Screening of Pavlova sp. and scale-up to outdoor photobioreactors

Joana Filipa da Silva Araújo outubro de 2019 Screening of Pavlova sp. and scale-up to outdoor photobioreactors Trabalho efetuado sob a orientação do Professor Doutor António Augusto Martins de Oliveira Soares Vicente da Doutora Joana Gabriela Laranjeira da Silva e do Doutor Hugo Galvão Caiano Pereira Dissertação de Mestrado Mestrado Integrado em Engenharia Biológica Universidade do Minho Escola de Engenharia ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii AGRADECIMENTOS Bem, agora que tudo terminou chega o momento dos agradecimentos a todos aqueles que, sem eles, não seria possível completar esta etapa tão importante! Em primeiro lugar, gostaria de agradecer ao meu orientador da Universidade do Minho, Professor Doutor António Vicente, e à minha orientadora da AlgaFarm, Doutora Joana Laranjeira. Ao professor Vicente, por ter aceite embarcar comigo neste projeto de “Pavlovas”, por se disponibilizar sempre que necessário e por me ter dado asas para voar. À Dra. Joana, por nos ter recebido tão bem na empresa, pela força e motivação que nos deu e por apoiar o nosso trabalho incondicionalmente. Um agradecimento especial ao Doutor Hugo Pereira que esteve sempre à distância de um telefonema ou uma mensagem no Whatsapp. Agradeço por me teres acompanhado neste trabalho, por me teres guiado e por todo o conhecimento que me transmitiste, tanto teórico como no terreno. Sem ti, este trabalho teria sido mais difícil. Quem diria que ia ter saudades de te enviar os updates por Whatsapp! Também, de uma forma especial, agradeço ao Filipe Maciel. Foi ele que, juntamente com o Hugo, idealizou o meu projeto. Obrigada por me teres motivado a fazer sempre mais e melhor e pelo apoio inicial quando sabia pouco ou nada do que estava a fazer. O maior dos agradecimentos vai para a minha equipa do UI&D: Inês, Pedro, Francisco, Susana, Rosângela e Sara. Que me fez sentir todos os dias em família e no melhor estágio que poderia ter. Convosco, senti-me realmente em casa e sem vocês esta tese não teria sido possível. Porque uma pessoa com 1.5 m não faz GW de 12 m!! Um agradecimento especial ao Pedro por ter perdido uns quantos jogos de futebol e umas idas ao Vale Furado a ajudar-me; à Inês pelas 10x que me explicou como funcionam as tubagens dos PBR e por estar sempre a correr comigo de um lado para o outro; ao Francisco que muitas vezes deixou o trabalho dele para ajudar a sua pequenita; à Rosângela por se disponibilizar vezes sem conta para me ajudar; à Susana que, sem perceber nada, esteve sempre do meu lado com duas mãos prontas a ajudar-me; e à Sara, pela amizade que sempre demonstrou e por ter sempre uma palavra amiga. Agradeço também, do fundo do coração, à Joana Teles que, de uma maneira ou de outra, sempre me ajudou a apagar os meus fogos tanto indoor como outdoor. Obrigada por me teres tornado uma pessoa mais capaz. À Margarida que, sem nos conhecer, sempre se disponibilizou a iv nos ajudar em tudo o que fosse possível ou mesmo impossível. Obrigada pela amizade. Também fazendo parte dos estagiários, obrigada à Mafalda pelas palavras reconfortantes que sempre nos deu e ao João Gonçalves que, com a sua experiência, muito me ajudou no trabalho outdoor. Também à malta da 42 – Bernardo, Quelhas e João Pereira – e à malta da 70 – Nuno, Joana e Fábio – pela ajuda e apoio que me deram na minha estadia tanto na AlgaFarm como na 70. Agradeço à Nádia a ajuda e o reconforto que me prestou no momento desesperante em que achei que tinha um fungo nas minhas culturas! Ah, e à Edna e à Inês Pedro por me disponibilizarem as balanças sempre que necessário, sei bem que precisaram de muita paciência para mim. Agradeço também às meninas do laboratório da fermentação – Ana Barros, Maria e Adriana. Por fim, aos operacionais de turno e a todas as outras pessoas da empresa que, de alguma forma, me ajudaram e contribuíram para este projeto! Ao professor João Varela e à sua equipa de investigação do CCMAR, estaremos sempre gratos pela disponibilidade com que nos receberam no seu laboratório – que já era apertado para eles, quanto mais para mais 7 de nós! Um especial agradecimento à Tamára que tão bem tomou conta dos seus “pequenos”. Um obrigada não chega pelo apoio que recebemos! Às minhas queridas amizades da universidade: Maria Inês, Ana Filipa, Nicole Oliveira, Sara Vilas, Cátia Carvalho, Joana Moreira, Fátima Ferreira e Patrícia Valadares. Sem vocês nada disto seria igual. Obrigada por estarem sempre lá quando foi preciso e mesmo quando não foi. O vosso incentivo e a vossa amizade foi essencial e, no final, festejamos! Por fim, mas não menos importante, agradeço aos meus pais e familiares por toda a compreensão que recebi deles. Bem sei que nem sempre foi fácil para eles não terem a sua menina em casa fins de semana sem conta, mas aguentaram sempre sem contestar e agradeçolhes por essa compressão. Ao meu namorado, Ricardo, pelo apoio incondicional que me deu antes, durante e após o meu estágio na AlgaFarm. Obrigada por estares do meu lado e por me ajudares, mesmo não percebendo nada de microalgas. v STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. vi ABSTRACT SCREENING OF PAVLOVA SP. AND SCALE-UP TO OUTDOOR PHOTOBIOREACTORS Microalgae are a promising source of several bioactive compounds such as proteins, lipids, carbohydrates, carotenoids and vitamins, which are valuable for commercial use and can be explored and used for diverse applications. Furthermore, microalgae are known producers of polyunsaturated (PUFA) omega-3 fatty acids, including eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids and, consequently, cultivation of microalgae as a supplement to marine animals is increasing. The marine microalgae Pavlova sp. is also widely used in aquaculture to feed crustacean, bivalves, and fish. In the present study, the growth of four Pavlova species ( P. lutheri, P. gyrans, P. pinguis and P. granifera ) was evaluated at lab-scale in order to select the most suitable species for outdoor and large-scale production. A preliminary trial comparing all strains showed that P. gyrans and P. pinguis had a higher final volumetric productivity, 0.087±0.005 g L-1 d-1 and 0.077±0.002 g L-1 d1, respectively. In accordance, higher specific growth rates and maximum volumetric productivity were achieved by these species. The biochemical composition of all strains understudy was analyzed and the amount of PUFA (DHA and EPA) demonstrated to be the same for all Pavlova species. In addition, 30% of inoculum revealed to be the optimum inoculation rate to obtain higher global productivity when compared to 5, 10 and 20%. Furthermore, a screening outdoor assay was carried in 55 L flat panels using P. gyrans and P. pinguis . Both demonstrated a similar growth and global volumetric productivity, 0.058±0.007 and 0.079±0.019 g L-1 d-1, respectively. The FAME profile showed a higher PUFA content of 48% of total fatty acids (TFA) in P. gyrans , followed by 37% of TFA in P. pinguis . Outdoor cultures proved to have an increased lipids content and a higher level of EPA (30-40% of TFA) and DHA (9% of TFA), when compared with lab-scale production. Different types of outdoor photobioreactors (PBR) were used to investigate and compare their impact on P. pinguis scale-up: flat panel, tubular PBR and raceway pond. The results showed that flat panel was the system with the highest global productivity of 0.077 g L-1 d-1 and, similarly, the highest specific growth rate of 0.316 d-1. Keywords: DHA, EPA, Growth optimization, Industrial-scale production, Pavlova vii RESUMO RASTREIO DA PAVLOVA SP. E AUMENTO DE ESCALA PARA FOTOBIORREATORES EXTERIORES As microalgas são uma fonte promissora de vários compostos bioativos como proteínas, lípidos, carbohidratos, carotenoides e vitaminas, valiosos para uso comercial e podem ser explorados e usados em muitos aplicações. Para além disso, as microalgas são as únicas produtoras dos ácidos gordos polinsaturados ómega-3, nomeadamente os ácidos eicosapentanóico (EPA) e docosahexanóico (DHA), e, consequentemente, o cultivo de microalgas como suplemento para os animais marinhos tem aumentado. A microalga marinha Pavlova sp. é amplamente usado em aquicultura para alimentar crustáceos, bivalves e peixes graças à sua acumulação de elevadas quantidades de ácidos gordos polinsaturados, especialmente EPA e DHA. O crescimento das quatro espécies Pavlova ( P. lutheri, P. gyrans, P. pinguis e P. granifera ) foi avaliado em escala laboratorial de forma a selecionar a melhor espécie para escala industrial. P. gyrans e a P. pinguis demonstraram valores maiores de produtividade global final, 0.087±0.005 g L-1 d-1 e 0.077±0.002 g L-1 d-1, respetivamente. Similarmente, obtiveram uma maior taxa especifica de crescimento e produtividade máxima. A composição bioquímica da microalga foi analisada e a produção de ácidos gordos polinsaturados (EPA e DHA) demonstraram ser iguais em todas as espécies Pavlova . Decorreu outro ensaio com a P. gyrans para testar o efeito da percentagem de inóculo no crescimento. Resultados mostraram que 30% é a concentração que potencia uma maior produtividade global, comparado com 5, 10 e 20 % de inóculo. Para além disso, um ensaio exterior decorreu em painéis de 55 L com P. gyrans e P. pinguis, mas ambas demonstraram um crescimento e produtividade global similar, 0.058±0.007 e 0.079±0.019 g L-1 d-1, respetivamente. O perfil de ácidos gordos mostrou um maior conteúdo de 48% do total de ácidos gordos na P. gyrans , seguido de 37% na P. pinguis . Culturas no exterior mostraram um aumento no conteúdo lipídico e um maior nível de EPA (30-40% do total de ácidos gordos) e DHA (9% do total de ácidos gordos), quando comparadas com o laboratório. Diferentes tipo de fotobiorreatores foram usados no aumento de escala da P. pinguis : painéis planos, fotobiorreator tubular e tanques abertos, para investigar e comparar o impacto dos sistemas no crescimento da Pavlova sp.. Resultados demonstraram uma maior produtividade nos painéis, 0.077 g L-1 d-1 e uma maior taxa especifica de crescimento 0.316 d-1. Palavras-chave: DHA, EPA, Otimização do crescimento, Pavlova , Produção industrial viii INDEX Direitos de autor e condições de utilização do trabalho por terceiros ........................................................ ii Agradecimentos ..................................................................................................................................... iii Statement of integrity .............................................................................................................................. v Abstract ................................................................................................................................................. vi Resumo ................................................................................................................................................ vii Index .................................................................................................................................................... viii List of figures .......................................................................................................................................... x List of tables ..........................................................................................................................................xii List of general nomenclature ................................................................................................................. xiii 1. Contextualization ............................................................................................................................ 1 1.1. Historical aspects of SECIL/ALGAFARM .................................................................................. 2 1.2. Thesis outline ......................................................................................................................... 4 2. Introduction .................................................................................................................................... 5 2.1. Microalgae ............................................................................................................................. 5 2.2. Microalgae cultivation ............................................................................................................. 7 2.2.1. Light intensity ................................................................................................................ 7 2.2.2. Temperature .................................................................................................................. 8 2.2.3. pH ................................................................................................................................. 8 2.2.4. Nutrients supply: macro and micronutrients ................................................................... 9 2.2.5. Mixture and aeration ....................................................................................................10 2.3. Large scale biomass production ...........................................................................................10 2.4. Microalgae biotechnological applications ...............................................................................15 2.5. Pavlova species: general overview and applications ...............................................................18 2.6. Research aims .....................................................................................................................20 3. Materials and methods .................................................................................................................21 3.1. Microalgae strain and inoculum preparation ..........................................................................21 ix 3.2. Lab-scale assays of Pavlova sp. ............................................................................................21 3.2.1. Pavlova sp. selection ....................................................................................................22 3.2.2. Effect of inoculum concentration in P. gyrans growth ....................................................22 3.3. Outdoor scale-up of Pavlova sp. ............................................................................................22 3.3.1. Scale-up of Pavlova sp. to outdoor flat panel reactors ....................................................23 3.3.2. Scale-up of Pavlova sp. to the outdoor tubular PBR .......................................................23 3.3.3. Scale-up of Pavlova sp. to the outdoor raceway .............................................................24 3.4. Growth assessment ..............................................................................................................24 3.5. Nitrate determination............................................................................................................25 3.6. Analytical determinations ......................................................................................................26 3.6.1. Total lipid determination ...............................................................................................26 3.6.2. Fatty acid methyl esters determination .........................................................................27 3.6.3. Elemental analysis (CHN) .............................................................................................28 3.6.4. Ash content .................................................................................................................28 3.6.5. Carbohydrates content .................................................................................................29 3.7. Statistical analysis ................................................................................................................29 4. Results and discussion .................................................................................................................30 4.1. Screening of Pavlova sp. and biochemical composition .........................................................30 4.2. Optimization of inoculum quantity of Pavlova gyrans in culture growth ...................................37 4.3. Outdoor screening of Pavlova sp. and biochemical composition .............................................40 4.4. Growth of Pavlova sp. on large scale PBR .............................................................................46 5. Conclusions and future perspectives .............................................................................................52 References............................................................................................................................................53 Annex A – Calibration curves .................................................................................................................64 Annex B – Screening of Pavlova sp. in laboratory scale ..........................................................................65 Annex C – Screening of Pavlova sp. at panel reactors ............................................................................66 Annex D – Cells morphology in the scale up of P. pinguis in tubular PBR ...............................................67 3 implementation of a microalgae production unit, known by AlgaFarm appears in this context. AlgaFarm has a mission to reduce CO2 emissions. The industrial-scale production unit resulted from the expansion of the initial pilot-scale unit and it is, currently, in commercial operation and directed to Chlorella vulgaris production for the food industry (Rabaçal et al., 2017). AlgaFarm unit is divided into four main fields: resources, production, processing, and control. Each sector includes various systems that, together, possess the largest microalgae closed production unit around the world, with a production volume of 1300 m3 (Rabaçal et al., 2017). Allmicroalgae is the supplier of Allma Chlorella , with continuous production in Algafarm, located in Pataias. Allmicroalgae’s products are focused on microalgae production in order to use them as supplements, food, animal feed, and cosmetic applications. In Algafarm unit, Chlorella vulgaris , Nannochloropsis oceanica , Phaeodactylum sp. and Tetraselmis sp. are grown in closed tubular systems and/or fermenters, minimizing contamination and ensuring maximum purity (Allmicroalgae, 2019). The Algafarm company, represented in Figure 1, is certificated by ISO 220000 in order to ensure product safety and quality. Figure 1 - Aerial view of Algafarm and SECIL facilities. 4 1.2. Thesis outline The present thesis dissertation is organized in 5 chapters, as follows: In Chapter 1, there is a contextualization of the thesis and a brief description of the history, vision, and work developed by SECIL and Algafarm. Chapter 2, the introduction, reviews the concept of microalgae and their importance for the industry, as well as the cultivation of Pavlova sp. and their possible applications. The main differences in open and closed systems and the challenges found in this area are also mentioned. Besides, a brief description of the most important parameters for microalgae cultivation is referred, together with a review of existing literature. To conclude this chapter, the main objectives of the present work are exposed. Chapter 3, material and methods, describes the methodology used in the laboratory followed by the scale-up experiments, the materials, and instruments required for the techniques described and the experimental procedure. The statistical tools used for data analysis are also presented, along with the tests used to obtains growth curves. Chapter 4, results and discussion, presents the results obtained from the standard protocols referred in the previous chapter, supported by a discussion of those results based on published literature. The final conclusions of the work are presented in Chapter 5, as well as the future perspectives. 5 2. INTRODUCTION 2.1. Microalgae Microalgae, defined as a polyphyletic group, are unicellular and eukaryote organisms which are capable of converting solar into chemical energy, through CO2 fixation. Known as thallophytes (lower plants), they possess similarities with superior plants since both have photosynthesis mechanisms (Priyadarshani & Rath, 2012; Wojciechowski et al., 2013). However, microalgae have been proved to be more efficient concerning to solar energy conversion due to their cellular simplicity, which consequently leads to tenfold increase in efficiency when compared with superior plants (Priyadarshani & Rath, 2012; Raja et al., 2008; Sathasivam et al., 2019). Microalgal metabolism involves numerous bioactive compounds of interest with application in nutrition, cosmetics and in the pharmaceutical industry (Das et al., 2011; Khan et al., 2018; Muller-Feuga et al., 2007; Richmond, 2004). Additionally to its cellular simplicity, microalgae present a wide geographic location resulting in metabolic plasticity that allows species to adapt to a range of environmental conditions (Khan et al., 2018; Muller-Feuga et al., 2007; Wojciechowski et al., 2013). They can be virtually found in all terrestrial conditions, even under extreme environments, with some species showing great resistance to dryness, high salinity, low light, among others (Evangelista et al., 2008; Wojciechowski et al., 2013). However, microalgae are mainly found in the aquatic environment playing an important role in the CO2 fixation, organic matter consumption and in the release of oxygen to the atmosphere (Wojciechowski et al., 2013). Microalgae classification is based on their photosynthetic pigments, macromolecules composition, and constituents of the cell wall. Their similarities to superior plants are due to the presence of CH, proteins, and photosynthetic pigments. Microalgae have chlorophyll a as a primary pigment and others such as carotenoids, phycocyanin, and phycoerythrin, whose distribution is limited. Furthermore, microalgae are also classified regarding cytologic and morphologic aspects such as flagellum structure, cellular division, nucleus formation, among others (Wojciechowski et al., 2013). Some microalgae species are represented in Figure 2. 6 Chinese were the first population resorting to microalgae ( Nostoc sp.) as food, in time of famine, about 2000 years ago. However, the biotechnology and commercialization related to microalgae just started to develop in the middle of the last century ( Chlorella sp. and Spirulina sp.) as healthy foods in countries such as Japan, Taiwan, and Mexico (Sathasivam et al., 2019). Nowadays, there are numerous microalgae applications, not restricted to the food supply. They are used to increase the nutritional value of food owing to their chemical composition; have a huge contribution to aquaculture and can be incorporated in cosmetics (Priyadarshani & Rath, 2012). These microorganisms are a source of polyunsaturated fatty acids (PUFA) that are added to infant formulas and nutritional supplements and pigments which are sold as natural dyes (Priyadarshani & Rath, 2012). Three relevant aspects give microalgae a huge potential to be converted into new products and investigated in different fields from commercial and biotechnological point of view: i) microalgae are a group of microorganisms with a wide range of physiological and biochemical characteristics; ii) they absorb 13C, 15N and 2H isotopes, used to facilitate the structure determination of proteins, CH and nucleic acids, and incorporate them into their biomass and into the various compounds that produce; iii) microalgae are part of a huge group of microorganisms under-explored and investigated and, consequently, could form a potential source of undeveloped products (Bux, 2013; Priyadarshani & Rath, 2012). Figure 2 - Microscopic image: (A) Chlorella vulgaris , (B) Scenedesmus obliquus , (C) Pavlova lutheri, and (D) Nannochloropsis oceanica . A B C D 7 For those reasons, cited before, it is important and urgent to proceed with microalgae investigation. 2.2. Microalgae cultivation Microalgae growing on an industrial scale aims to contribute, in a sustainable way, to industrial development through biomass production and products with high commercial value and a good price-benefit ratio. Nevertheless, information about some microalgae strains with high industrial interest is still needed in order to improve these processes. These microorganisms can be cultivated using different methods and conditions attending to their general and specific needs. Microalgae can be grown in the presence of light in photoautotrophic culture, or in the presence of both light and fixed organic carbon in mixotrophic culture, or, in certain cases, in the presence of organic carbon in darkness, in heterotrophic culture (Das et al., 2011). In the particular case of photoautotrophic cultivation, they require a light energy source so that they can convert absorbed water and CO2 into biomass, through photosynthesis. To grow, they need nitrogen and phosphorus as main nutrients and others such as sodium, magnesium, calcium, potassium (Cai et al., 2013; Walker, 1954). Those nutrients, as well as micronutrients, can be provided from residual waters that are considered a good source to the same and will contribute to microalgae feeding (Cai et al., 2013). Nitrogen and phosphorus are the nutrients with a higher importance in the culture medium, the N:P rate is one of the most important factors when designing culture media (Cai et al., 2013). 2.2.1. Light intensity Light intensity is one of the most important confining factors in autotrophic microalgae growing (Khan et al., 2018; Muller-Feuga et al., 2007). The photoperiod and light intensity directly affect microalgae photosynthesis and impact its biochemical composition and biomass growth efficiency (Boussiba et al., 1987; Brown et al., 1996; Liang et al., 2006; Roessler, 1990). The ideal light intensity should be determined experimentally in order to maximize CO2 absorption. The optimal level of light intensity, for the most microalgae, is usually found between 200-400 µmol m2 s-1 (Schuurmans et al., 2015). 8 A balance between periods of light and absence of light must be maintained for microalgae photosynthesis. Periods of light results in ATP (adenosine triphosphate) and NADPH (nicotinamide adenine dinucleotide phosphate) synthesis while in absence of light are produced carbon skeletons (Khan et al., 2018; Sun et al., 2018). 2.2.2. Temperature Temperature is one of the most important abiotic factors in microalgae growth and it influences directly its metabolism. It is important to highlight that each species possess its own optimal temperature that improves its cellular growth (Khan et al., 2018). However, increasing or decreasing this optimal temperature leads to a reverse performance, delaying or even stopping exponential growth. Above optimal value of the temperature of each species, the rate of viability loss increases. This increase can be explained by the fact that algal death is most likely due to degradation of key enzymes and membrane denaturation (van Boekel, 2002). Moreover, a lower temperature will affect photosynthesis through decreasing in carbon assimilation, whereas if it is too high it will reduce photosynthesis, affecting negatively cell energy balance and inactivating photosynthetic proteins. It also contributes to cell size reduction and affects their respiration as well (Khan et al., 2018). Generally, the optimal temperature of most microalgae species is between 20 and 30 °C (Singh & Singh, 2015). 2.2.3. pH Another parameter affecting microalgae growth is pH, whereas its optimal value varies with the microalgae strain being cultured (Khan et al., 2018). The majority of microalgae has an optimal pH between 6 and 9 and are sensitive to pH variations, in contrast to C. vulgaris , which is able to withstand a wide range of pH values (Khan et al., 2018; Lam & Lee, 2012). Unusual variations in pH result in chemical modifications of some substances such as CO2, phosphate, iron, among others, and it will directly interfere in microalgae metabolism, affecting membrane permeability, ionic transport, and the enzymatic reactions (Khan et al., 2018)(Wojcechowski et al., 2013). pH and CO2 concentration are directly related. When dissolved in the water, CO2 forms carbonic acid (H2CO3), helping to acidify the culture media. If there is not a supply of CO2, this compound is consumed by microalgae from the culture medium and the pH increases 9 (Wojciechowski et al., 2013). Alkaline media may result in toxic ammoniacal production through dissolved salts, which may inhibit microalgae production (Chisti, 2013). The use of nitrate (NO3-) supply as a source of nitrogen helps controlling the pH however, microalgae appear to have a preference for ammonia (Scherholz & Curtis, 2013). A CO2 injection system is often designed and installed in the reactor in order to control the pH during the various stages of microalgae production (Chisti, 2013). 2.2.4. Nutrients supply: macro and micronutrients Different microalgae species vary in their nutrient needs, however, nitrogen, phosphorus, and carbon are the macronutrients required for the growth of any microalgae and a deficiency in these inorganic nutrients affects directly microalgae growth, resulting in lower biomass productivity (Cai et al., 2013; Khan et al., 2018; Walker, 1954). Nitrogen is an important element in the microalgae metabolism, being required in high quantities, immediately after carbon. It integrates the molecular composition of proteins, enzymes, and nucleic acids (Wojciechowski et al., 2013). Usually, nitrogen obtained through NO3and urea constitutes a good and economical source when compared with other inorganic sources (Khan et al., 2018). Phosphorus also plays important roles in microalgae growth and energy (ATP/ADP) production, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) structure and it is part of the plasmatic membrane (phospholipids) (Wojciechowski et al., 2013). The main phosphorus forms are dihydrogen phosphate (H2PO4-) and hydrogen phosphate (HPO4-), and they can be incorporated into organic components through phosphorylation with the release of energy (Cai et al., 2013). Microalgae are able to store an excess amount of phosphorus, in the form of polyphosphate (Larsdotter, 2006). Carbon can be added to microalgae cultures in organic form, glycerol, and acetate, or inorganic form, CO2. However, at the industrial scale, it is recommended to use environmental CO2 as a carbon source, which is more economical and allows CO2 fixation (Khan et al., 2018). Although atmospheric CO2 concentration is not enough and extra injection of CO2 is often necessary (Chisti, 2016). Microalgae are composed of 50% carbon, which makes it an important element to microalgae growth and the main when considering autotrophic metabolism (Chisti, 2016). 10 Regarding micronutrients, such as manganese, potassium, sodium, cobalt, iron, magnesium, boron, zinc, among others, they are required in vestigial quantities (Walker, 1954; Wojciechowski et al., 2013). At high concentrations, micronutrients are toxic to the majority of microalgae species (Khan et al., 2018; Monteiro et al., 2011). Usually, they are added to the culture media together with a chelating agent, which avoids them to achieve toxic concentrations (Larsdotter, 2006). Micronutrients play roles related to enzymatic activity and microalgae structure. However, their addition is not always mandatory and shows to be more relevant in synthetic media (Khan et al., 2018; Wojciechowski et al., 2013). 2.2.5. Mixture and aeration The standardization between nutrients, air and CO2 in microalgae culture is achieved through mixture and aeration of the culture. This procedure allows nutrients dissolution and uniform distribution of light inside microalgae culture and, at the same time, avoid biomass settlement and its consequent aggregation. In case of absence of mixture and air supply, biomass productivity is significantly minor and, therefore, microalgae culture must be continuously mixed (Khan et al., 2018). 2.3. Large scale biomass production Microalgae cultivation is increasingly being researched and explored and has shown remarkable signs of progress. There are several configurations that contribute to the success of the mass cultivation of microalgae, according to the species (Khan et al., 2018; Thukral & Sharma, 2015). The nutritional value of microalgae and their application should be an important parameter to consider when proceeding to large scale biomass production. The selection of the accurate culture system should take into account some parameters such as light efficiency, pH and temperature control, productivity, hydrodynamic stress, the need for an axenic culture, harvesting, and feasibility of scale-up (Guedes & Malcata, 2012; Tamiya, 1956). Nowadays, microalgae cultivation at large scale can be carried out in open and closed cultivation systems (Carvalho et al., 2006). The first one allows contact with the atmosphere and the other does not allow this contact (Fonseca et al., 2016). The choice of the most suitable system 11 is a decision dependent on the species to be produced and the final purpose intended, these two parameters play a major role in this decision (Carvalho et al., 2006). There are five main types of open systems: water tanks, raceway ponds, circular ponds, thin layer cascade, and shallow big ponds (Fonseca et al., 2016; Narala et al., 2016). The first step in the direction to microalgae production was achieved through a raceway pond (Figure 3) and, since then, there was extensive research in microalgae cultivation in open systems (Borowitzka, 1999; Carvalho et al., 2006; Johnson et al., 1988). These systems have some economic advantages when compared with closed systems, not requiring high capital and operating costs, are easier to clean and do not need much energy for culture mixture (Narala et al., 2016). However, open systems present also space issues, a higher probability of occurring contaminations, are dependent on favorable climate, have evaporation losses, CO2 diffuses easily into the atmosphere, present an inefficient use of light and lower productivity rates (Fonseca et al., 2016; Narala et al., 2016). Raceway ponds are known by their channels with an oval and closed-form, designed to flow the culture in a loop, with a paddlewheel paddling the microalgae culture in one direction. The paddlewheel should be in continuous operation to prevent sedimentation and to ensure proper mixing of the nutrients (Brennan & Owende, 2010; Show et al., 2017). Therefore, the mixture and Figure 3 - Raceway pond cultivation systems. (A) Schematic design (Chisti, 2007) (B) Photography of a raceway pond in AlgaFarm facilities. A B 12 circulation are needed in order to stabilize and maximize microalgae growth and productivity. The depth of the system is usually between 0.2 and 0.5 m in order to enable the entrance of light (Brennan & Owende, 2010). In this type of reactors, CO2 injection is usually done from the surface air, but submerged aerators may be installed to enhance CO2 absorption (Terry & Raymond, 1985). On the other hand, closed systems proved to be more efficient than the previous and allow better control of the culture parameters. Inside of the closed systems, mainly known as photobioreactors (PBR), they can appear with different configurations: tubular reactors, and flat panel reactors (Carvalho et al., 2006). The difference between these PBR lies in aspects such as manufacturing material, layout, the passage of light and the aeration. Wherefore, the operation of each system is influenced by the species concerned, climate conditions and associated costs (Carvalho et al., 2006; Narala et al., 2016). Closed systems are used to overcome the open systems' disadvantages, allowing better efficiency and achieving higher biomass concentrations, as far as the operating conditions can be easily controlled and contaminations can be prevented. Nevertheless, the huge problem associated with this type of reactors is the higher cost of using them (Narala et al., 2016). Configurations of tubular reactors are mostly vertical and horizontal tubular reactors. Vertical PBR can be categorized into airlift and bubble column reactors (Figure 4) based on their liquid flow patterns inside the PBR (Mirón et al., 2000). Bubble columns and airlift PBR can be useful for culturing phototrophic organisms. Light availability in bubble columns and airlift reactors is influenced by aeration rate, gas holdup, and culture mixing (Mirón et al., 2000). They are normally built-in polyethylene or glass tubes that have sufficient transparency to allow the penetration of the light in the reactor (Carvalho et al., 2006). And as they are manufactured with common and non-expensive materials, are considered financially accessible. In these bioreactors, the air is bubbled from the bottom of the reactor and, resorting to a gas sparger system, the inlet air is converted into tiny bubbles. This way, reactors with this configuration can provide good mixture, enough O2 removal, and CO2 supply (Carvalho et al., 2006; Thukral & Sharma, 2015). The difference between a bubble column and an airlift reactor is the draft tube that allows culture circulation (Figure 4B) (Behin, 2012). 19 different growth conditions in P. lutheri . However, although the growth of P. lutheri was inhibited under heterotrophic conditions, it was enhanced in mixotrophy, when compared to microalgae growth under light intensity alone. Organisms belonging to Haptophyta phylum are very rich in FA and a large amount of these FA may correspond to the valuable omega-3 PUFA, namely eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids (Eikrem et al., 2016). It is well known the importance of PUFA in human health and, although the major source of these FA is incorporated in a diet composed by fish, it is known that fish, by themselves, synthesize EPA and DHA only to a limited extent (Dyerberg, 1986; Grima et al., 1993; Pereira et al., 2012). Therefore, these compounds must be supplemented in their feed, including PUFA-rich microalgae (Borowitzka, 1997; Guedes & Malcata, 2012; Grima et al., 1993). Therefore, Pavlova sp. can become one of the most important feed sources in aquaculture and is currently used in mariculture hatcheries to feed crustacean, bivalves, and fishes (Meireles et al., 2003). Microalgae are one of the most promising sources of EPA and DHA and, consequently, cultivation of microalgae, especially Haptophytes, as a supplement of fish and other marine animals is increasing day by day (Eikrem et al., 2016). The first species of microalgae used Figure 8 - The most frequently observed shapes of P. gyrans (Butcher, 1952). 20 in aquaculture were selected because they presented a natural growth in the marine environment and, therefore, exhibited to be the easiest microalgae to grow for this purpose. Subsequently, other species were investigated and showed to be most nutritionally efficient than the previously used ones (Richmond, 2004). 2.6. Research aims The main objective of this Master’s thesis was to study the growth of four species of Pavlova at lab-scale and determine the one which demonstrates better performance under the same lightregime, salinity, and temperature conditions, and at a given inoculum concentration. Furthermore, to discover if the biochemical composition of microalgae is easily modified through different system configurations, light intensity and photoperiod, an assay using P. gyrans and P. pinguis cultures was carried out in outdoor scale. Finally, to study and evaluate the influence of the cultivation system in Pavlova sp. cultures growth, an assay was carried out on a large scale: flat panel, tubular PBR, and raceway pond. 21 3. MATERIALS AND METHODS The growth assessment experiments were performed at Algafarm facilities, located in Pataias, Portugal, between February 06th and August 23th 2019. The biochemical profile of produced biomass was performed at MarBiotech, a group belonging to the Centre of Marine Sciences (University of Algarve) between the July 24th and August 08th 2019. 3.1. Microalgae strain and inoculum preparation Pavlova granifera, Pavlova gyrans and Pavlova pinguis were obtained from Roscoff Culture Collection, while Pavlova lutheri was obtained from AlgaFarm Culture Collection. All autotrophic experiments were carried out using non-axenic cultures as inoculum. Pavlova sp. cultures have grown in t-flasks expose to natural light and at room temperature. These cultures were later scaled-up to 1.5 L and 5 L reactors which were grown at room temperature, under continuous light irradiance of ~100 µmol m-2 s-1, with aeration and injection of CO2. Guillard’s F/2 (10 mmol L-1 of NO3-), supplemented with iron (Fe2+) and Conway’s vitamin solutions, was used as culture medium, with a salinity adjusted to 30 g L-1 (Tompkins et al., 1995). The pH of all cultures was daily adjusted to 8 (Carvalho & Malcata, 2005; Carvalho & Malcata, 2000; Shah et al., 2014). All Pavlova sp. cultures were operated in batch mode and were used to inoculate all the autotrophic reactors used in the present work. These conditions were used in all the assays performed, except when mentioned. 3.2. Lab-scale assays of Pavlova sp. The autotrophic assays were conducted in 1 L bubble column reactors set with constant aeration of 600 L min-1, containing 1% CO2 used to manually maintain pH at 8. Cultures were kept at 24 °C under continuous LED radiation exposure of 700 µmol m-2 s-1. Samples were taken every two days together with the addition of water to compensate for evaporation losses. The water used in the inoculation and in the evaporation losses was local water provided by SECIL group and was 22 chemically treated with sodium hypochlorite and neutralized with thiosulfate. All experiments were conducted in triplicates. 3.2.1. Pavlova sp. selection The first laboratory assay consisted of testing the growth of the different species of Pavlova . Accordingly, P. lutheri , P. granifera , P. gyrans and P. pinguis were grown in 1 L bubble column reactors. The assay was intended to start at, nearly, 0.20 g L-1 of biomass. The culture conditions used in this assay were the same previously mentioned. In the first 12 days, cultures were maintained at continuous radiation of ~100 µmol m-2 s-1 and then the light exposure was increased to ~700 µmol m-2 s-1. All experiments were done in triplicate. 3.2.2. Effect of inoculum concentration in P. gyrans growth The assay was performed in 1 L bubble column reactors. P. gyrans was inoculated at 0.15, 0.23, 0.35 and 0.49 g L-1, corresponding to 5, 10, 20 and 30% inoculum. Samples were taken every two days during the trial, where the growth performance (optical density at 750 nm), pH, salinity and NO3concentration were monitored. 3.3. Outdoor scale-up of Pavlova sp. Culture systems used in this study are represented in Figure 9, with increasing culture volumes: A) 1.5 and 5 L balloon reactors; B) 600 L flat panels; C) 2500 L tubular PBR; D) 3500 L raceway. Microalgae cultures were grown in 1.5 and 5 L laboratory balloon reactors. The reactors were continuously aerated with 0.2 µm filtered air and CO2. pH was maintained around 8. The reactors were under continuous light irradiance of ~100 µmol m-2 s-1 and at room temperature. 23 3.3.1. Scale-up of Pavlova sp. to outdoor flat panel reactors Four 5 L reactors were used as pre-inoculum for the outdoor 60 L flat panel and successive scale-ups were made until the achievement of a 600 L flat panel reactor. Optical density, salinity, and NO3consumption were daily monitored. The sterilization of the air inlet was carried out with 0.2 µm polypropylene filters (WhatmanTM, United Kingdom). pH was registered using a pH meter (Combo pH/Conductivity/TDS Tester, Hanna instruments, Portugal) three times a day and maintained around 8 by the manual control of CO2 pulses, injected by the same system used in the air inlet. The temperature was maintained below 25 °C by a system of spraying water over the flat panel surface. 3.3.2. Scale-up of Pavlova sp. to the outdoor tubular PBR Two 600 L flat panels were used to inoculate a 2500 L horizontal tubular PBR with P. gyrans , which started at 0.4 g L-1. Another 2500 L tubular PBR was inoculated with P. pinguis using a 600 L flat panel at 0.4 g L-1. pH was controlled by an automated system and CO2 injected in order to keep the pH at the optimal value of 8. Besides, culture mixing was achieved using a centrifugal pump at, maximum, 18 Hz. One sample was taken per day during the trial, where the growth performance, salinity, and NO3consumption were monitored. Figure 9 - Pavlova sp. growing in different PBR at AlgaFarm facilities: (A) 1.5 and 5 L laboratory balloon reactors, (B) 600 L flat panels, (C) 2500 L tubular PBR and (D) 3500 L raceway pond. A B C D 24 3.3.3. Scale-up of Pavlova sp. to the outdoor raceway A preliminary growth assay was performed in a 28.8 m2 raceway pond. In this trial, a 600 L flat panel reactor of P. pinguis was used to inoculate a 3500 L raceway at 0.25 g/L. The water line was kept at 10.5 cm and the paddlewheel operating at 6 Hz. One sample was taken per day during the trial. Every two days, water was added to cover evaporation losses. 3.4. Growth assessment The culture growth was determined by cell density and DW . As an indirect measurement of the growth of the culture, optical density ( OD ) was determined in a Zuzi spectrophotometer (model Zuzi 4251/50, Spain) at 750 nm. DW was determined by filtering a known volume of culture and then washed twice with ammonium formate 35 g L-1 in 0.7 µL glass microfiber filters (VWR, Portugal)(Guihéneuf & Stengel, 2017). After that, the filters were dried on a moisture analyzer (MA 50.R, RADWAG, Poland). Then, the final value of DW was obtained in a precision balance. This procedure was to establish the calibration curve of OD at 750 nm versus DW , present in Equation 1. OD750nm=2.7067 DW (g L-1) - 0.1464 R2 = 0.9377 (1) The calibration curve generated is present in Annex A. The specific growth rate of culture (μ) was estimated by the quotient between the growth rate ( r X) and the concentrations of biomass ( X ) at that moment. The development of the integral results in the simplified equation that is the fraction between the Neo-logarithmic of biomass concentration ( X Y) on time later ( t Y) divided by biomass concentration ( X X) on time previous ( t X) and the time interval ( t Y less t X). The specific growth rate was measured using Equation 2. μ(day-1)=rX X=1 X dX dt =ln(XyXx ⁄) 𝒕y− 𝒕x (2) 25 Global volumetric productivity ( P ) during the logarithmic phase was calculated through Equation 3 and was determined by the division of DW in g L-1 differences ( X 2 and X 1) and the time interval ( t 1 and t 2 represent the total time in d-1 of the trial). P(g L-1day-1)=X𝟐X𝟏 ⁄ 𝒕𝟐− 𝒕𝟏 (3) Productivity per unit of occupied-land area per unit of time ( P a) was determined by multiplying the volumetric biomass productivity by the volume of the PBR in L ( V ) and divided by the area of the PBR, in m2 ( A ). Pa was calculated through Equation 4. Pa(g m-2day-1)=P x V 𝑨 (4) The photosynthetic efficiency was determined as the ratio between the higher heating value ( HHV ) and the solar irradiation that reached the reactor. The solar radiation was measured using a meteorological station (RM Young) and an Apogee Logan UT SP-110 pyranometer. HHV was calculated according to a previous correlation reported by Callejón-Ferre et al. (2011), shown in Equation 5. HHV (kJ g-1) = - 3.393 + 0.507 C - 0.341 H + 0.067 N (5) C is the percentage of carbon, H the percentage of hydrogen and N the percentage of nitrogen. 3.5. Nitrate determination Samples collected were centrifuged for 20 min at 3500 rpm (MiniStar silverline, VWR, Portugal). NO3were determined according to Armstrong (1963). Briefly, the collected supernatant was diluted (ratio of 1:80) and an hydrochloric acid solution (1 mol L-1) was added. Samples were 26 measured at 220 and 275 nm. NO3concentration was calculated from a previously established calibration curve. 3.6. Analytical determinations After cultivation, the microalgal biomass obtained during the growth phase was harvested by centrifugation at 3500 rpm for 15 min. The obtained biomass pellet was frozen at -18 °C and was then freeze-dried in a Telstar LyoAlfa 15 until a constant weight was achieved. 3.6.1. Total lipid determination Total lipid content (Figure 10) was determined following the Blingh and Dyer (1959) method, with a few modifications by Pereira et al. (2012). Lyophilized biomass was weighed ( w i) in glass tubes and 0.8 mL of distilled water, 2 mL of methanol and 1 mL of chloroform was added, followed by homogenization with an IKA Ultra-Turrax disperser (IKA-Werke GmbH, Staufen, Germany) on ice during 60 s. Thereafter, 1 mL of chloroform and 1 mL of distilled water were added one at a time to the mixture and homogenized for two intervals of 30 s. Afterwards, the mixture was vortexed and centrifuged at 2500 g for 10 min and, with a Pasteur pipette, the chloroform phase (lower layer) was extracted to a new glass tube. A well-known volume of chloroform (0.5-1 mL) was transferred to previously weighed tubes ( w 1), which were placed in a dry bath at 60 °C to evaporate the chloroform. After that, the tubes were put in the desiccator and then weighed in a precision balance. The resulting dried residue was weighed ( w 2) in a precision balance and the percentage of lipids in each sample was calculated according to Equation 6. Figure 10 - Total lipid quantification procedure: (A) Lipids extraction on UltraTurrax, (B) Lipids extraction with phase separation after centrigugation (chloroform is present in the lower layer) and (C) Chloroform extraction. A B C 27 % Total lipids= (w2-w1) * V total of chloroform V evaporated chloroform (wi) (6) 3.6.2. Fatty acid methyl esters determination FA (Figure 11) were converted into the correspondent fatty acid methyl esters (FAME) following the Lepage and Roy method (1984) with a few modifications by Pereira et al. (2012). Lyophilized biomass was weighed (5-10 mg) to derivatization vessels and 1.5 mL of a methanol/acetyl chloride solution (20:1, v/v) was added. The mixture was homogenized on ice for 90 s with an Ultra-Turrax disperser. Afterwards, 1 mL of hexane was added and the vessels were placed in a water bath at 70 °C, for 60 min, and then in ice, for 15 min. Next, 1 mL of water and 4 mL of hexane were added to the mixture and then centrifuged at 2000 g for 5 min. With a Pasteur pipette, the hexane phase (higher layer) was extracted to another vessel. The previous two steps were repeated (just the hexane fraction). The extracted hexane was dried with the addition of anhydrous sodium sulfate (Na2SO4) and was filtered (0.22 µm) to new tubes. After that, the hexane was evaporated under nitrogen gas flow until dryness. The FAME were resuspended in 500 µL gas chromatography-grade hexane and transferred to vials for GC-MS analysis. Figure 11 - Some steps of fatty acid methyl esters procedure: (A) Samples in ice for 15 min; (B) PUFA extraction with phase separation after centrifugation; (C) Samples with anhydrous sodium sulfate (D) Evaporation of the hexane fraction under nitrogen gas flow. A B C D 28 3.6.3. Elemental analysis (CHN) Elemental analysis of carbon, hydrogen, and nitrogen was achieved using a vario EL III (Vario EL, Elementar Analyser system, GmbH, Hanau, Germany). The liophylized biomass (1 mg) was weighed in specific aluminium caps (Figure 12). Total protein was estimated by multiplying the N content by 4.59 (Lourenço et al., 2004). 3.6.4. Ash content Biomass was weighed and placed in small ceramic cups and burned for 5 h at 550 °C using a furnace. The ceramic cups were stored in a desiccator. Total ash was determined by the weight difference before and after burning the produced biomass (Figure 13). Figure 12 - (A) Precision balance to weigh the aluminum capsules with 1 mg of biomass and (B) Vario EL, Elementar Analyser system, GmbH, Hanau, Germany). Figure 13 - (A) Initial biomass wheiged in ceramic cups and and (B) Ashes obtained after biomass burned at 550 °C. A B A B 35 Table 6 - Fatty acid methyl esters (FAME) composition of Pavlova sp. in 1 L bubble column reactor. All data represent the mean (±sd) of three individual replicates FAME % of total FAME P. granifera P. gyrans P. pinguis P. lutheri Saturated (SFA) C14:0 8.75±0.52 24.83±1.37 30.71±1.04 10.97±1.05 C16:0 30.61±0.40 22.88±2.54 14.76±2.06 33.06±3.26 Sum 39.36±0.67 47.71±1.35 45.47±1.02 44.04±2.25 Monounsaturated (MUFA) C16:1 33.97±1.37 28.39±0.93 34.44±2.64 26.95±4.7 C18:1 0.16±0.22 - - 1.46±0.70 Sum 34.13±1.53 28.38±0.93 34.44±2.64 28.41±4.73 Polyunsaturated (PUFA) C18:3n-3 0 0 0 0.09±0.13 C18:2n-6c 0 0 0 0.64±0.61 C20:5n-3 (EPA) 17.74±2.11 17.85±0.94 14.72±1.18 21.70±5.39 C22:6n-3 (DHA) 8.76±0.86 6.05±0.69 5.38±1.10 5.13±1.81 Sum 26.51±1.67 23.9±0.51 20.1±2.21 27.55±6.93 ∑ (n-3) 26.51±1.67 23.9±0.51 20.1±2.21 26.92±7.17 ∑ (n-6) 0 0 0 0.64±0.61 ∑ (n-3) / ∑ (n-6) - - - 9.68±7.36 PUFA/SFA 0.67±0.05 0.5±0.02 0.44±0.05 0.64±0.19 Previous analyses of FAME showed some differences between the constituents detected. FA such as octadecatrienoic (C18:3n-3), octadecadienoic (C18:2n-6c), octadecenoic (C18:1), octadecanoic (C18:0) and eicosatetraenoic (C20:4n-6), reported in the literature as minor constituents (Guihéneuf et al., 2009; Guihéneuf & Stengel, 2017; Patil et al., 2007; Ponis et al., 2006b) were not possible to quantify in the present study. The biochemical composition of biomass produced by different species of Pavlova showed some differences between SFA, MUFA and PUFA fractions compared to previous studies (Patil et al., 2007; Ponis et al., 2006b; Volkman et al., 1989). Relatively to SFA, P. gyrans and P. pinguis ( p ≥0.05) demonstrated higher C14:0 accumulation, while P. granifera and P. lutheri ( p ≥0.05) showed to have tendency to C16:0 accumulation. Guihéneuf et al. (2009) obtained a C14:0 content 36 of 8-10% of total fatty acids (TFA) in P. lutheri , which is similar to those obtained in the present study for P. granifera and P. lutheri , but a lower content of C16:0 (16% of TFA) compared with this study (14-33% of TFA). Another study carried by Guihéneuf et al. (2011) observed in P. lutheri cultures higher values of C14:0 and C16:0, 22% and 28% of TFA, respectively. Hexadecanoic acid (C16:1) was about 30% of TFA in all tested strains ( p ≥0.05) and it is the MUFA present at the highest percentage in Pavlova sp.. Although the results in this study are higher than the 17-23% of TFA previously reported, C16:1 is always reported as the major MUFA (Patil et al., 2007; Ponis et al., 2006b; Volkman et al., 1989). Differences should be explained by differences in the light intensity, since MUFA levels are increased with the increase of light intensity (Guihéneuf et al., 2009). Several studies investigating the biochemical composition of microalgae with the aim of improving the nutritional value of algal foods (aquaculture) and/or to boost PUFA production (pharmaceutical industry) are exclusively dedicated to Pavlova sp., specially to P. lutheri (Brown et al., 1997; Guihéneuf et al., 2009). Many of these studies focus on the use of algae as feed in mariculture (Ponis et al., 2006a; Tatsuzawa & Takizawa, 1995; Volkman et al., 1989). Pavlova sp. presents both EPA and DHA in its composition (Carvalho & Malcata, 2005). In this study, the production of DHA and EPA obtained was between 15-21% and 6-9% of TFA, respectively. No statistical differences in EPA and DHA content were detected in Pavlova species ( p ≥0.05). Previous studies measured a DHA content of 10-13% of TFA in P. lutheri , while 3% and 4% of TFA were verified in P. gyrans and P. pinguis , respectively (Patil et al., 2007; Ponis et al., 2006b; Volkman et al., 1989; Yang & Hur, 2012). However, other studies quantified DHA around 6.6% of TFA under the maximum light and temperature tested (18 °C and 200 µmol m-2 s-1) (Guihéneuf & Stengel, 2017). Those differences between the current study and the previous ones should be related to combined effects of temperature and light intensity affecting the lipid content and the FA profile of Pavlova sp. (Guihéneuf et al., 2009; Tatsuzawa & Takizawa, 1995). However, it has been shown that the PUFA content, especially EPA, was significantly higher under low light intensity, whereas SFA content and DHA levels were significantly higher under high light intensity (Guihéneuf et al., 2009; Thompson et al., 1990). In contrast, EPA content found in this study was very similar to previous studies, on which the percentage of EPA was about 20% of TFA in P. lutheri and 15% of TFA in P. pinguis, although the light intensity used was lower (Ponis et al., 2006b; Volkman et al., 1989). However, EPA content found in P. gyrans and P. lutheri was higher in this 37 study, 18% and 21% of TFA, respectively, that what obtained by other authors, 8% EPA of TFA, under simultaneous high temperature and light intensity (Guihéneuf & Stengel, 2017; Yang & Hur, 2012). Marine algae are major producers of omega 3-PUFA (EPA and DHA), while algae from fresh water are predominantly producers of SFA and MUFA (Patil et al., 2007). In this study, there was a higher quantity of SFA and MUFA and a lower content of PUFA ( p <0.05), which is not in agreement with previous studies that observed a higher content of PUFA in Pavlova sp. (Dunstan et al., 1993; McCausland et al., 1999; Patil et al., 2007; Ponis et al., 2006b; Volkman et al., 1989). However, similar results to the present work were obtained by Guihéneuf & Stengel (2017), which combination of the highest temperature and continuous light intensity resulted in a higher SFA (40%) and MUFA (35%) content and decreased PUFA levels (22%). Another study carried by Guihéneuf & Stengel (2013) obtained a FA profile composed by 45% SFA, 32% MUFA and 21% of PUFA, in cultures grown under nutrient depletion. According to Roessler (1990), higher levels of MUFA and SFA appear to be synthesized as an early response to growth under conditions when energy input – light intensity – exceeds the cellular capacity for energy utilization such as cell growth and division. Besides, many microalgae species have tendency to produce those FA under stress conditions, such as pH and salinity nonoptimal and/or NO3depletion (Breuer et al., 2012; Guihéneuf et al., 2015; Guihéneuf & Stengel, 2013; Hu et al., 2008b)(Breuer et al., 2012; Guihéneuf & Stengel, 2013; Q. Hu et al., 2008). In this study, P. gyrans and P. pinguis MUFA and SFA amounts could be related to nutrient depletion since NO3were almost fully consumed (Annex B) (Breuer et al., 2012; Guihéneuf & Stengel, 2013). In the particular case of P. granifera and P. lutheri the high amount of those FA should be associated to higher quantities of light received, since P. lutheri has been proven to grow faster under lower light intensities (Guihéneuf & Stengel, 2017; Roessler, 1990) . 4.2. Optimization of inoculum quantity of Pavlova gyrans in culture growth The present assay was performed with only one species of Pavlova . P. gyrans was the species chosen to continue the rest of the trials because of AlgaFarm’s interests. Four different concentrations of P. gyrans were submitted to a batch assay to compare the microalgal growth performance among them. In Figure 15 the growth curves of P. gyrans cultures are presented. In all cultures, microalgae started their exponential growth in day 0, being not possible to see a lag 38 phase. Cultures reached a stationary phase at day 6, with the exception of the culture which started with 20% of P. gyrans , which extended the growth two more days. All cultures showed a decline phase at day 10 of the experimental assay. The different inoculum percentages showed different total biomass productions ( p <0.05). The highest biomass concentration was achieved by the 30% inoculum volume of P. gyrans (1.544±0.007 g L-1). Similar results were obtained in the study of the effects of initial population density on growth of Nannochloropsis sp., in which the microalgae cultured with higher concentration resulted in a higher biomass concentration (Chen et al., 2012). The specific growth rates and the biomass productivities values for each culture of P. gyrans are presented in Table 7. For the range of inoculum size tested, there were no significant differences comparing the specific growth rate of cultures starting 5 and 10% of P. gyrans and 20 and 30% ( p ≥0.05). The specific growth rate was lower when starting with 30 (0.191±0.002 d-1) and 20% of P. gyrans inoculum (0.209±0.003 d-1), while it was higher using 5% (0.273± 0.022 d-1) and 10% of inoculum (0.247±0.013 d-1). Accordingly, Wang et al., (2015) studied the influence of inoculum size in growth culture of Nannochloropsis sp. and found cultures with the lowest initial biomass demonstrated the highest specific growth rate. These results should be because cell concentration 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0246810 DW /(g L-1) t /d Figure 15 - Screening of the growth of P. gyrans in 1 L bubble column reactors using 5% ( ), 10% ( ), 20% ( ) and 30% ( ) of inoculum. Each point represent a mean of three replicates and respective standard deviation (n=3; mean±sd). 39 in microalgae cultures affects the amount of light that reaches the individual cells of culture. Therefore, the higher cell density, the lower the specific growth rate to be expected in these culture systems (Boussiba et al., 1987). Table 7 - Volumetric productivity on biomass during the whole assay, maximum value, and specific growth rate on the exponential phase for each percentage of inoculum in the study. All data represent the mean (±sd) of three individual replicates % inoculum Maximum volumetric productivity (g L-1 d-1) Global volumetric productivity (g L-1 d-1) Specific growth rate (d-1) 5% P. gyrans 0.187± 0,055 0.108±0.020 0.273± 0,022 10% P. gyrans 0.178±0.031 0.130±0.012 0.247±0.013 20% P. gyrans 0.208±0.027 0.134±0.013 0.209±0.003 30% P. gyrans 0.248±0.008 0.175±0.002 0.191±0.002 There were no statistical differences in the value of maximum productivity, according to different inoculum concentrations ( p ≥0.05). Experimental results obtained by Li et al., (2017) in C. vulgaris indicate that high inoculum size was more favorable to increased daily biomass productivity. Results showed by Bohutskyi et al., (2016) also demonstrated a positive effect on volumetric productivity using higher concentrations of inoculum. On the other hand, a study conducted by Dogaris et al., (2015) in Picochlorum oculatum cultures did not observe significant differences in the maximum biomass production and productivity value when inoculum sized varied from 10% to 15% and 20%. According to global volumetric productivity, in the present work, significant differences were found when starting with 30% inoculum or the other tested concentrations ( p <0.05). Based on these data, it will be of greater interest to start the cultures with 30% of inoculum, leading to a higher biomass in less time (0.175±0.002 g L-1 d-1). On the other hand, there were no differences in the other inoculum sizes ( p ≥0.05), which suggest that the use of a culture with a less percent of inoculum, such as 5 %, could be a feasible option to reduce the costs associated with larger inoculum size preparation, being particularly important when considering industrial scales (Dogaris et al., 2015). 40 4.3. Outdoor screening of Pavlova sp. and biochemical composition After the growth of Pavlova sp. at the lab-scale, a scale-up to 55 L flat panel reactors with initial biomass of 0.4 g L-1 was carried out. The species used to compare their growth in outdoor PBR were the ones that demonstrated higher growth in lab-scale: P. gyrans and P. pinguis . Figure 16 resumes Pavlova sp. in outdoor reactors. Cultures had grown exponential for 11 days, having a 1 day lag phase. The growth ended after 12 days of assay and it was not possible to see a stationary phase. P. gyrans and P. pinguis had consumed the available NO3in culture, remaining 3.7 and 0.1 mmol L-1 after 12 days, respectively (Annex C). Triplicates were performed during the first half of June, with a maximum daily temperature of 23 °C and light exposure of 1592 µmol m-2 s-1. P. gyrans and P. pinguis have shown a similar total biomass during the outdoor experiment ( p ≥0.05) and the maximum biomass obtained during this assay was 1.33 g L-1 by P. pinguis and 1.09 g L-1 by P. gyrans . This result is lower than the values reported in the literature. A study in 4 L flat panel operating in batch and then in semi-continuous regime carried out by Ponis et al., (2008) during 35 days, achieving a final biomass of 3.5 g L-1 in P. lutheri cultures. Another study involving P. lutheri cultivation in 4 L flat panel PBR, under continuous light of 125 µmol m-2 s-1, reached in 7 days a density of 1.9 g L-1 (Ponis, et al., 2006a). Differences related to the size of 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 2 4 6 8 10 12 DW /(g L-1) t /d Figure 16 - Growth of P. gyrans ( ) and P. pinguis ( ) in 55 L flat panel reactors. Each point represent a mean of three replicates and respective standard deviation (n=3; mean±sd). 41 the reactor and the continuous lightening can explain the low biomass obtained during the present study. Furthermore, the culture in outdoor flat panels reached a higher biomass concentration than that obtained in bubble column reactors under 100 µmol m-2 s-1. Flat panels allowed to obtain 1.33 g L-1 P. pinguis and 1.09 g L-1 P. gyrans , whereas lab-scale achieved 0.70 g L-1 and 0.61 g L-1 by P. pinguis and P. gyrans , respectively. Similar results were obtained in previous studies that demonstrated higher growth in cultures growing in flat panel reactors (Ponis et al., 2006a). Volumetric productivity and specific growth rate were calculated and are present in Table 8. Table 8 - Volumetric productivity on biomass during the whole assay, maximum value, specific growth rate on the exponential phase, and photosynthetic efficiency for each Pavlova sp. in outdoor flat panel reactors. All data represent the mean (±sd) of three individual replicates Species Maximum volumetric productivity (g L-1 d-1) Global volumetric productivity (g L-1 d-1) Specific growth rate (d-1) Photosynthetic efficiency (%) P. gyrans 0.083±0.001 0.058±0.007 0.152±0.011 0.453±0.053 P. pinguis 0.120±0.055 0.079±0.019 0.166±0.052 0.606±0.156 In accordance to the reported laboratory assays, P. gyrans and P. pinguis showed no differences in total biomass ( p ≥0.05), such as in global and maximum productivities ( p ≥0.05). Similarly, no significant differences were found in the growth rate of each species ( p ≥0.05). P. gyrans showed a lower global and maximum productivity in outdoor conditions compared to laboratory conditions at 700 µmol m-2 s-1 ( p <0.05), however, it is important to consider the differences in system geometry and culture conditions, such as temperature, pH, light intensity and light-regime, have a strong influence on biomass productivity (Ryu et al., 2012). Comparing the global productivity obtained in flat panel and lab-scale at 100 µmol m-2 s-1, the cultures growing in PBR attained to a higher productivity value. Ponis et al., (2006a) obtained the same results, under continuous light of 150 µmol m-2 s-1, in which cultures grown in flat panel PBR attained a higher global productivity of 0.25 g L-1 with respect to that in carboys cultures, that achieved 0.10 g L-1. Differences in photosynthetic efficiency were not noticed in Pavlova sp. ( p ≥0.05). 42 Once the growth of these microalgae species was statistically similar in both laband outdoor scale, it was important to evaluate if biochemical profile suffered differences. Results are resumed in Table 9. Table 9 - Proximate composition of batch cultures grown in flat panel reactors comparing different species growth. All data represent the mean (±sd) of three individual replicates Species Proteins (%) Lipids (%) Ashes (%) Carbohydrates (%) P. gyrans 40.13±0.58 19.54±1.75 12.22±2.47 28.11±2.05 P. pinguis 42.10±0.88 22.04±4.29 10.13±1.25 25.74±2.80 For the different Pavlova species tested in outdoor flat panels, there were no significant differences in the biochemical content ( p ≥0.05), showing similar contents of proteins, lipids, ashes and CH. However, statistical differences in biochemical content of lipids and CH were detected between growth in lab-scale and growth in outdoor scale for both Pavlova species ( p <0.05), such as the protein content of P. pinguis ( p <0.05). No differences in ashes content of both Pavlova sp. were found comparing the growth of lab-scale with the growth in outdoor flat panels ( p ≥0.05). The results of this study indicated that the biochemical composition was influenced by the culture system and the conditions to which they were subjected, such as temperature, light intensity and light-regime, as suggested by other studies (Carvalho et al., 2009; Richardson et al., 1983; Uslu et al., 2009). Nevertheless, a previous study about the effect of the culture system and culture technique on biochemical characteristics of P. lutheri did not show to induce differences in the microalgae biochemical profile cultivated in flat panel reactor, comparing to lab-scale (Ponis et al., 2006a). However, a continuous illumination was used in both lab-scale and flat panel, whereas in the present work a light/dark cycle was used in outdoor reactors. A study realized by Wahidin et al. (2013) observed significant differences in the lipid composition of Nannochloropsis sp. under different light/dark cycles and the maximum total lipid content was obtained when the cultures were exposure to 18:6 light/dark, being the lowest content at 24:0 light/dark. Therefore, and as described in the literature, light stands out as a key factor in microalgae growth and biochemical profile (Guihéneuf et al., 2015). Richardson et al. (1983) reported in their study that different light intensities and light-regimes exhibited various responses in the growth of microalgae and in their chemical composition. Moreover, it is proven that different light intensities 43 and light/dark cycles affect especially lipid profile of various microalgae species (Harwood, 1998). All of these should explain the differences obtained in lipid content of outdoor and indoor growth conditions. In the present work, the maximum total lipid content in P. gyrans and P. pinguis (1922% of DW ) was obtained while the cultures were exposed to a light regime of 18:6 (L:D) and a daily maximum light intensity of 1592 µmol m-2 s-1, under outdoor conditions. Total lipid content decreased under 24:0 (L:D) regime, presenting a lipid content of 12-14% of DW and under a lower light intensity of 700 µmol m-2 s-1. The results obtained are in accordance with the study realized by Wahidin et al. (2013), which studied the influence of photoperiod and light intensity on lipid content of Nannochloropsis sp. and proved to be an important factor. Furthermore, during outdoor cultivation, there was a decreased in CH content. This should be associated to the existence of a light/dark regime, once it is described that polysaccharides are synthesized during the light period and consumed in the dark phase (Terry et al., 1985). The FA profile of microalgae growing in outdoor flat panels was also investigated and are listed in Table 10. The FA showing the higher difference between lab-scale and flat panel was C16:0 ( p <0.05). Outdoor cultivation led to a drastic decline in the value of this SFA, 23 and 15% of TFA in P. gyrans and P. Pinguis at lab-scale, respectively, and 2 and 5% of TFA at large scale. According to Wahidin et al. (2013), light is essential for triacylglycerol production and the light and regime appropriated vary with the species and for this reason Pavlova sp. growing at a lab-scale, in the absence of a dark period, produce higher quantities of C16:0. Furthermore, there was an increase in DHA levels, which could represent the lower accumulation of C16:0. According to Sahin et al., (2018), a decrease in SFA may be an indicator of a metabolic pathway that results in DHA production. A study of P. lutheri growing in flat panels found C16:0, C16:1, EPA and DHA as the major FA presents. The amount of the major SFA and MUFA present in those cultures made up a total of 37% of TFA while in this study a total of 17% was detected (Ponis, Parisi, et al., 2006). However, the assays were carried under continuous light of 150 µmol m-2 s-1, while in the present study a different photoperiod and light intensity were used. 44 Table 10 - FAME composition of P. gyrans and P. pinguis on 55 L flat panel reactors. All data represent the mean (±sd) of three individual replicates FAME % of total FAME P. gyrans P. pinguis SFA C14:0 34.29±0.91 31.98±0.68 C16:0 1.62±0.51 5.33±1.13 Sum 35.91±1.03 37.31±1.75 MUFA C16:1 16.06±0.98 25.50±1.63 Sum 16.06±0.98 25.50±1.63 PUFA C20:5n-3 (EPA) 39.97±0.08 28.40±0.83 C22:6n-3 (DHA) 8.06±0.51 8.79±0.52 Sum 48.03±0.45 37.19±0.71 ∑ (n-3) 48.03±0.45 37.19±0.71 ∑ (n-6) 0 0 ∑ (n-3) / ∑ (n-6) - - PUFA/SFA 1.34±0.05 1.00±0.06 Furthermore, there was a decrease in SFA amounts of outdoor Pavlova sp. cultures compared to indoor conditions, which is not supported by bibliography. An increase in SFA content was observed in the study about the effect of different light intensities in the biochemical characteristics of P. lutheri , in which SFA increased from 24 to 30% of TFA with increasing light from 20 to 340 µmol m-2 s-1 (Guihéneuf et al., 2009). According to Roessler (1990), increasing light intensity commonly results in an increase in triacylglycerol levels. This differences should be justified with the absence of a light/dark cycle in the lab-scale, that was always under a 24 h photoperiod (Guihéneuf et al., 2009; Guihéneuf & Stengel, 2017). Studies conducted by Guihéneuf et al. (2009 and 2017) observed differences in SFA content using cultures under a continuous light intensity of 200 µmol m-2 s-1 (40%) and cultures under 340 µmol m-2 s-1 with a 14:10 (L:D) photoperiod (30%). The C16:1 was detected at higher amounts in P. pinguis cultures ( p <0.05) while it was not possible to conclude when the assays were performed in bubble column reactors ( p ≥0.05). 51 productivity was also obtained in Tetraselmis sp. cultures cultivated in tubular PBR but according to literature, higher values of productivity are frequently associated with these reactors (Narala et al., 2016; Show et al., 2017). Similar results were obtained in Nannochloropsis sp. cultures, by De Vree et al., (2015), in which the raceway pond productivities were the lowest due to the long culture depth in the system. Horizontal tubular PBR are the most commonly used for commercial production of microalgae because they are associated with higher productivities and their tubes diameter ensures an efficient light penetration (Show et al., 2017). Furthermore, they are capable of operating huge volumes with a lower risk of contamination under controlled parameters (Lee & Pirt, 1981; Pirt et al., 1983). However, these reactors consume more energy and require a large amount of capital investment (Carvalho et al., 2006). In contrast, open systems, such as raceway pond, have lower energy requirement but generally produce a lower amount of biomass for the same area and are more susceptible to contamination (Carvalho et al., 2006; Chisti, 2013; Mazlan & Hashim, 2016). However, no contamination was found in the present study. The divergence between theoretical potential associated with tubular PBR and the biomass productivity obtained in P. pinguis is due to hydrodynamic stress caused on cells by pumping (Gudin & Chaumont, 1991). Hu et al. (2008a) tried scaling-up Pavlova viridis using a 60 L tubular PBR, which demonstrated primarily a similar growth as laboratory assays, under high temperatures and light intensity. However, a second outdoor assay was carried out but with a lower temperature and light intensity, which caused slower growth in P. viridis culture. Therefore, Pavlova sp. cultivation can be cultured either in open (raceway pond) or closed systems (tubular PBR and flat panel). However, the comparison between Pavlova sp. cultivated in different systems demonstrated the best performance of flat panel reactor operating in batch mode in terms of volumetric and productivity per unit of area per unit of time, as well as in specific growth rate. This type of reactors, which operate under controlled conditions, showed to be effective in the production of highly concentrated microalgal biomass and proved to be feasible for P. pinguis culture, maintaining high daily productivity and specific growth rate. 52 5. CONCLUSIONS AND FUTURE PERSPECTIVES Pavlova sp. is rich in EPA and DHA and for that reason it is important to access different species growth and industrial potential. A comparison in lab-scale of four different Pavlova species – P. granifera, P. gyrans, P. pinguis and P. lutheri – was performed in this work. Results demonstrated a higher total biomass production in P. gyrans and P. pinguis , independent of the light intensity used. The biochemical composition of microalgae was analyzed and the production of PUFA confirmed to be similar for all Pavlova species. Besides, an optimization of the inoculum size of P. gyrans was performed and 30% of inoculum was the concentration with higher global productivity but no differences were observed between the other inoculum volumes tested – 5, 10 and 20%. A scale-up to pilot flat panels was performed using P. gyrans and P. pinguis . cultures. Growth in outdoor PBR did not show differences between species, however, it evidenced a higher total biomass production than in lab-scale, during the same period of time, achieving the highest volumetric productivity and specific growth rate. Moreover, the biochemical composition and FAME profile of the produced biomasses exhibited some differences in lipid and CH contents. A higher level of EPA and DHA was obtained in cultures growing in outdoor PBR, showing that flat panels were effective for the production of Pavlova sp.. 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