Thesis (M.S.)--Mississippi State University. Department of Biological Sciences, 2006. / Title from Web page (viewed on Sept. 16, 2008). Includes bibliographical references (p. [30-35]).

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Στην παρούσα ερευνητική εργασία μελετήθηκε η σύσταση σε λιπαρά οξέα των λιπιδίων (ολικών, φωσφολιπιδίων, γλυκολιπιδίων και σφιγγολιπιδίων, ουδετέρων) των μικροφυκών Nannochloropsis oculata και Chlorella sp. και μεικτών καλλιεργειών των ανωτέρω στελεχών καθώς επίσης και τροχοζώων του είδους Brachionus plicatilis καλλιεργούμενων σε βιοαντιδραστήρες μεγάλης κλίμακας υπό διαφορετικές συνθήκες αύξησης. Η καλλιέργεια των μικροφυκών πραγματοποιήθηκε σε βιοαντιδραστήρες βιομηχανικής κλίμακας 300 L, ο φωτισμός ήταν συνεχής και η θερμοκρασία παρέμενε σταθερή στους 28±3 οC κατά την περίοδο της άνοιξης και στους 22±3 οC κατά την περίοδο του φθινοπώρου και του χειμώνα. Το Chlorella sp., συνέθεσε λιπίδια πλούσια σε α-λινολενικό και παλμιτικό οξύ, ενώ στα λιπίδια του Nannochloropsis oculata τα υπερέχοντα λιπαρά οξέα ήταν τα οξέα παλμιτικό, εικοσιπεντανοϊκό (EPA) και παλμιτελαϊκό. Μετά από κλασμάτωση των λιπιδίων του Chlorella sp. βρέθηκε ότι το κλάσμα των ουδέτερων λιπιδίων (NL) ήταν το σημαντικότερο ποσοτικά, σε όλες τις φάσεις αύξησης. Στις πρώτες φάσεις αύξησης τα φωσφολιπίδια (P) υπερείχαν των γλυκο- και σφιγγο- λιπιδίων (G+S) ενώ καθώς η καλλιέργεια εξελισσόταν τα ποσοστά των P μειωνόταν προς όφελος των G+S. Τέλος, στη μεικτή καλλιέργεια όπου επικρατές στέλεχος ήταν το Nannochloropsis oculata τα λιπαρά οξέα με τα υψηλότερα ποσοστά ήταν τα οξέα παλμιτικό, παλμιτελαϊκό και EPA. Μεταξύ των λιπιδιακών κλασμάτων η αναλογία των NL ήταν σημαντικά υψηλότερη, ακολουθούσε εκείνη των G+S και τέλος των P. Τα τροχόζωα καλλιεργήθηκαν σε βιοαντιδραστήρες βιομηχανικής κλίμακας 2,500 L. Για τη διατροφή τους χρησιμοποιήθηκαν καλλιέργειες μικροφυκών (Nannochloropsis oculata, Chlorella sp.), ζύμη αρτοποιίας και διάφορα εμπλουτιστικά περιέχοντα σε υψηλά ποσοστά EPA και εικοσιδυεξανοϊκό (DHA). Η ανάλυση των λιπιδίων των τροχοζώων έδειξε ότι η σύσταση τους σε λιπαρά οξέα εξαρτάται από την τροφή την οποία καταναλώνουν. Διατροφή με καλλιέργειες μικροφυκών είχε ως αποτέλεσμα τη σύνθεση λιπιδίων που περιείχαν σε σημαντικά ποσοστά EPA. Μεταξύ των λιπιδιακών κλασμάτων, η αναλογία των NL ήταν σημαντικά υψηλότερη, ακολουθούσε των P και τέλος των G+S. Με τη χρήση εμπλουτιστικών τα λιπίδια των τροχοζώων που συσσωρεύτηκαν ήταν πλούσια σε EPA και DHA.Το λιπιδιακό κλάσμα με τα υψηλότερα ποσοστά ήταν το κλάσμα των NL, ακολουθούσε το κλάσμα των P ενώ αυτό των G+S εμφανίστηκε σε σημαντικά χαμηλότερα ποσοστά. / In this MSc thesis, fatty acids composition of lipids (total, phospholipids, glyco- and sphingolipids, neutral) synthesized by Nannochloropsis oculata, Chlorella sp. and mixed cultures of the above mentioned microalgae as well as by the rotifer Brachionus plicatilis, cultivated in industrial scale bioreactors under several growth conditions, were studied. Microalgae were cultivated in industrial scale bioreactors 300 L, under full sunlight and artificial light during night-time and at a constant temperature, 28±3 οC at sprint and 22 ±3 οC at autumn and winter. In Chlorella sp. the most abundant fatty acids were α- linolenic and palmitic acids, whereas in Nannochloropsis oculata palmitic, eicosapentaenoic (EPA) and palmitoleic acids were predominant. Neutral lipids (NL) fraction was found in higher proportions. Among polar lipids Phospholipids (P) fraction was higher at the initial growth steps while Glyco- plus Sphingolipids (G+S) fraction predominates as growth proceeded. In the mixed culture, where Nannochloropsis oculata was the dominant strain palmitic, palmitoleic and EPA were the major fatty acids. NL was the major fraction followed by G+S and P. Rotifers were cultivated in 2,500 L tanks. Rotifers fed with microalgae’s culture (Nannochloropsis oculata, Chlorella sp.), baker’s yeast and artificial diets with high level of EPA and docosahexaenoic acids (DHA). Rotifers fatty acid composition depended on the fatty acid composition of the feed. Rotifers fed microalgae cultures were rich in EPA, while when artificial diets were used, lipids rich in EPA and DHA were accumulated. Among lipid fractions, the proportion of NL was the highest, followed by P and G+S.

A busca por segurança energética tem feito com que a maioria dos países empenhe-se na busca por fontes alternativas de energia, procurando mitigar problemas econômicos, sociais e ambientais. Espera-se que a biomassa, disponível de forma geograficamente dispersa, venha a tornar-se um dos principais recursos renováveis na produção de alimentos, materiais, produtos químicos, combustíveis e energia. Nesse cenário, o desenvolvimento de biorrefinarias representa a chave para uma produção integrada, combinando rotas de conversões químicas, bioquímicas e termoquímicas, no processamento da biomassa, visando à utilização otimizada dos recursos disponíveis. As biorrefinarias com estruturas descentralizadas são uma alternativa à centralização de produções em grandes plantas industriais e à monocultura, pois utilizam biomassas disponíveis regionalmente, integram sistemas de produção, potencializando os recursos locais, reduzem custos com logística e impactos ambientais, além de melhorarem a distribuição da renda. O presente trabalho apresenta uma revisão da disponibilidade de biomassa no Brasil e no mundo, com especial interesse no aproveitamento de microalgas e resíduos orgânicos, dos principais produtos de interesse e conceitos de biorrefinarias existentes. O objetivo é discutir qual é o conceito que melhor se adapta às necessidades do cenário brasileiro, bem como propor um modelo com estrutura descentralizada em duas ou três etapas, visando à otimização de um processamento sustentável de biomassa para obtenção de vários produtos comerciáveis e energia, além de delinear diretrizes para investimentos na área. É apresentada uma metodologia para a tomada de decisões na concepção e análise de viabilidade do projeto conceitual de uma biorrefinaria, considerando-se também as restrições de ordem ecológica, econômica e tecnológica. Como estudo de caso, é proposta uma biorrefinaria a partir de microalgas. A escolha por microalgas como matéria-prima é baseada nas vantagens que sua utilização apresenta frente à de outras biomassas, dentre elas cita-se a capacidade de produção rápida e durante todo o ano, a captura do CO2 necessário ao seu crescimento, a necessidade de menos água do que plantas terrestres, são cultiváveis em água salobra e terras não aráveis, apresentam elevado teor de óleo, seus nutrientes podem ser obtidos a partir de águas residuais, sua composição bioquímica pode ser modulada por diferentes condições de crescimento e são capazes de produção fotobiológica de bio-hidrogênio. Das alternativas de rotas possíveis para seu processamento, são apresentadas duas que apontam como sendo as mais promissoras: o uso da microalga como substrato de algum outro microorganismo, visando à obtenção de compostos com maior valor agregado, tais como biopolímeros, e o uso do processo de pirólise rápida para obtenção de bio-óleo, que deve ser processado posteriormente, visando à especificação em biocombustível. As vantagens da otimização do cultivo, colheita, rotas viáveis de processamento e a análise do potencial econômico desse modelo, indicam uma excelente oportunidade para obtenção de um espectro de produtos de alto valor agregado e energia e um grande potencial de aplicação. / The search for energy security has been doing with that most of the countries strive to seek alternative sources of energy that allows mitigating economic, social and environmental problems. It is expected that biomass, available on a geographically dispersed way, will become one of the major renewable resources for food, materials, chemicals, fuels and energy production. Additionally, the development of biorefineries represents the key to an integrated production, combining chemical, biochemical and thermochemical conversion routes for biomass processing, aiming the optimized use of available resources. Biorefineries with decentralized structure are an alternative to the centralization of production in large industrial plants and to the monoculture because it uses biomass regionally available, integrates production systems potentiating local resources, reduces logistics costs and environmental impacts, as well as improves income distribution. This work presents a review of: i) biomass availability in Brazil and in the world, focus on the use of microalgae and organic wastes; ii) the main products of interest; and; iii) concepts of existing biorefineries. The objective is to discuss which one is the concept that better fits the needs of the Brazilian scenario and propose a new model with decentralized structure in two or three stages, seeking to an optimized and sustainable biomass processing to obtain various marketable products and energy, and delineate guidelines for investment in the area. It is presented a methodology for making the conceptions decisions and feasibility analysis of the conceptual design of a generic refinery, considering also the ecological, economic and technological constraints. As a case study, it is proposed a biorefinery from microalgae. The choice of microalgae as a raw material is based on the advantages that their use offers over the others biomasses. Among the advantages it can be cited the ability for rapid production and during all the year, the capture of CO2 for its growth, the need for less water than land plants, they can be cultivated in brackish water and non-arable land, have a high oil content, nutrients can be obtained from waste water, their biochemical composition can be modulated by different growth conditions and are capable of producing photobiology bio-hydrogen. Of the possible alternative routes for microalgae processing, are presented two as the most promising: the use of microalgae as a substrate for some other microorganism in order to obtain compounds with high added value, biopolymers as an example, and use the process of fast pyrolysis for obtaining bio-oil, which will be processed later in order to specify biofuels. The advantages of the cultivation optimization, harvesting, processing viable routes and analyzing the economic potential of this model indicate an excellent opportunity to obtain a spectrum of value-added products and energy and its great potential for application.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Lisa R. Wilken / Microalgae are a potential feedstock for renewable and sustainable bioproducts and energy but there are significant scientific and engineering challenges to address before widespread acceptance of this platform. In particular, biorefining microalgae serves to maximize biomass valorization and minimize waste to improve process economics. The overall goal of this dissertation was the development of a biological-based microalgae biorefinery to enhance the economic feasibility of Chlamydomonas reinhardtii as a source of multiple products including native proteins and lipids. Specific objectives included accumulating biomass enriched in target biomolecules and determining processing strategies that eliminated the need to dry biomass, employed mild conditions to maintain extractability and quality, and minimized application of petroleum-derived and toxic solvents during extraction. The microalgae biorefinery developed included biomolecule accumulation, biomass harvesting, and targeted enzymatic degradation of the cell wall and organelles for release of native proteins and lipids. Biomass was cultivated, and kinetic studies indicated that 48 h nitrogen deprivation was adequate for protein and lipid accumulation. Four lytic enzymes were screened for their ability to permeate the C. reinhardtii cell wall and the C. reinhardtii-produced enzyme, autolysin, led to >85% cell disruption. TEM imaging confirmed cell disruption and retention of lipid droplets in organelle remnants indicating that protein, lipids, and starch could be distinctly partitioned and recovered. A design of experiments optimization study determined that incubation of disrupted biomass at pH 12 for 4 h at 45°C resulted in up to 65% of total protein released from disrupted biomass followed by 40-50% protein recovery with isoelectric precipitation. The cell disruption and protein extraction steps were subsequently integrated to minimize unit operations, processing time, and energy inputs. Secondary application of trypsin led to release of ~73% of total lipids (enriched in triacylglycerols) from the disrupted biomass. Characterization by thin layer chromatography and GC-FID of released lipids revealed similar profiles of enzymatically released lipids as compared to those released by conventional extraction procedures. Finally, the composition of released lipids indicated favorable combustion behavior, high oxidation stability, and suitability as biodiesel. The developed biological-based biorefinery is a promising step towards adoption of microalgae as a source of bioproducts to provide energy and food to meet the needs of a growing population. The second focus of the work was mitigation strategies for isolation of critical impurities (or potential co-products) while processing microalgae and plant hosts. Specific emphasis was placed on evaluating the impact of proteases, polysaccharides, phenolic compounds and pigments, phytic acid, and host cell proteins on the processing of microalgae and other plant hosts for extraction, recovery, and purification of therapeutic proteins. This review served as evaluation of the broader implications of application of the biorefinery to transgenic microalgae and other plants.

The threats of global warming attributed to fossil fuel combustion, combined with increasing energy demands and a growing population, have generated interests in diversifying the world energy mix. Biofuels from microalgae offer a sustainable renewable option and do not suffer the sustainability issues associated with early forms of bioenergy. However, research efforts of nearly 5 decades have not resulted in any significant gains and have motivated further investigation into novel techniques. The dilute nature of microalgae suspensions often requires dewatering and drying, which adds to energy intensity and costs associated with recovery processes. Curiously, the conventional recovery techniques do not consider the characteristic tendency of microalgae cells for surface attachment. This behaviour of cells, coupled with the discovery of a non-porous adsorbent material, NyexTM particles, has brought to the fore an exciting prospect. This has motivated the underpinning question behind this research; does the non-porous characteristic of the NyexTM particles presents an opportunity to recover microalgae cells from suspension using an adsorption technique?Using Chlamydomonas reinhardtii as a model microalgae strain, preliminary batch studies revealed a rapid recovery of the cells onto the NyexTM particles; nearly 90% recovery was attained within one minute, which depends on suspension concentration. At a correlation coefficient, R2 = 0.99, the Freundlich isotherm was found to give a better description of batch systems than the Langmuir isotherm, which suggests that cell coverage onto the NyexTM particles may not be a simple monolayer adsorption. Although a low adsorptive capacity of 0.55 mg/g was measured, the equilibrium parameter (1⁄) of about 0.6 was well within the range for favourable adsorption (i.e. 0 - 1). Further studies undertaken suggest that the recovery of cells could be driven by a hydrophobic-hydrophobic interaction, electrostatic forces of attraction and the flocculating behaviour of the NyexTM particles. Fixed bed studies showed that the lack of pores led to an early breakthrough. However, findings demonstrated that unlike most column studies, the bed capacity was a more valuable parameter to assess the column performance. Unexpectedly, depressed breakthrough curves, where bed exhaustion never attained Ct/C0 = 1.0, were observed. Nonetheless, the modified dose response (MDR) model was found to predict the experimental bed capacity to a greater degree of accuracy than other models. Furthermore, this research exploited the logistic features of the Bohart-Adams and the Clark models to adapt them to the experimental data. The adapted models significantly improved the accuracy of predictions with R2 values > 0.99 for the depressed breakthrough curves. The conductive nature of NyexTM particles was explored to electrochemically regenerate the adsorbent and reuse it to recover more cells. A current density of 32 mA.cm-2 was sufficient to inactivate the cells, regenerate the adsorbent and attain a maximum percentage recovery. Interestingly, scanning electron micrograph showed that the membranes of the adsorbed cells were ruptured, during NyexTM regeneration, potentially leading to lipid release. The maximum lipids extracted into a hexane solvent was estimated as 30 μg/mL at a current density of 64 mA.cm-2.Overall, the potential to recover microalgae cells onto a non-porous adsorbent has been demonstrated. The prospect of rupturing membranes of adsorbed cells offers the opportunity to use this technique to recover microalgae cells for potential biofuel applications. The results obtained from this research can serve as the impetus to further exploit this novel procedure. Future work should consider high lipid producing varieties of microalgae strains, develop a robust protocol to account for all forms of lipids released and undertake an energy and cost analysis to develop the technology further.

The study introduces and demonstrates the viability of the continuous flow reactor (CFR) system for the production of bio-crude oil (BCO) from wet microalgae. Preliminary experiments conducted in the CFR system in hot compressed water (HCW) were successful in converting wet microalgae into liquid BCO. However, the synthesis and aggregation of high boiling point (HBP) components of BCO and the accumulation of char in the tubular piping of CFR system were identified as the limiting factor to the viability of the system. The aggregation of HBP components and the accumulation of char result to system blockage which prevents the continuous flow of the liquefaction product mixture in the CFR system. Inhibiting the reactions leading to the formation of HBP components and char will improve the performance of the CFR system. Therefore, the study seeks to incorporate co-solvents in the liquefaction reaction media in an attempt to inhibit or minimize the prevalence of HBP components of BCO. As such, different co-solvents were screened for their influence on improving the quality of BCO with respect to its boiling point profile (BPP), initial and final boiling point, as well as the amount of char recovered from each experiment. Only one co-solvent was chosen for further exploration in the CFR system. Batch liquefaction reactor’s (BLR) made up of stainless steel were used to carry out the co-solvent screening experiments. These experiments were carried out at a constant temperature (280 °C), pressure (75 bar), and co-solvent concentration (10 wt.%), at varying residence times. Solvent extraction with dichloromethane (DCM) was performed on the liquefaction product mixture to separate the products, viz. BCO, char and water soluble components. The extracted BCO was analysed through simulated distillation (SimDis) to obtain the BPP. The BPP properties of the BCO samples, from different liquefaction media, and the amount of char recovered were highly influenced by the addition of a co-solvent. The final boiling point (FBP) of tetralin, heptane, and n-octanol BCO products were significantly reduced to below 500 °C for all tested residence times except at 20 minutes. The residence time also proved to be influential in the processing of wet microalgae. n-Octanol was selected as the optimal performing co-solvent and was used for the continuous liquefaction of wet microalgae in the CFR system. The CFR system was modified by adding a co-solvent feed line into the continuous system since n-octanol was insoluble in water. The n-octanol pump was set at different flow rates, 0.2, 0.3, and 0.4 g/min, which resulted in a concentration of about 10 wt.% in the reactor feed. The concentration of n-octanol had a significant influence on the BPP of BCO components. The FBP’s were reduced with an increase in n-octanol concentration. The initial boiling point (IBP) of n-octanol BCO was increased to just above 100 °C which was required for the stability of the BCO product. The components of BCO were identified by GCMS. n-Octanol also proved to affect the composition of the BCO with respect to its components. HCW BCO components were significantly different from those identified from n-octanol BCO. A second co-solvent (tetralin) was used to prove whether the difference on the components of BCO was affected by n-octanol. The results proved that indeed the addition of different solvents in liquefaction reaction media favours the formation of different components. The amount of char formed was also reduced when using a co-solvent. A decrease in the oxygen/nitrogen compounds was also observed in the presence of a co-solvent, thus improving BCO properties.

A busca por segurança energética tem feito com que a maioria dos países empenhe-se na busca por fontes alternativas de energia, procurando mitigar problemas econômicos, sociais e ambientais. Espera-se que a biomassa, disponível de forma geograficamente dispersa, venha a tornar-se um dos principais recursos renováveis na produção de alimentos, materiais, produtos químicos, combustíveis e energia. Nesse cenário, o desenvolvimento de biorrefinarias representa a chave para uma produção integrada, combinando rotas de conversões químicas, bioquímicas e termoquímicas, no processamento da biomassa, visando à utilização otimizada dos recursos disponíveis. As biorrefinarias com estruturas descentralizadas são uma alternativa à centralização de produções em grandes plantas industriais e à monocultura, pois utilizam biomassas disponíveis regionalmente, integram sistemas de produção, potencializando os recursos locais, reduzem custos com logística e impactos ambientais, além de melhorarem a distribuição da renda. O presente trabalho apresenta uma revisão da disponibilidade de biomassa no Brasil e no mundo, com especial interesse no aproveitamento de microalgas e resíduos orgânicos, dos principais produtos de interesse e conceitos de biorrefinarias existentes. O objetivo é discutir qual é o conceito que melhor se adapta às necessidades do cenário brasileiro, bem como propor um modelo com estrutura descentralizada em duas ou três etapas, visando à otimização de um processamento sustentável de biomassa para obtenção de vários produtos comerciáveis e energia, além de delinear diretrizes para investimentos na área. É apresentada uma metodologia para a tomada de decisões na concepção e análise de viabilidade do projeto conceitual de uma biorrefinaria, considerando-se também as restrições de ordem ecológica, econômica e tecnológica. Como estudo de caso, é proposta uma biorrefinaria a partir de microalgas. A escolha por microalgas como matéria-prima é baseada nas vantagens que sua utilização apresenta frente à de outras biomassas, dentre elas cita-se a capacidade de produção rápida e durante todo o ano, a captura do CO2 necessário ao seu crescimento, a necessidade de menos água do que plantas terrestres, são cultiváveis em água salobra e terras não aráveis, apresentam elevado teor de óleo, seus nutrientes podem ser obtidos a partir de águas residuais, sua composição bioquímica pode ser modulada por diferentes condições de crescimento e são capazes de produção fotobiológica de bio-hidrogênio. Das alternativas de rotas possíveis para seu processamento, são apresentadas duas que apontam como sendo as mais promissoras: o uso da microalga como substrato de algum outro microorganismo, visando à obtenção de compostos com maior valor agregado, tais como biopolímeros, e o uso do processo de pirólise rápida para obtenção de bio-óleo, que deve ser processado posteriormente, visando à especificação em biocombustível. As vantagens da otimização do cultivo, colheita, rotas viáveis de processamento e a análise do potencial econômico desse modelo, indicam uma excelente oportunidade para obtenção de um espectro de produtos de alto valor agregado e energia e um grande potencial de aplicação. / The search for energy security has been doing with that most of the countries strive to seek alternative sources of energy that allows mitigating economic, social and environmental problems. It is expected that biomass, available on a geographically dispersed way, will become one of the major renewable resources for food, materials, chemicals, fuels and energy production. Additionally, the development of biorefineries represents the key to an integrated production, combining chemical, biochemical and thermochemical conversion routes for biomass processing, aiming the optimized use of available resources. Biorefineries with decentralized structure are an alternative to the centralization of production in large industrial plants and to the monoculture because it uses biomass regionally available, integrates production systems potentiating local resources, reduces logistics costs and environmental impacts, as well as improves income distribution. This work presents a review of: i) biomass availability in Brazil and in the world, focus on the use of microalgae and organic wastes; ii) the main products of interest; and; iii) concepts of existing biorefineries. The objective is to discuss which one is the concept that better fits the needs of the Brazilian scenario and propose a new model with decentralized structure in two or three stages, seeking to an optimized and sustainable biomass processing to obtain various marketable products and energy, and delineate guidelines for investment in the area. It is presented a methodology for making the conceptions decisions and feasibility analysis of the conceptual design of a generic refinery, considering also the ecological, economic and technological constraints. As a case study, it is proposed a biorefinery from microalgae. The choice of microalgae as a raw material is based on the advantages that their use offers over the others biomasses. Among the advantages it can be cited the ability for rapid production and during all the year, the capture of CO2 for its growth, the need for less water than land plants, they can be cultivated in brackish water and non-arable land, have a high oil content, nutrients can be obtained from waste water, their biochemical composition can be modulated by different growth conditions and are capable of producing photobiology bio-hydrogen. Of the possible alternative routes for microalgae processing, are presented two as the most promising: the use of microalgae as a substrate for some other microorganism in order to obtain compounds with high added value, biopolymers as an example, and use the process of fast pyrolysis for obtaining bio-oil, which will be processed later in order to specify biofuels. The advantages of the cultivation optimization, harvesting, processing viable routes and analyzing the economic potential of this model indicate an excellent opportunity to obtain a spectrum of value-added products and energy and its great potential for application.

abstract: The ability of microalgae to be mass cultivated and harvested for production of pharmaceuticals, nutraceuticals, and biofuels has made microalgae a focal point of scientific investigation. However, negative impacts on production are essentially inevitable due to the open design of many microalgae mass culture systems. This challenge generates a need for the consistent monitoring of microalgae cultures for health and the presence of contaminants, predators, and competitors. The techniques for monitoring microalgae cultures are generally time-intensive, labor-intensive, and expensive. The scope of this work was to evaluate the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a viable alternative for the characterization of microalgae cultures. The studies presented here evaluated whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels, 2) characterize simple mixtures of microalgae, 3) detect changes in a single microalgae culture over time, and 4) characterize growth phases of microalgae cultures. This research required the development of a MALDI-TOF MS microalgae analysis protocol for organism characterization. The results yielded in this research showed that MALDI-TOF MS was just as accurate, if not more so, than molecular techniques for the identification of microalgae at the species and strain levels during its logarithmic growth phase. Additionally, results suggest that MALDI-TOF MS is sensitive enough to characterize simple mixtures and detect changes in cultures over time. The data presented here suggests the next logical step is the development of protocols for the near-real time health monitoring of microalgae cultures and detection of contaminants using MALDI-TOF MS. / Dissertation/Thesis / Masters Thesis Biology 2016

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / HÃ, presentemente, grande esforÃo de pesquisa no sentido de se substituir, parcial ou totalmente, a farinha de peixe das dietas artificiais por fontes protÃicas vegetais, de menor custo e maior previsibilidade de produÃÃo. Os resultados experimentais obtidos em alguns trabalhos evidenciam o bom potencial da farinha de Spirulina como aditivo impulsor do crescimento em dietas para camarÃes marinhos. Apesar de sua qualidade nutricional, nenhum trabalho foi conduzido atà agora sobre o uso da farinha de Spirulina como aditivo em dietas completas para L. vannamei. O presente trabalho objetivou o estudo do desempenho zootÃcnico e da preferÃncia alimentar de juvenis de Litopenaeus vannamei criados em laboratÃrio sob condiÃÃes controladas alimentados com diferentes dietas suplementadas ou nÃo com farinha de Spirulina. Juvenis de L. vannamei (3,89  0,25 g) foram estocados intensivamente em 28 tanques circulares de polietileno de 500 L, na densidade de 44 camarÃes/tanque (64 juvenis/m2) por 71 dias. As dietas foram suplementadas com 0,5% de um atrativo comercial para raÃÃes de camarÃo (MAC e BAC) ou com 0,5% de farinha de Spirulina (MSP e BSP). Em MAC e MSP e em BAC e BSP houve reduÃÃes de 25% e 50% do nÃvel de inclusÃo de farinha de peixe, respectivamente. No segundo experimento, as dietas foram oferecidas aos animais em duas bandejas claramente identificadas, em quantidades iguais, colocadas no mesmo momento, em lados opostos dentro de cada tanque e foram recolhidas no mesmo instante. A raÃÃo remanescente em cada bandeja de alimentaÃÃo foi coletada e pesada para calcular o peso seco posteriormente. O ganho em peso semanal dos camarÃes alimentados com a raÃÃo MSP (0,89  0,03 g) nÃo foi significativamente diferente daqueles que se alimentaram com a dieta MAC (0,89  0,01 g) e P (0,95  0,08 g). O fator de conversÃo alimentar e a taxa de eficiÃncia protÃica dos camarÃes alimentados com MSP nÃo foram estatisticamente diferentes daqueles observados em CN1 e P. O peso corporal final e a sobrevivÃncia dos camarÃes alimentados com MSP nÃo foram significativamente diferentes daqueles observados em CN1 e P. O estudo da atratividade mostrou que MSP teve preferÃncia significativamente maior pelos camarÃes do que a dieta MAC. Pode ser concluÃdo que a farinha de Spirulina adicionada a 0,5% em uma dieta completa para juvenis de L. vannamei atuou como um atrativo alimentar, e foi capaz de promover os mesmos efeitos de um aditivo comercial. Trabalhos futuros sÃo requeridos para avaliar outros nÃveis de inclusÃo, tÃcnicas de produÃÃo baratas, compostos responsÃveis pela atratividade da farinha de Spirulina etc / At present, there is a great effort to partially or completely replace fishmeal in artificial diets for less costly and more predictable vegetable protein ingredients. Experimental results of some studies indicate the potential of the Spirulina meal as a growth impeller in diets for marine shrimp. Despite its nutritional quality, no work has been conducted so far about the use of Spirulina meal as an additive in complete diets for Litopenaeus vannamei. The present work aimed at investigating the growth performance and feeding preference of L. vannamei raised under controlled laboratory conditions fed diets supplemented or deprived with Spirulina meal. Juveniles of L. vannamei (3.89  0.25 g) were stocked under intensive conditions at 44 shrimp/tank (64 juveniles/mÂ) in 28 polyethylene tanks of 500 L and raised for 71 days. Diets were supplemented with 0.5% of a commercial feed attractant for marine shrimp (MAC and BAC) or with 0.5% of Spirulina meal (MSP and BSP). In MAC and MSP and BAC and BSP, inclusion of fishmeal was reduced at 25% and 50%, respectively. In a second study, diets were offered to animals in a two feeding trays clearly identified, in equal amounts, delivered at the same time in the opposite sides of each tank and simultaneously recovered. Feed remaining in each feeding tray was collected and weighted to calculate the dry weight. Weekly shrimp weight gain fed diet MSP (0.89  0.03 g) was not significantly different from those fed diet MAC (0.89  0.01 g). Feed conversion rate and protein efficiency rate for shrimp fed diet MSP were not significantly different from those seen for CN1 and P. Final body weight and survival of shrimp fed diet MSP were not significantly different from those observed for CN1 and P. The attractability study demonstrated that MSP showed a significantly higher preference compared to MAC diet. It can be concluded that Spirulina meal added at 0.5% in a complete diet for juveniles of L. vannamei acted as a feeding attractant capable of promoting the same effects of a commercial additive. Further work is required to evaluate others inclusion levels, cheap production techniques, compounds responsible for attractabilityâs Spirulina meal etc