This project investigates the potential of the 27 Everglades green algal strains for producing biodiesel. The five potential strains chosen by measuring the neutral lipid content using the Nile red method were Coelastrum 46-4, Coccoid green 64-12, Dactylococcus 64-10, Stigeoclonium 64-8 and Coelastrum 108-5. Coelastrum 108-5 and Stigeoclonium 64-8 yielded the same amount of lipids in both Gravimetric and Nile red method. A linear relationship between algal biomass and lipid accumulation was seen in Coelastrum 46-4, Coccoid green 64-12, Stigeoclonium 64-8 and Coelastrum 108-5 indicating that increase in algal biomass increased the lipid accumulation. Nitrogen and phosphorous stress conditions were also studied where higher lipid accumulation was observed significantly (p < 0.05) in 64-8 Stigeoclonium and 64-12 Coccoid green. Collectively, it could be summarized that Coelastrum 108-5, Coccoid green 64-12 and Stigeoclonium 64-8 were promising in some aspects and could be used for further studies.

Background and Aim: Microalgae are a potential economical source of omega-3 fatty acids, which are known for their health benefits. Omega-3 fatty acids derived from microalgae are however still expensive due to high costs involved in production. These costs can partly be attributed to cultivation of microalgae in unialgal cultures, which are prone to contamination, difficult to scale up and require stringent control of growth conditions. This study therefore is aimed to investigate the feasibility of using a mixed microalgae culture for the production of omega-3 fatty acids. Methods and materials: The microalgae were grown under uncontrolled conditions in a hanging-bag photo-bioreactor, with only the phosphorus-nitrogen ratio (P:N-ratio) varied. Variations in the different population proportions in the culture were studied and related to the biomass, total fatty acid and omega-3 fatty acid productivity of the culture. Finally, a comparison was made between the results obtained in this study and similar studies found in literature. Results and discussion: The mixed culture used was composed of several green microalgae, whose proportions varied with P:N-ratio, season, and age of the culture. Different interactions between the populations were observable, and these were thought to influence the culture’s biomass, total fatty acids (TFAs) and omega-3 fatty acid productivity. The TFA content of the culture was ~10-20% by the end of the exponential phase, depending on the nutrients supplied. The fatty acids were composed of mostly unsaturated fatty acids, the majority of which were omega-3 fatty acids; C18:3ω3 (up to 50% of TFA), C16:4ω3 (up to 15%) and C18:4ω3 (up to 5%). Other fatty acids observed included C16:0 (up to 25%), C18:1ω9/C18:1ω8 (up to 20%), C18:2ω6 (up to 5%), traces of C18:0, C18:3ω6, C17:0, C16:3ω3 and C16:1ω7 were also detected. The presence of these fatty acids was found to vary depending on season, P:N-ratio, and age of the culture. When compared to studies in the literature, the mixed culture was found to perform better than, or as well as reported studies, where unialgal cultures were cultivated under controlled laboratory conditions. Conclusion: In view of the reported advantages of cultivating mixed cultures, the production of omega-3 fatty acids via such cultures appears attractive. More research however is needed to optimise the culture; study interactions between the organisms and their effect on biomass, total fatty acid and omega-3 fatty acid productivity. Finally, the economic implications of using mixed cultures need to be evaluated as well.

Data on 75 species from 124 quadrats collected from the intertidal and subtidal regions in the Flat Top Islands area of Georgia Strait in late spring to early autumn, 1972, were subjected to a number of community analysis techniques including a Williams and Lambert association analysis, a species constellation diagram, a cluster analysis of quadrats, an inverse analysis of species, a Zurich-Montpellier analysis, and a Bray and Curtis ordination. Association analysis and cluster analysis classification of quadrats delimited four communities: an intertidal, an upper subtidal, a lower subtidal, and a sandy bottom community. Species constellation diagram and inverse analysis classification of species delimited six communities: an intertidal, a shallow red algal, a laminarian, a turf, a deep water, and a sandy-bottom community. The Zurich-Montpellier analysis and the Bray and Curtis ordination did not produce clearly definable groups of quadrats. The results of all the analyses indicate that marine benthic algae form broadly overlapping distributions from which communities of varying affinities can be extracted. / Science, Faculty of / Botany, Department of / Graduate

Cell wall structure was investigated in 20 different red algae. Representatives from all 4- families of the order Ceramiales and one family of the order Gigartinales were investigated. Of these, 3 genera, Polysiphonia, Pterosiphonia and Antithamnion were investigated with regards to both the cellulosic and mucilaginous portions of the cell wall. A new staining technique utilizing a combination of ruthenium red and osmium tetroxide as a postfixation was used in the latter portion of the study. The ultrastructure of pit connections was examined in all algae. The inner cellulosic portion of the cell wall consists of a reticulate pattern of microfibrils which appear densely stained, In Pterosiphonia this cellulosic portion was found to consist of 2 layers; an inner layer of microfibrils which ensheathed individual cells and an outer layer of microfibrils which ensheathed the entire thallus and was in contact with the mucilaginous coat. The microfibrils in the inner layer appear nearly cross-sectioned, while those in the outer layer appear more longitudinally oriented to the plane of sectioning. The outer mucilaginous coat covers the entire thallus. It consists of 4 layers. The first or outermost layer consists of loose bunches of microfibrils extending out from the second layer. The second layer consists of a zone of medium electron density approximately 750 A in thickness. The third layer is wholly contained within the second layer. It is composed of a densely staining band of microfibrils extending from a similarly staining membrane-like structure. The fourth layer is a densely stained membrane-like structure in contact with the cellulosic portion of the cell wall. An additional layer, the D layer, is sometimes found in the cell wall. When present it is found in the outermost portion of the cellulosic wall and obscures the fourth layer of the mucilaginous coat. It consists of a densely staining amorphous material. Investigation of the pit connection showed the occurrence of 2 stages of one basic pit structure. One stage, the single disc stage-pit structure, has been found in all algae investigated. It consists of a solid, lenticular, membrane-bound plug situated within an aperture in the cell wall. The plug consists of a granular material surrounded by a zone of densely staining amorphous material. The other stage, the double disc stage pit structure, is a modification of the single disc stage. It is not found in young cells near the apex of the thallus, but only in cells which have, or are undergoing, rapid elongation and vacuolation. This pit structure has only been observed in axial cells of the family Ceramiaceae in the order Ceramiales. The double disc stage pit structure differs from the single disc stage in that the granular material of the plug is segregated into 2 regions or plates, one on either side of the plug. The central region of the plug at first appears clear but later appears to be partially occupied by a granular to fibrillar material. The differentiation of the double disc stage pit structure from the single disc stage has been described. These observations are thought to support and confirm the earlier work of Jungers (25). However, his observations have been extended through the use of electron microscopy in this study. It has been proposed that the terms used in this study, single disc stage- and double disc stage pit structures, replace the terms Polysiphonia and Griffithsia pits used by Jungers. / Science, Faculty of / Botany, Department of / Graduate

A study of the red algal order Gelidiales in British Columbia and northern Washington was conducted. Gelidiales are represented in the study area by four species, Gelidium coulteri, G. purpurascens, G. vagum and Pterocladia caloglossoides, and a key to identify the taxa is provided. Earlier reports of G. crinale, G. pusillum, G. robustum and G. sinicola from British Columbia and northern Washington have been shown to be misidentifications of the other taxa and are excluded from the flora. The occurrence of G. vagum in the British Columbian flora is significant because it appears to have been introduced from the northwest Pacific and now has a disjunct distribution, occurring in Japan and China and on two islands in the Strait of Georgia, British Columbia. Gelidium vagum and the Gulf of California species, G. johnstonii, are considered to be sister species. They share the unique character of monoecy (i.e. unique in the genus) and a morphological resemblance, but differ in several other characters, including arrangement of cortical cells, shape and flattening of tetrasporangial stichidia, shape of stichidial apices and position of the apical initial with respect to the adjacent cortex, presence/absence of an apical furrow and sterile margin in tetrasporangial stichidia and presence/absence of intact third order filaments in mature cystocarps. The life history of Gelidium vagum was completed in culture, only the second Gelidium species for which this has been accomplished. Gelidium vagum has a triphasic Polysiphonia-type life history with isomorphic (monoecious) gametophytes and tetrasporophytes, and carposporophytes growing on gametophytes. The rare occurrence (G. purpurascens, G. coulteri) or absence (Pterocladia caloglossoid.gametophytes in the field and the lack of successful completion of a life history in culture suggests that, whereas these species may occasionally complete a Polysiphonia-type life history in situ, vegetative or apomictic mechanisms are more important in maintaining field populations. Chromosome counts of n = 14 - 15 were made on undivided tetrasporangia of G. vagum. The pattern of spore germination in Gelidium coulteri, G. purpurascens and G. vagum is similar for carpospores and tetraspores. A single germ tube grows from the spore, the entire cytoplasmic contents of the spore evacuate into the germ tube and a wall cuts the germ tube off from the empty spore. The germ tube divides unequally to form a concave and a fusiform cell. The primary attachment rhizoid forms from a derivative of the concave cell and the sporeling apical cell forms from a fusion cell derivative. As apical organization is established, the subapical cell produces two lateral periaxial cells. Carpogonium and carposporophj'te development was followed in Gelidium purpurascens and G. vagum. The functional carpogonium is intercalary. Non-functional carpogonia divide transversely with a concave wall and become sessile and degenerate. The fertilized carpogonium consistenty forms a fusion cell by fusing with adjacent cortical cells. Fusion cell lobes cut off gonimoblast filaments that send out processes that fuse with haploid gametophyte cells of nutritive chains. Carposporangia are produced terminally and laterally by gonimoblast cells and protrude into two locules that have formed as the cortex is pushed away from the plate of second order filaments. Carpospores are released through a single ostiole in each locule. Spermatogenesis, tetrasporogenesis and rhizine morphology were studied ultrastructurally for the first time in Gelidiales. In spermatangial development, apical chloroplasts in the spermatangial mother cell are cut off by a furrow, a mechanism previously not reported in red algae for exclusion of chloroplasts from spermatangia. As the spermatangium matures, spermatangial vacuoles form from the coalescence of vesiculated endoplasmic reticulum and dictyosome-derived vesicles. Spermatangial vacuoles are discharged from the spermatangium just prior to the release of the spermatium. Stages of tetrasporangium development are marked by wall development. Prior to karyokinesis, the young tetrasporangium is dominated by a large nucleus. Following karyokinesis, chloroplasts, starch grains and dictyosomes increase in abundance, a wall layer is deposited, and dictyosomes undergo a series of morphological changes from small and flat, to large and flat producing fibrillar vesicles, to hemispherical and producing cored vesicles. At cytokinesis an electron dense wall layer is deposited around the tetrasporangium and in the cleavage furrow, and cored vesicles secrete a mucilage-like material inside this wall layer. / Science, Faculty of / Botany, Department of / Graduate

Seven species of marine phytoplankters assigned to different taxonomic divisions were tested for toxic responses to two different molecular species of selenium known to be prevalent in seawater, selenite and selenate. Selenate proved to be more toxic than selenite, although severe toxicity was only observed at high concentrations (10⁻² and 10⁻³ M) of both selenate and selenite. At these concentrations, growth was completely or severely inhibited in most species tested. In some of the species that remained viable, both the percentage of motile cells and their swimming speed were drastically reduced. Scanning electron microscopy revealed that, under these circumstances, Dunaliella tertiolecta cells possessed much shorter flagella compared to the controls, while those that became non-motile lacked flagella altogether. Despite these striking alterations in both growth and morphology, cells of Amphidinium carterae, Dunaliella tertiolecta and Pavlova lutheri showed, after prolonged exposures, signs of adaptation to high selenium concentrations. Lower concentrations of selenium were generally non-toxic and frequently even stimulatory to growth. These observations suggest that for meaningful inferences on selenium toxicity both the concentration range and the length of the studies must be considered and the potential for adaptation to high selenium concentrations taken into consideration. The main ultrastructural and physiological changes in cells of Dunaliella tertiolecta, Pavlova lutheri and Amphidinium carterae treated with selenite or selenate involved the cell coat, mitochondria, chloroplasts as well as the respiratory and photosynthetic rates. Other changes were observed in the nucleus, lipids, vacuoles, nitrogen and carbon contents, but these showed greater variability among the microalgae studied. The major alterations suggested that energy transducing systems were severely affected by selenium toxicity. These led to significant decreases or even elimination of storage products which were indicative of severe shortage in energy and produced major reductions in growth. These occurred later upon exposure to the toxicant and coincided with the loss of cell coat material, suggesting that the shedding of cell surface material might play a major role in the detoxification and adaptation of the microalgae to toxic concentrations of selenium. / Science, Faculty of / Botany, Department of / Graduate

A comparative, year long (15 month) study of three lakes (Whonock, Munday, Como) in the Lower Fraser Valley, British Columbia was undertaken to gain information on the physicochemical and seasonal limits of scale-bearing Synuraceae (Chrysophyta). In addition, sporadic collections were made in different lakes in this region to augment the information provided from the three main lakes. The synuracean taxa are reviewed with worldwide distribution lists, and environmental and seasonal information are discussed. Thirty-one taxa of Chrysophyceae were identified, of which 20 are in the Synuraceae. Eight taxa within this family are new records for British Columbia, viz. : Mallomonas cratis var. asmundiae Wujek et Van der Veer Mallomonas papillosa Harris et Bradley Mallomonas doignonii var. tenuicostis Asmund et Cronberg Mallomonas transsylvanica Peterfi et Momeu Mallomonopsis ouradion (Harris et Bradley) Harris Paraphysomonas vestita (Stokes) de Saedeleer Synura splendida Korshikov Synura glabra Korshikov It was found that the Synuraceae are not restricted to cold waters, as often assumed. Some taxa are present throughout the year. They do seem to favour acidic and sometimes dystrophic environments, and it is suggested that factors such as pH or water chemistry should be investigated as probable controlling factors for synuracean populations. Of the 11 non-synuracean Chrysophyceae noted', six are new records for the Province: Bicoeca kepneri, Chrysococcystis elegans, Dinobryon crenulatum, Epipyxis lauterbornii, Epipyxis utriculus var. acuta, and Eusphaerella turfosa. / Science, Faculty of / Botany, Department of / Graduate

This dissertation reports a study of the fungi and 'protists' (Labyrinthulids, Thraustochytrids, Hyalochlorella marina) associated with the intertidal red algae Rhodoglossum affine and Gelidium coulteri. Research focused on laboratory isolations from algal thalli collected from in situ populations. Different isolation techniques and isolation media were employed to evaluate the abundance and diversity of fungi and protists associated with these red algae. Algal tissue surface sterilization and rigorous rinsing procedures were used to remove and/or enumerate surface-associated microbes. The results obtained from the different isolation techniques and algal tissue pretreatment procedures are compared and discussed in terms of their usefulness for each member of the algal-associated microbiota. Natural populations of affine and coulteri support a rich fauna of marine protists. The most prevalent members of this protist fauna were Labyrinthula spp. resembling the "Vishniac Strains" and Thraustochytrium motivum. Schizochytrium aggregatum, a new species of Labyrinthulid designated Labyrinthuloides sp. 1, and Hyalochlorella marina were also common depending upon the isolation method utilized. These protists appear to be associated with the surfaces of the algal thalli, and exist as saprobes and/or perthophytes rather than biotrophic parasites of the algal tissues. Isolations from field-collected algal tissues also yielded actinomycetes, yeasts, and a high diversity of imperfect fungi. Overall isolation frequencies for individual fungal taxa were low. Most of the mycelial fungi isolated are considered to be of terrestrial origin and of questionable 'significance' in the intertidal habitat. Only four, possibly five, are presently considered marine. The mycelial fungi most commonly isolated include: Acremonium sp. 019-78, Cladosporium cladosporioides, Dendryphiella salina, Penicillium spp., Phoma sp. (Group 1), Sigmoidea littoralis sp. nov., and Unidentified hyphomycete 044-78. Certain of these fungi may grow saprobically (as pertho-phytes) on reproductive and/or senescing algal tissues in the intertidal habitat, but their activities appear to be limited. Field-collected thalli of Rhodoglossum affine and Gelidium coulteri were allowed to decompose in mesh bags placed in the intertidal. The succession of higher fungi associated with the decomposing algae was followed by plating representative bimonthly subsamples of the algal tissues onto a Base Mineral Medium. Rhodoglossum affine deteriorated completely after 52 days of exposure, while a small amount of Gelidium coulteri remained after 71 days. Qualitative aspects of the mycobiota associated with the two algal species were similar, however fungi were isolated more frequently from coulteri. A dominant mycobiota was apparent after 36 days of exposure on the beach. Acremonium sp. 019-78, Dendryphiella salina and Sigmoidea littoralis sp. nov. were active colonizers of the decomposing algal tissues, and their isolation frequencies increased as decomposition proceeded. Several species of bacteria capable of utilizing the cell wall polysaccharides of red algae (agar, carrageenan) were also present on the decomposing algae. It is possible that the activities of these bacteria enhanced fungal development. Thraustochytrium motivum, Schizochytrium aggregatum and Ulkenia sp. RC02-80 were placed into sterile seawater cultures with surface-sterilized tissues of Rhodoglossum affine and Gelidium coulteri. After 72 hours of incubation, positive growth associations were examined using scanning electron microscopy. The three Thraustochytrids displayed luxuriant growth on all algal tissue types, and produced extensive ectoplasmic networks on the algal surfaces which functioned in attachment and, presumably, in the absorption of dissolved nutrients. Ectoplasmic net elements were resolved down to 0.02 pm in diameter, but no obvious 'penetration' of the algal tissues could be discerned. All of the protists (Labyrinthulids, Thraustochytrids, Hyalochlore11a marina) isolated from these red algae are described and illustrated. Certain commonly encountered and/or poorly known mycelial fungi are also described, including a new species of marine hyphomycete, Sigmoidea littoralis sp. nov. / Science, Faculty of / Botany, Department of / Graduate

Harmful algae studies, in particular toxic dinoflagellates, and their response to global warming in the Red Sea are still limited. This study was aimed to be the first to characterize the identity, thermal responses and toxin production of four Prorocentrum strains isolated from the Central Red Sea, Saudi Arabia. Morphological and molecular phylogenetic analysis identified the strains as P. elegans, P. rhathymum and P. emarginatum. However, the identity of strain P. sp.6 is currently unresolved, albeit sharing close affinity with P. leve. Growth experiments showed that all species could grow at 24-32°C, but only P. sp.6 survived the 34°C treatment. The optimum temperatures (Topt) estimated from the Gaussian model corresponded to 27.17, 29.33, 26.87, and 27.64°C for P. sp.6, P. elegans, P. rhathymum and P. emarginatum, respectively. However, some discrepancy with the Topt derived from the growth performance were observed for P. elegans and P. emarginatum, as thermal responses differed from the typical Gaussian fit. The Prorocentrum species examined showed a sharp decrease after the optimum temperature resulting in very high activation energies for the fall slope, especially for P. elegans and P. emarginatum. The minimum critical temperature limit for growth was not detected within the range of temperatures examined. Subsequently, high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis revealed all species as non okadaic acid (OA, common toxin of the Prorocentrum genus) producers at any temperature treatment. However, other forms of toxin (i.e. fast acting toxins) not examined here could be produced. Therefore, further investigations are required. The results of this study provided significant contribution to our knowledge regarding the presence, thermal response and toxin production of four Prorocentrum species from the Central Red Sea, Saudi Arabia.

abstract: Phosphorus (P) is a limiting nutrient in ecosystems and is mainly used as fertilizer to grow food. The demand for P is increasing due to the need for increased food supply to support a growing population. However, P is obtained from phosphate rock, a finite resource that takes millions of years to form. These phosphate rock deposits are found in only a few countries. This uneven distribution of phosphate rock leads to a potential imbalance in socio-economic systems, generating food security pressure due to unaffordability of P fertilizer. Thus, the first P-sustainability concern is a stable supply of affordable P fertilizer for agriculture. In addition, improper management of P from field to fork leaves an open end in the global P cycle that results in widespread water pollution. This eutrophication leads to toxic algal blooms and hypoxic “dead zones”. Thus, the second P-sustainability concern involves P pollution from agriculture and cities. This thesis focuses on P flows in a city (Macau as a case study) and on potential strategies for improvements of sustainable P management in city and agriculture. Chapter 2 showed a P-substance-flow analysis for Macau from 1998-2016. Macau is a city with a unique economy build on tourism. The major P flows into Macau were from food, detergent, and sand (for land reclamation). P recovery from wastewater treatment could enhance Macau’s overall P sustainability if the recovered P could be directed towards replacing mined P used to produce food. Chapters 3 and 4 tested a combination of P sustainability management tactics including recycling P from cities and enhancing P-use efficiency (PUE) in agriculture. Algae and biosolids were used as recycled-P fertilizers, and genetically transformed lettuce was used as the a PUE-enhanced crop. This P sustainable system was compared to the conventional agricultural system using commercial fertilizer and the wild type lettuce. Chapters 3 and 4 showed that trying to combine a PUE-enhancement strategy with P recycling did not work well, although organic fertilizers like algae and biosolids may be more beneficial as part of longer-term agricultural practices. This would be a good area for future research. / Dissertation/Thesis / Doctoral Dissertation Biology 2020