococcus sp. (Synechococcales) decayed the slowest under cold, anoxic conditions, followed by Trichormus (Nostocales), then Microcystis (Chroococcales), suggesting differential preservation of DNA. I then compared the quantitative performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the analysis of sedDNA and found that the ddPCR results were more consistent with the known history of the lakes. Furthermore, ddPCR showed that cyanobacterial abundance increased over the past century in all study lakes, but the greatest increase was observed in experimentally fertilized L227. HTS revealed shifts in the cyanobacterial community towards Nostocales dominance and a decrease in alpha diversity in response to phosphorus-only additions. An increase in abundance of the mcyE gene (indicative of microcystin producing taxa) was uniquely observed in L227 when nitrogen additions ceased. Heating degree days were important in explaining variation in the cyanobacterial community composition in all lakes, but nutrients had a greater influence on the L227 community. When sediment data were compared to historical surface water phytoplankton records, moderate to strong correlations between the two archives were found, validating the use of sedDNA. This research demonstrated that sedDNA can elucidate cyanobacterial trends at the community, population, and species level over multidecadal timescales in response to environmental change.

Saudi Arabia is highly accessible to marine water, receives year-round availability of sunlight and generates a high annual carbon dioxide emission, all of which are justifications that merits the deployment of cyanobacterial cell factories. However, industrial cyanobacterial strains capable of thriving in conditions of the Arabian Peninsula are currently lacking. Given the fact that native cyanobacteria from the Red Sea are adapted to the local conditions, they are therefore good cell factory candidates where their inherent attributes can be harnessed. In this dissertation, an isolation and screening pipeline was developed to specifically identify physiologically robust cyanobacterial strains from the central Red Sea. Seventeen unicellular cyanobacterial strains were extensively cataloged through a series of physiological characterization and their evolutionary relationships were ascertained through phylogenetic analyses. Arising from this survey work, a high light, thermo- and halo-tolerant Synechococcus sp. RSCCF101 was selected for metabolic analysis under various growth conditions to assess its suitability as a platform for cell factory development. Significant metabolic changes were observed in cells subjected to different light regimes. High phycocyanin and chlorophyll a content were obtained under the low-light growth (50 μmol photons.m-2.s-1) while high biomass was accumulated, along with an increase external nitrate demand, under the high light growth (200 μmol photons.m-2.s-1). A genomic and transcriptomic approach was undertaken to elucidate the molecular signatures of Synechococcus sp. RSCCF101. Synechococcus sp. RSCCF101 contains a small genome (3 Mbp) that is rich in guanine cytosine content (68%) and harbors genes that encode for compatible solutes biosynthetic pathway and phycobilisome subunits which may account for its halo-tolerant and phycocyanin rich phenotype. Upon high-light treatment, the light harvesting machineries of Synechococcus sp. RSCCF101 was downregulated while the photosystem protection and carbon fixation capacity were upregulated. Taken together, the findings of this research will facilitate in the development of a new model system for industrial applications in high-light, high temperature and high salinity environments in general and Saudi Arabia in particular.