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A thesis submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, Republic of South Africa, in fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering. Johannesburg, 2016 / The process of photosynthesis is of great importance as it is the reaction of carbon dioxide (CO2) and water with the help of light, ’free’ energy from the sun, to form useful carbohydrates and oxygen. Photosynthesis is therefore useful both in carbon dioxide mitigation and growing bio-feedstocks towards making biofuel. This thesis aims to address two areas for analysing the photosynthesis process: 1. Looking at the physical limits of the growth; and 2. Improving the production rate of some aquatic plants, such as duckweed and microalgae. To address the first aim, the fundamental concepts of thermodynamics were used to analyse the photosynthetic process. It was found that the theoretical minimum number of moles of photons (NP) required (9–17) is less than the values reported by other researchers, suggesting that the photosynthesis process is highly irreversible and inefficient (operating at 35% efficiency or less). This is because the number of moles of photons will increase with greater process irreversibility (when the entropy generated is greater than zero). If the photosynthesis process is indeed that irreversible then the removal of heat (the heat not used by other cellular processes) by the plant becomes a major problem. It is suggested that transpiration, and other cellular processes, are the processes by which that is done, and it is shown that the water needs of the plant for transpiration would dwarf those needed for photosynthesis. Knowing the fundamental limits to growth could also be of use because if an organism was growing at a rate close to this value there would be no advantage to try to do genetic modification to improve its rate. Following the ideas presented above a spectrophotometer was used not only to obtain the absorption spectrum of algae, but it was also used to grow small samples at specific light wavelengths. The algae species researched was Desmodesmus spp., which, for example, is used to remediate waste water or as a source of feedstock for biofuel production. It also tolerates high CO2 concentrations. This simple experimental method demonstrated that a specific light wavelength (in particular the Secomam Prim spectrophotometer) 440 nm was preferred for the algae growth. It was recommended that this specific light wavelength would be best for growth. It might also be useful to know this fact particularly when designing photobioreactors, as this could reduce the amount of heat released into the surroundings and thus make the process more energy efficient. Interestingly, the wavelength for maximum growth corresponded to one of the peaks in the absorption spectra but there was no increase in growth rate corresponding to any of the other peaks. To address the second aim, the author determined how well predictions on improving the growth of algae (Desmodesmus spp. for example), based on a theoretical model, would work when tested experimentally. What the researcher found was that the method improved algae production, using the same set of equipment. The production was improved by a factor of 1.28 and 1.26 (at product concentrations 1000 mg/L and 600 mg/L respectively) when retaining 40% of the algae suspension. The method may be particularly useful when large amounts of biomass are required as there is no extra cost of purchasing additional equipment. The same model was applied to a growth profile of duckweed (Spirodela polyrhiza 8483, which is convertible into biofuel or a source of food), and the author showed that the model could work if the duckweed was provided with an added carbon source. In order to find an economical and reliable alternative to bridge the scale gap between laboratory and industrial production, the author checked if duckweed species (Spirodela polyrhiza 8483, Spirodela polyrhiza 9509, Lemna gibba 8428, Lemna minor DWC 112, Wolffia cylindracea 7340 and Wolffia globosa 9527) could be cultivated in media less expensive than the basal laboratory medium (Schenk and Hildebrandt). The author found that duckweed can be cultivated more efficiently, and in a more cost-effective manner, in the alternative media types, while maintaining growth rates, RGR 0.09 day-1, and starch contents, 5– 17%(w/w), comparable with that obtained with the conventional laboratory media. Thus, by looking at the photosynthesis process thermodynamically and experimentally, it is shown to be possible to improve the process by using concepts presented in this thesis. / MT2017

O uso intensificado de combustíveis fósseis como fonte de energia, vê-se a necessidade do desenvolvimento de novas tecnologias, principalmente as renováveis, como o hidrogênio, que possui vantagens por ser elemento abundante no universo, ser renovável e não poluente. A utilização de microalgas e cianobactérias é uma alternativa para a produção de biohidrogênio a partir da quebra da água e de compostos orgânicos. De acordo com isso, nesta pesquisa foram testados diversos fatores físico-químicos e nutricionais nas condições de cultivo de cepas de Chlamydomonas spp. e Anabaena spp. Para tanto, cepas selecionadas foram cultivadas em duas fases experimentais, a primeira aeróbia e a segunda anaeróbia, para proporcionar produção de hidrogênio por biofotólise direta anaeróbia, via hidrogenase, sob privação de enxofre para a clorofícea, e de nitrogênio para a cianobactéria, estimulando para esta também a produção por biofotólise indireta, via nitrogenase. A cepa com melhor produtividade de hidrogênio, de cada gênero, foi selecionada para a etapa de otimização das fases experimentais de cultivo. Durante os ensaios foram realizadas análises de produção máxima, velocidade de produção, volume e produtividade de hidrogênio, além de análises de concentração de biomassa, físico-químicas, bioquímicas e geração de subprodutos. O método utilizado foi eficiente para produção de hidrogênio e ficou comprovada a diferença de produção de hidrogênio entre diferentes cepas. Anabaena sp. obteve produtividade média de hidrogênio quatro vezes maior, aproximadamente de 76,8 µmol.L-1.h-1, comparada a C. reinhardtii, com média de 18,6 µmol.L-1.h-1. / The intensifying use of fossil fuels as energy source, one sees the need to develop new technologies, especially renewable, such as hydrogen. This has advantages because hydrogen is an abundant element in the universe, be renewable and non-polluting. The use of microalgae and cyanobacteria is an alternative for the production of bio-hydrogen of breaking water and organic compounds. Accordingly, in this study were tested several physic-chemical factors and nutrition in growing conditions of Chlamydomonas spp. and Anabaena spp. strains. For this purpose, strains selected were cultured in two experimental phases, first aerobic and second anaerobic, to hydrogen production by direct biofotolise anaerobic, via hydrogenase, under sulfur deprived to chlorofycea, and nitrogen to cyanobacterium, for this also to production by indirect biofotolise, via nitrogenase. The strain with highest productivity of hydrogen, of each gender, was selected for the optimization of the experimental stages of cultivation. During the tests were analyzes of maximum production, velocity, volume and productivity of hydrogen, and analysis of biomass concentration, physic-chemical, biochemical and generation of by-products. The method used was efficient for the production of hydrogen and was different between strains. Anabaena sp. obtained average yield four times highest, approximately 76.8 µmol. L-1.h-1compared to C. reinhardtii, averaging 18.6 µmol. L-1.h-1.

O crescimento acelerado da aqüicultura no estado de São Paulo, ou seja, a implantação de pesque-pagues e pisciculturas pode estar causando uma série de problemas ambientais. A contribuição para o processo de eutrofização é uma das conseqüências desses empreendimentos, pois tanto os tanques utilizados na piscicultura como os afluentes em torno desses estabelecimentos, estão sendo eutrofizados pelo excesso de nutrientes. Uma das conseqüências da eutrofização é o aparecimento de florações de cianobactérias, e a principal preocupação está nas toxinas liberadas por estas cianobactérias, que se ingeridas pelos seres humanos e animais, podem causar efeitos de intoxicação, como fraqueza, cefaléia, vômito e dependendo da concentração ingerida pode levar à morte. Desta forma é necessário que haja um programa de controle da qualidade da água dos tanques e reservatórios e também dos peixes que ali são criados, pois florações de cianobactérias vêm sendo encontradas em diversos corpos d\'água. Este estudo teve como foco a determinação da cianotoxina microcistina-LR, empregando técnicas como a extração em fase sólida e a cromatografia líquida para a detecção e quantificação da microcistina-LR em amostras de florações de cianobactérias. Os testes feitos com a extração em fase sólida demonstraram que esse procedimento não se faz necessário para todas as amostras, pois houve casos em que não se obteve diferença nos picos interferentes mais próximos ao tempo de retenção do analito de estudo. Como as matrizes desse tipo de amostras são muito complexas e variam muito conforme o meio em que se encontram, recomenda-se que sejam avaliados caso à caso a necessidade de se promover a extração em fase sólida, pois o mesmo é um processo que demanda um tempo maior de análise e conseqüente aumento nos custos. Foi determinado e validado um método cromatográfico considerado capaz de fornecer dados reproduzíveis e confiáveis, por meio de testes de seletividade, limite de detecção e de quantificação, linearidade, precisão, exatidão e recuperação, conforme critérios de aceitação da Resolução n°899 de 2003, da ANVISA. O limite de detecção do método ficou estipulado em 0,1 µg mL-1, e o limite inferior de quantificação em 0,5 µg mL-1, determinados conforme a relação sinal-ruído proposta pelo Guia de Validação de Métodos Bioanalíticos da ANVISA. A quantificação da microcistina-LR foi feita utilizando o método de superposição de matriz, que minimiza e/ou compensa o efeito de matriz ou de possíveis interferentes presentes na amostra, e a curva analítica obtida y = 1,5888+21,849 x, com um coeficiente de correlação de 0,997 mostra uma boa linearidade. Foram analisadas amostras de florações de cianobactérias, coletadas em pesque-pagues e pisciculturas situadas na região do Alto Mogi (subdivisão da bacia do Mogi Guaçu), conforme o método de extração e análise estudado. / The rapid growth of aquaculture in the state of São Paulo may be causing a number of environmental problems. The contribution to the eutrophication process is among the consequences of these undertakings, given that the tanks used in fish farming as well as the changes around these establishments are becoming eutrophic systems due to excessive nutrients. A frequent consequence of eutrophication in waters is the massive development of cyanobacteria.The occurrence of these blooms induces severe problems, as Microcystis aeruginosa, the most widespread distributed cyanobacteria, which can produce microcystin-LR. Toxic effects of MC have been described in liver, lungs, stomach, and intestine. Deaths in wildlife, livestock and human beings were also associated with microcystin exposition, which can occur directly by ingestion, inhalation, contact, intravenous inoculation of contaminated water (hemodialysis) or indirectly, by the consumption of animals, as fish and mollusks, the major ingestors of cyanobacteria and its toxins. Thus we need a program to control the quality of water tanks and reservoirs and also the fish breeded there, as cyanobacteria blooms have been found in various water bodies. This study focused on the determination of the cyanotoxins microcystin-LR, using techniques such as solid phase extraction and liquid chromatography for the detection and quantification of microcystin-LR in samples of cyanobacteria blooms. Tests performed with solid phase extraction showed that this procedure is not necessary for all the samples because there were cases where no difference was obtained in interfering peaks near the retention time of the analyte studied. As the parent of such samples are very complex and vary greatly, because the extracts contained too much coextrated material that interfered in the LC-UV detection, and depending on the way in which it is recommended to be assessed, case by case, the solid phase extraction needs to be promoted, because it is a process that demands a longer period of analysis and consequently an increase in costs. A liquid chromatography method was established and validated, which is deemed capable of providing reproducible and reliable data, by testing for selectivity, limit of detection and quantification, linearity, precision, accuracy and recovery, in accordance with the acceptance criteria of Resolution No. 899 of 2003 of ANVISA. The detection limit of the method was set at 0.1 µg mL-1, and the lower limit of quantification at 0.5 µg mL-1 determined according to the signal to noise ratio proposed by the Validation Guide of Bioanalytical Methods, ANVISA. Quantification of microcystin-LR was performed using the matrix-matched method, which minimizes and/or offsets the effect of possible matrix interference or present in the sample. The analytical curve obtained y = 1.5888 + 21.849 x, with a coefficient of correlation of 0.997 shows a good linearity. Real aquaculture samples were analyzed that were detected and quantified according to the method developed.

Green macroalgae represent an attractive source of renewable carbon. Conversion of algal biomass to useful products requires depolymerization of the cell wall polysaccharides cellulose and ulvan. Cellulose saccharification has been widely studied and involves synergistic action of endoglucanases, exoglucanases, and β-glucosidases. The enzymatic depolymerization of ulvan has not received the same attention and additional studies are required in order to fully understand the mechanisms involved in its biodegradation. Synthetic biology offers the possibility of importing modules such as biomass-degrading systems and biofuel producing pathways from different organisms into a genetically tractable host such as Escherichia coli. In this study it was shown that E. coli expressing the glycosidase CHU2268 of Cytophaga hutchinsonii grows well on cello-oligosaccharides such as cellohexaose, and co-expression with the endoglucanase CenA of Cellulomonas fimi allows growth on untreated crystalline cellulose. Moreover, a model for ulvan utilization was built for the first time based on a polysaccharide utilization locus from the alga-associated flavobacterium Formosa agariphila. It was also shown that F. agariphila, is able to grow using biomass from the green macroalga Ulva lactuca as its sole carbon source, and enzymes with ulvanase activity are induced by the presence of this alga in the culture medium. Enzymes for ulvan depolymerization from F. agariphila, including an ulvan lyase, xylanases and rhamnosidases, were cloned using the PaperClip DNA assembly method and expressed in active form in E. coli. Furthermore, a secretion system based on the use of the Antigen 43 was successfully used to secrete an active ulvan lyase using E. coli and ribosome binding sites of different strengths were studied and used to optimize the system. These results represent a first step for the design of a microorganism capable of utilizing green macroalgal biomass for the production of biofuels and other valuable bio-products.

This pilot study was conducted to determine if an Electron Magnetics Oxygen and Hydrogen (EMOH) device can increase the dissolved oxygen (DO) concentration of a residential surface water. By using EMOH, DO concentration will increase and allow bacteria to remove the substrate that creates blue-green algae for which the City of Boynton Beach (City) receives complaints. Those complaints center on odors and the visual appearance of the ponds. The study was conducted in-situ at the INCA Pond system in the City of Boynton Beach, Florida with data collection taking place bi-weekly, using surface aeration techniques. Water sampling was conducted in the INCA Pond system via a handheld water sensor. Primary variable monitored included: water temperature, barometric pressure, DO concentration, and DO saturation (DOSAT). Biomass of dead algae at the bottom of the pond was also monitored to determine if increased DO concentration aided the biological digestion of the organic matter. Data analysis shows that exposure to EMOH treatment allowed the relationship between DO and temperature to change from a negative correlation (the expected relationship) to a positive trend. Furthermore, pressure and DOSAT became less correlated after exposure to EMOH effluent. In all, EMOH was shown to be an effective means of treating hypoxic pond water. The optimal EMOH effluent discharge is determined to be deep in the subject pond. Backed by research on the surface-air water and bubble-water oxygen transfer coefficients, DO concentration in the subject pond was 110% higher when effluent was directed down toward the floor of the pond. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 +/- 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 +/- 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 +/- 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600?1700 cm^-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm^-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm^-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells.

Cyanobacterial toxins present a real and growing threat to humans and animals due to the projected increases in algal blooms resulting from increasing global temperature and pollution. Wild animals, livestock, pet animals and humans can be poisoned from contaminated drinking water. With the discovery of cyanobacterial toxins present in nutritional supplements, a new concern looms over consumers with threats of neurotoxin and hepatotoxin related damage from exposure to these products. To this end, work on the development of a freeze dried algal reference material was pursued for future use in environmental and nutritional supplement analysis. The first stage of the project was to prepare needed calibration standards, starting with homoanatoxin a, a homologue of the highly neurotoxic anatoxin-a compound. The resulting reference material (RM-hATX) had a homoanatoxin-a concentration of 20.2 ± 0.7 ?M, and proved to be stable while stored at temperatures of 80°C. Reference samples for dihydro and epoxy analogues of anatoxin-a and homoanatoxin-a were then prepared by semi-synthesis. The second stage of the project was the development of new analytical methods for the anatoxins. A derivatization reaction in which dansyl chloride was coupled with a novel cleanup step produced anatoxin derivatives suitable for liquid chromatography (LC) with mass spectrometry (MS) or fluorescence detection (FLD). Limits of quantitation were 60 ng L-1 and 1.6 ?g L-1 for the developed LC-MS/MS and LC-FLD methods, respectively, with the limit of quantitation significantly better than that of a previously developed method for the underivatized toxins based on HILIC MS/MS. Quantitative results for anatoxins in various algal samples using all three methods of analysis of were compared and it was found that there were no significant differences between the three methods. Unfortunately, experiments showed that the various toxin analogues did not elicit equimolar responses in either LC-MS/MS or LC FLD, thus indicating the importance of having individual calibration standards for quantitative analysis. The LC-MS/MS and LC-FLD methods were paired with a previously developed method for the analysis of hepatotoxic microcystins to screen a small number of nutritional supplement samples for cyanobacterial toxins. Microcystins were detected in all five Aphanizomenon flos-aquae samples examined. This method involved a fifteen-fold pre-concentration using a solid phase extraction cartridge, which gave a 98% recovery of microcystins. The third phase of the project was the preparation and testing of a preliminary algal matrix reference material as a feasibility study for the eventual production of a CRM. After selecting various algal cultures and samples that could be blended together, a freeze dried algal reference material was prepared and packaged. This material (RM-BGA) was then characterized using several methods including the two new dansylation-based procedures.