This diploma thesis (Heterotrophic incorporation of selenium in Chlorella sp.) focuses on characterisation of the genus Chlorella and large-scale use of algae for biofuel and starch production. Besides that, the thesis contains an up-to-date summary of forms of autotrophic algae cultivation ranging from large-volume tanks covering the area of dozens of hectares down to laboratory cultivation in photobioreactors and heterotrophic cultivation of algae in fermenters. Experimental part of the thesis focuses on cultivation and observation of the heterotrophic growth of algae Chlorella vulgaris in relation to the concentration of added selenium which was performed on glucose substrate with varying concentrations and forms of selenium. Selenium was applied in a form of sodium selenite Na2SeO3 and sodium selenate Na2SeO4. Numerous experimental repetitions suggest a significantly important effect of the form of the added selenium on growth rate and vitality of Chlorella sp. While the algae did not thrive on sodium selenite medium, they prospered on sodium selenate and exhibited faster growth compared to that on the media without additional selenium.

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Many experiments have been performed with the Warburg apparatus, or variations of this manometric technique, since Warburg's experiments (52, 53) where the effects of cyanides upon dark reactions and of urethanes upon light reactions of photosynthesis were demonstrated. The same basic techniques were utilized in this research in attempting to determine the effects of some coordination complexes upon the apparent photosynthetic rate of Chlorella pyrenoidosa. A second goal of the present paper was to investigate the potential of the Warburg apparatus as a tool for screening algicidal compounds.

Purpose of the study: to assess chlorella impact on productivity of sows and on speed of piglets’ growth. This research of master degree was accomplished in 2004-2006 in Animal Nutrition Department of Lithuanian Veterinary Academy, in feed research laboratories of Agrochemical Investigations Centre of Lithuanian Institute of Agriculture and joint stock company AB “Kauno Grūdai“ and in breeding-ground of farmer G. Charitonovas (Žadeikonių village, Pasvalio region) with the swine of the Swedish Yorkshire breed of the first group. Research revealed that: 1. during suckling period mass reduction in sows, that were given a suspension of chorella during pregnancy period, was lower by 14.5% compared to the control group containing sows that were fed by combined feeds only (p<0.05); 2. prolificacy of the test group sows was by 10.0% higher (p>0.05), piglets produced by them were 9.6% heavier (p<0.05) and families of born piglets where less varied when compared to the control group; 3. sows of the test group that were given a suspension of chorella were by 10.98 kg or 22.4% milkier when compared to the sows of control group (p<0.05); 4. chorella had a positive impact on change of piglets mass: piglets at birth were by 9.6%, on the 21st day of life – by 8.4%, on the 35th day of life, i.e. at the wean off – by 7.6% heavier when compared to the piglets of control group (p<0.05); 5. in the test group retention of piglets was as follows: on the 10th day of life – by 2.2%, on the 21st day... [to full text]

Immunocytochemical analyses have demonstrated that the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is predominantly localized in the pyrenoid region of chloroplasts of evolutionarily diverse algae. That Rubisco remains pyrenoid-localized at photosynthetically-saturating irradiance in the green alga Chlorella pyrenoidosa indicates a catalytic, rather than storage function for pyrenoid-localized Rubisco. This is further supported by the immunolocalization of Rubisco activase to the pyrenoids of two species of green algae. The exclusion of phosphoribulokinase from the pyrenoids of a red and a green alga indicates that pyrenoids do not possess the full complement of Calvin cycle enzymes. / Thylakoid lamellae traverse the pyrenoids of many algae. The absence of light-harvesting phycoerythrin and of photosystem (PS) II activity, but not PSI activity, from the intrapyrenoid thylakoids of the red alga Porphyridium cruentum indicates a structural and functional heterogeneity between these lamellae and those located in the chloroplast stroma. In contrast, the intrapyrenoid thylakoids of cryptomonads, algae whose chloroplast is thought to have evolved from red algae, possess both PSI and PSII protein complexes. These results are discussed with reference to Rubisco being mainly pyrenoid-localized in these algae.

Driven by the increase in industrialization and population, the global demand of energy and material products is steadily growing. Microalgae have come into prominence in the past several decades due to their ability to utilize solar energy to fix atmospheric carbon dioxide, and produce biomass and lipids at productivities much higher than those possible with terrestrial biomass. The main objective of this research is to maximize the biomass and lipid production of Chlorella vulgaris by varying different external conditions so as to achieve the ideal feedstock for the production of commodity chemicals and implement wastewater treatment. The effects of various culture medium compositions on Chlorella vulgaris growth and lipid production were investigated using batch culture. Thirteen culture media: Modified Chu’s No. 10, Bold basal, BG-11, Modified BG-11, N-8, M-8, RM, Modified Spirulina, F-si, Fogg’s Nitrogen free, Fog, F/2, and Johnson medium were compared in terms of optical density, biomass production, specific growth rate and lipid production. Following a 10-day culture in a temperature controlled environment, Bold basal medium was found to have the highest average biomass productivity of 48.056 ± 2.097 mg L -1 day -1 , with overall specific growth rate of (d -1 ): 0.211 ± 0.003 and lipid productivity of 9.295 mg L -1 day -1 among the selected media. This is a basis for the optimization of different cultivating medium to enhance algal lipid production. In order to maximize the quality and quantity of the algal biomass and lipid content in Chlorella vulgaris, different strategies were used using different ratios of nitrogen and phosphorus source in the modified Bold basal medium (BBM). In the 12-day batch culture period, the highest biomass productivity obtained was 72.083 mg L -1 day -1 under Bold basal medium with Nitrogem control Phosphorus limited conditions. The highest lipid content, lipid concentration and lipid productivity obtained were 53.202%, 287.291 mg/L and 23.449 mg L -1 day -1 respectively, under Bold basal medium with Nitrogen Control Phosphorus Deprivation conditions. Nitrogen starvation was found to be the critical factor affecting the biomass production and lipid accumulation while the starvation of phosphorus induced a higher total lipid content and affected the lipid composition of Chlorella vulgaris cultures. Recently, as the demand for pure microalgae strains for the production of algal lipid as a feedstock of renewable energy has been increasing, the designation of an effective photobioreactor (PBR) for mass cultivation is essential to assure stability in the amount of feedstock. Various PBRs design such as bubbling, air-lift, porous air-lift was compared. In general, the bubbling design is a better PBR designs than the others, having the highest biomass concentration of 0.78 g/L during the culture time. Besides, it was observed that the 35 cm draft tube of the porous air-lift PBR had shorter mixing time (24.5 seconds) and higher biomass concentration (0.518 g/L) than the 50 cm air- lift design. The bubbling PBR with the highest gas flowrate of 2.7 L/min produced the highest biomass production of 0.74 g/L within the cultivation time. The information is shown to be a useful guide for determining the optimal condition of the PBRs. Light wavelengths and intensities were determinant factors in affecting the growth and lipid content of autotrophic organisms such as C. vulgaris. The experiment investigated the effect of algal lipid production by using LEDs (Light Emitting Diodes) with different wavelengths. C. vulgaris was grown in the effluent for 10 days under the photoperiod of 18:6 h Light/Dark cycles with different visible light sources (cool white, blue and red) and intensities (50 μmol m -2 s -1 ) at 25°C. The overall maximum dry biomass of 1353.33 mg/L was observed at 50 μmol m -2 s -1 cool white light during 10th culture day, with the highest overall productivity of dry biomass production (117.23 mg/L d -1 ) within cultivation time. The highest lipid content (34.06 %) was obtained with the blue color due to light efficiency and deep penetration to the photosynthetic pigments (chlorophyll) in C. vulgaris. However, the highest lipid productivity was observed in cool white light of 318.63 mg/L during the 10th culture day. The effect of light intensity toward the lipid productivity was further investigated by increasing the light intensity of cool white light. The highest lipid productivity was observed at 110 μmol m -2 s -1 in a light intensity of 658.99 mg/L during the 10th culture day. In high irradiance (110 μmol m -2 s -1 ), the proportion of poly unsaturated fatty acid (C18:1 and C18:2) contributed most of the fatty acid methyl ester (FAME) content in the collected sample, irrespective of all treatments. The next study optimized the harvesting rate of algae by using an electro- coagulation-flotation (ECF) harvester, which combines the electrochemical reaction in the electrodes and the dispersion of hydrogen gas to allow floatation of microalgae cell for surface harvesting. The response surface methodology model (RSM) was employed to optimize different ECF parameters: electrode plate material, electrode plate number, charge of electrode, electrolyte concentration and pH of the solution. The model revealed that aluminum was the best electrode material for the ECF process. It was also found that a three electrode plates setup with one anode and two cathodes had the best performance for harvesting. Additionally, sodium chloride (NaCl) at 8 g/L in harvesting medium could increase the flocculant concentration and reduce electric power consumption. Also, having the culture medium at pH 4 also had a significant effect on improving the flocculant production. Combining these optimal conditions, the highest flocculant concentration reached 2966 mg/L in 60 mins; a 79.8% increase in flocculant concentration, based on the tested conditions. The results of this study show the significance of different parameters affecting the coagulation and flocculation of C. vulgaris and provide a reference for the design of a large-scaled harvester for microalgae harvesting in the further study. To conclude, this research comprises a study on the use of indigenous algae for the production of algal lipid, which is used to produce commodity chemicals. Details on the use of nutrient sources, the techniques of cultivation and the optimization of cell harvesting were included so as to remove nutrients from effluents to minimize the occurrence of eutrophication in harbor, thereby providing economic advantages. Thus, the optimization of these processes is very adequate and offers significant advantages for the wastewater treatment. The developing of algal cell biotechnology is necessary to further enhance algal lipid production in an attempt to apply it commercially.

Many contaminants, such as industrial chemicals, fertilizers, herbicides, pharmaceuticals and heavy metals are released to e environment. 3,4-dichloroaniline（3,4-DCA） originated from degradation of some herbicides such as diuron, propanil and linuron, is toxic to aquatic organisms and affects human being immune system. Triclosan, widely used as antimicrobial agent in pharmaceuticals and personal care products (PPCPs), has been detected as contaminat in various aquatic environments. In this work, green algae were isolated from local environment, then applied for the removal and biodegradation of 3,4-DCA and triclosan. Two axenic pure algae were isolated using the solid agar method. One of the algae was identified morphologically as Desmodesmus sp. based on the experimental results. The other one was identified morphologically as Chlorella pyrenoidosa by accredited authority. At the same time, alga S. obliqnus was obtained commercially. All the three green algae were cultured in tris-acetate-phosphate (TAP) medium. Firstly, the alga C. pyrenoidosa was applied to remove and biodegrade 3,4-DCA with a concentration of 4.6 μg/mL for 7 d. A removal percentage of 78.4% was obtained over a 7-d period. Two major metabolites with less toxicity were identified as 3,4-dichloroformanilide and 3,4-dichloroacetanilide using HPLC-ESI-ion trap-MS. iii Secondly, all the three green microalgae species including C. pyrenoidosa, Desmodesmus sp., and S. obliqnus, were compared in the removal and biodegradation of triclosan in aqueous medium. When triclosan with concentration of 400 ng/mL was cultured with the three algal species separately, triclosan was quickly eliminated from medium in the 1 d cultivation by algae with removal percentages of 62.4%, 92.9% and 99.7% for C. pyrenoidosa, Desmodesmus sp. and S. obliqnus, respectively. The dominant mechanism for the removal of triclosan by C. pyrenoidosa was determined as cellular uptake. Biotransfromation of triclosan involved hydroxylation and methylation, glucose conjugation was determined as the predominant mechanisms for the removal of triclosan by algae Desmodesmus sp. and S. obliqnus. The intermediates from hydroxylation, reductive dechlorination, or ether bond cleavage were immediately subjected to glucosylation and/or methylation via the hydroxyl group of triclosan or introduced, which served as detoxification mechanisms of the chlorinated aromatic chemicals. In order to find the intermediates in the metabolic pathway of triclosan by algae, Desmodesmus sp. was exposed to 400 ng/mL triclosan. 2,4-DCP was detected during the cultivation period 3-12 h using ultra performance liquid performance (UPLC)-ESI-MS/MS. The metabolites from multi metabolic reaction like the glucose conjugate of hydroxylated triclosan were detected in the first 30 min after exposure. The metabolites as products from glucosylation and consecutive hydroxylation and methylation of triclosan or 2,4-DCP were detected after 3 h iv cultivation. To provide more information about the reductive capability of C. pyrenoidosa, the reaction between C. pyrenoidosa and triclosan was investigated. When C. pyrenoidosa was exposed to triclosan with concentration from 100 to 800 ng/mL, more than 50% of triclosan was eliminated by algal uptake from the culture medium during the first 1 h exposure. In the biodegradation experiments, a major metabolite from the reductive dechlorination of triclosan was identified by using liquid chromatography (LC)-ESI-MS. The ability of reductive dechlorination of C. pyrenoidosa might potential application for bioremediation of polychlorinated biphenyls (PCBs) that with similar chemical structure to triclosan, but belonging to the catagory of persistent organic pollutants (POPs). Through the TEM observation, it was found that the triclan treatment resulted in the disruption of the chloroplast of algal cells, which indicated that triclosan may affect membrane metabolism.

Submitted by Maykon Nascimento (maykon.albani@hotmail.com) on 2015-04-29T19:06:00Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Gustavo Benevides.pdf: 380662 bytes, checksum: e22589abe3e8dc1a64213785a64889e5 (MD5) / Approved for entry into archive by Elizabete Silva (elizabete.silva@ufes.br) on 2015-04-30T20:12:01Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Gustavo Benevides.pdf: 380662 bytes, checksum: e22589abe3e8dc1a64213785a64889e5 (MD5) / Made available in DSpace on 2015-04-30T20:12:01Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Gustavo Benevides.pdf: 380662 bytes, checksum: e22589abe3e8dc1a64213785a64889e5 (MD5) Previous issue date: 2015-04-29 / FAPES - Fundação de Amparo a Pesquisa do Espírito Santo / O uso de lipídios obtidos a partir da biomassa de microalgas tem sido descrito como uma alternativa promissora para a indústria petro-diesel e envolve etapas como o cultivo de microalgas, separação da biomassa e extração de lipídios. Para viabilizar a produção em larga escala, é necessário selecionar as espécies mais produtivas, diminuir os custos de produção e determinar as condições ideais de cultivo. Os gêneros Chlorella, Desmodesmus e Ankistrodesmus apresentam características favoráveis à produção comercial, tendo sido então selecionada uma espécie de cada no presente trabalho. O objetivo do estudo foi avaliar diferentes condições de cultivo de Ankistrodesmus fusiformis, Chlorella vulgaris e Desmodesmus spinosus visando o aumento da produtividade em biomassa e lipídios totais. As algas foram identificadas e cultivadas em laboratório, em condições controladas de temperatura a 26ºC (±1), aeração por borbulhamento à pressão ambiente e luminosidade fornecida por lâmpadas fluorescentes, com intensidade de 47,25 μmol de fótons m-2.s-1 (3500 lux), fotoperiodo de 12h e pH 7, sob duas concentrações estressantes de nitrato de sódio (0,10 g/L e 0,05g/L). Os cultivos duraram em média 16 dias, sendo as curvas de crescimento construídas com dados de espectrofotometria óptica coletados a cada 48h, e a biomassa obtida ao final do cultivo por centrifugação e liofilização de cada unidade experimental. Para extração dos lipídios totais, foi utilizada a mistura de clorofórmio: metanol (1:2), segundo a metodologia de Bligh & Dyer (1959). Os tratamentos de estresse em D.spinosus resultaram em maior acúmulo lipídico, com aumento de até 149,7%, porém com drástica diminuição do crescimento e biomassa. Em C. vulgaris, nos tratamentos de estresse, verificou-se apenas ligeiro aumento do peso seco e teor de lipídios, não havendo diferença significativa entre os tratamentos e o controle. Da mesma forma, A.fusiformis não mostrou respostas significativas ao estresse pela redução de nitrato de sódio do meio, havendo ligeira diminuição do conteúdo lipídico e aumento do crescimento e biomassa. Com respostas diferentes para cada espécie estudada, evidencia-se a necessidade do conhecimento da fisiologia e autoecologia da cepa a ser cultivada em escala comercial visando à produção de ácidos graxos para fins de biodiesel. / The use of lipids from microalgae biomass has been described as a promising alternative to the petro-diesel industry and involves steps such as microalgae cultivation, biomass separation and lipid extraction. To facilitate the large-scale production, it is necessary to select the most productive species, reduce production costs and determine optimal growing conditions. Chlorella, Desmodesmus and Ankistrodemsus are genus with favorable characteristics for commercial production, being selected then, one species from each one in the present work. The aim of the study was to evaluate different growth conditions in Ankistrodemsus fusiformis, Chlorella vulgaris and Desmodesmus spinosus aiming to enhance the biomass production and total lipid content. The algae were identified and grown in the laboratory under controlled temperature conditions of 26 ºC (±1), aeration by bubbling at ambient pressure and light provided by fluorescent lamps at an intensity of 47,25 μmol photons m-2.s-1 (3500 lux), 12 hours photoperiod and pH 7, under two stressful concentrations of sodium nitrate (0.10 g/L and 0.05 g/L). The cultures lasted an average of 16 days, with growth curves constructed with optical spectrophotometry data collected every 48 hours, and the biomass was obtained at the end of the cultures by centrifugation and lyophilisation of each experimental unit. For extraction of total lipids, were used the mixture chloroform: methanol (1:2) according to Bligh & Dyer (1959) method. The stress treatments in D. spinosus resulted in increased lipid accumulation, an increase of up to 149.7%, although with a drastic decrease in growth and biomass. In C. vulgaris, the stress treatments were found to only slightly increase the dry weight and lipid content, with no significant difference between the treatments and the control. Similarly, A. fusiformis showed no significant responses to stress by reduction of sodium nitrate in the medium, with a slight decrease of lipid content and increased growth and biomass. With different answers for each studied species, we highlight the need for knowledge of both the physiology and autecology of the strain to be grown on a commercial scale aimed at the production of fatty acids for biodiesel purposes.

Submitted by Milena Dias (milena.dias@ufpe.br) on 2015-03-11T18:49:15Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Páblo Eugênio da Costa e Silva.pdf: 1498936 bytes, checksum: 4eb542fe966b2a53e862d6b33ee0195d (MD5) / Made available in DSpace on 2015-03-11T18:49:15Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Páblo Eugênio da Costa e Silva.pdf: 1498936 bytes, checksum: 4eb542fe966b2a53e862d6b33ee0195d (MD5) Previous issue date: 2013 / CAPES / As doenças cardiovasculares são as principais causas de morte em todo o mundo e podem ser causadas pelo o acúmulo de fibrina nos vasos sanguíneos. Vários micro-organismos produtores de enzimas fibrinolíticas já são descritos na literatura, dentre eles bactérias e fungos. O objetivo do trabalho foi produzir enzimas fibrinolíticas a partir da microalga Chlorella vulgaris utilizando glicerol e milhocina, resíduo agroindustrial do processamento do milho, como fonte de carbono e nitrogênio, respectivamente. No cultivo utilizando 0,5 % de milhocina em meio Bold’ Basal (BBM) foi observado uma produção de 745 U mL-1 de enzima fibrinolítica pela microalga. Após a seleção do meio de cultura, foi realizado um delineamento estatístico utilizando Metodologia de Superfície de Resposta (RSM). A concentração celular foi otimizada, e o valor estimado pela equação foi de 1,52 g L-1, utilizando 0,9 % de glicerina e 1,2 % de milhocina. A produtividade celular, produção de protease e enzima fibrinolítica não conseguiram ser otimizadas, no entanto alcançaram níveis de 232 mg L-1 dia-1, 416 U mL-1 e 704 U mL-1, respectivamente. O presente trabalho mostrou que C. vulgaris cultivada em meio de cultura utilizando glicerol e o resíduo agroindustrial milhocina, pode ser viável para obter altos níveis de concentração de enzima fibrinolitica, bem como ser utilizada como um recurso no combate a doenças cardiovasculares.