Optimization of culture media for industrial scale production of microalgae. João Diogo Dias Correia Bernardo. Biological Engineering

Optimization of culture media for industrial scale

production of microalgae

João Diogo Dias Correia Bernardo

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisor(s):

Dr. Edgar Tavares dos Santos

Professor Frederico Castelo Alves Ferreira

Examination Committee

Chairperson: Professor Miguel Nobre Parreira Cacho Teixeira

Supervisor: Dr. Edgar Tavares dos Santos

Member of the Committee: Professor Marília Clemente Velez Mateus

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This work was performed on the Póvoa de Santa Iria production site (ALGATEC) of A4F – AlgaFuel S.A., from February to August 2019, under the supervision of Dr. Edgar Tavares dos Santos. The thesis was co-supervised at Instituto Superior Técnico by Prof. Frederico Castelo Alves Ferreira.

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Declaration

I declare that this document is an original work of my own authorship and that it fulfills all the requirements of the Code of Conduct and Good Practices of the Universidade de Lisboa.

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Acknowledgments

A realização desta dissertação de mestrado contou com o apoio, ajuda e incentivo de muitos, sem os quais eu não teria alcançado e concluído esta etapa da minha vida, aos quais estou muito grato.

Quero desde já agradecer à A4F – AlgaFuel pela oportunidade de poder desenvolver não só este trabalho na ALGATEC na área das biotecnologias que eu tanto gosto, assim como o estágio de verão nas Instalações Piloto do Pólo do Lumiar em 2018.

Ao Doutor Edgar Santos por todo o seu contributo, nas críticas e opiniões durante a realização deste trabalho, assim como pela sua disponibilidade para me ajudar com qualquer dúvida ou problema que tive neste trajeto. Quero também agradecer à Doutora Sara Cabral pelo apoio científico e pela ajuda no tratamento das amostras da experiência.

Agradeço também ao professor Frederico Ferreira do Departamento de Bioengenharia do IST por toda a ajuda que me prestou, especialmente na parte final deste projeto.

A todos os meu colegas de trabalho da A4F – AlgaFuel pelo auxílio prestado na realização das minhas experiências, companheirismo prestado durante o estágio, e pela companhia nas nossas longas viagens de bicicleta e fantásticas matraquilhadas.

Ao Filipe Semião, à Nádia Veiga e ao Sérgio Castanheira, por, no estágio de verão, me terem ensinado todas as bases, procedimentos e conceitos que eu vim depois a aplicar na minha tese.

A toda a minha família que sempre se preocupou comigo, com especial agradecimento à minha Mãe e Irmã, por me terem aturado lá em casa, alimentado, ajudado quando estava mal e todo o apoio incondicional que me prestaram. Sem a minha família não teria conseguido nada disto.

À minha namorada por me ter ajudado a melhorar como pessoa e estudante e por toda a ajuda, apoio e companheirismo que me deu. Nesta nota, obrigado a todos os meus amigos por me tentarem ajudar a descomprimir e rir quando eu precisava.

Ao Kite, por toda a companhia que me deu não só enquanto escrevi esta dissertação, mas também durante todo o meu percurso universitário.

Finalmente, mas não menos importante, à minha avó Guiomar. Obrigado por tudo o que me ensinaste na vida, por me teres educado e por teres sido a melhor avó que alguém pode desejar. Tenho saudades tuas.

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Resumo

Este trabalho experimental teve como objetivo o estudo do comportamento de uma cultura de microalgas de Tetraselmis spp. produzida em fotobiorreatores a uma escala industrial após a aplicação de recirculação de meio de cultura.

Para tal, foram realizados quatro ciclos de recirculação de meio de cultura, com aproveitamento de 70% do meio e renovações na ordem de 13% a 70%, usando com fotobioreatores alimentados com meio fresco como controlo. Estes resultados foram posteriormente comparados quer ao nível de produtividade da microalga em estudo, bem como dos elementos constituintes do meio de cultura. Foi igualmente realizada a análise elementar dos sobrenadantes das culturas de modo a determinar elementos essenciais ao crescimento das microalgas em falta, mas também elementos acumulados que possam inibir o crescimento da microalga. Os fatores ambientais, como a radiação solar e a temperatura atmosférica foram igualmente tidos em consideração para a análise do crescimento da cultura.

Em conclusão, a microalga Tetraselmis spp. pode ser cultivada à escala industrial com recirculação de meio, mantendo uma produtividade aceitável, fazendo-se dois ciclos de recirculação de meio.

Palavras-chaves:

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Abstract

This experimental work focused on the study of the behaviour of a Tetraselmis spp. microalgae culture, produced in photobioreactors at an industrial scale after being subjected to a culture medium recirculation strategy.

To do it so, four culture medium recirculation cycles were performed, using 70% of the culture medium and with renewal rates of 13% to 70%, while photobioreactors fed with fresh medium were used as control. These results were subsequentially compared regarding both microalgae productivity and the culture medium elements. It was also performed an elemental analysis on the culture supernatant in order to determine the missing essential elements and the ones that were accumulating and could potential be hazardous for microalgae growth. The environmental factors, such as solar radiations and atmospheric temperature, were also taken in consideration for the culture growth analysis.

In conclusion, the microalgae Tetraselmis spp. can be cultivated in an industrial-scale with medium recirculation, performing two recirculation cycles, while yielding acceptable results.

Keywords:

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1.1. Microalgae history and use ... 12

1.2. Microalgae in Portugal ... 14

1.3. Overview of Microalgae Biotechnology ... 16

1.3.1. Microalgae general characteristics ... 16

1.3.2. Tetraselmis sp. ... 16

1.4. Microalgae cultivation parameters and strategies ... 18

1.4.1. Cultivation systems ... 18

1.4.2. Culture parameters ... 19

1.4.3. Medium recirculation strategy ... 22

1.5. Biomass harvesting ... 23

1.5.1. Harvesting methods ... 23

1.6. Framework and Goals ... 24

2. Practical experiment design ... 25

2.1. Equipment ... 25

2.2. Reagents and solutions ... 26

2.2.1. Culture Medium Composition ... 26

2.2.2. Other solutions... 27

2.3. Harvesting and recirculation ... 28

2.4. Analytical Methods ... 29

2.4.1. Culture concentration ... 29

2.4.2. Culture productivity ... 29

2.4.3. Mean and Standard deviation ... 29

2.4.4. Percentage of variation ... 30

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3.1. Biomass growth and productivity analysis of Tetraselmis spp. culture ... 31

3.2. Impact of weather conditions ... 33

3.3. Elemental analysis of fresh and recycled culture medium ... 35

3.3.1. 1st _{cycle analysis ... 36}

3.3.2. 3rd cycle analysis ... 38

4. Conclusions ... 42

5. Bibliography ... 43

6. Attachments ... 46

6.1. Annex A –Radiation and temperature values collected by local weather station ... 46

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List of Figures

Figure 1 – Potential locations for microalgae biorefineries that fall within the imposed criteria [14] ... 15

Figure 2-Bayesian inference tree of species of the Chlorodendrophyceae class inferred using 18S rDNA sequences [21] ... 17

Figure 3- Two different of open systems. On the left a conventional raceway pound [26], on the right a recent variation of the raceway, cascade raceway [27] ... 18

Figure 4-Three different types of photoreactors. On the left the greenwall photobioreactor [29], in the middle a column photobioreactor [30] and on the right tubular photobioreactor [31] ... 19

Figure 5- Industrial centrifuge used in microalgae harvesting [48] ... 23

Figure 6 -Simplified schematic representation of the recirculation method used... 28

Figure 7 – Control vs test dry weight progression throughout the various cycles. ... 32

Figure 8 – Daily average solar radiation and its daily range throughout the experiment ... 34

Figure 9 –Average daily temperature throughout the experiment duration, as well daily temperature variation. ... 35

Figure 11 – Table regarding response of Tetraselmis spp. to the present of mercury. Table taken from [43] ... 39

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List of Tables

Table 1 – Accepted scientific classification for Tetraselmis spp. [18]. ... 16

Table 2 – List of equipment used. ... 25

Table 3 – Main constituents present in the composition of the nutritive medium. ... 26

Table 4 – List of reagents and solutions used in disinfection and sterilization. ... 27

Table 5 – Renewal cycles used with the photobioreactors. ... 32

Table 6 – Average productivity for all cycles, along with its standard deviation. ... 32

Table 7 – 3rd _{cycle (from day 29 to 46) intermediate productivity results. ... 34}

Table 8 – Nutritive medium element consumption. ... 36

Table 9 – Percentage of element accumulation of the 1st _{cycle. ... 37}

Table 10 – Elemental consumption on both 3rd _{cycle control and test cultures. ... 38}

Table 11 – Difference comparison between the 1st _{cycle fresh culture medium and the 3}rd _{cycle test} culture medium. ... 40

Table 12 – Daily values for average solar radiation measured throughout the experiment, and the maximum radiation value registered. The minimum solar radiation is always zero corresponding to the night period. ... 46

Table 13 – Daily maximum, average and minimum temperatures registered throughout the duration of this work... 47

Table 14 – Elements consumption in the 1st cycle ... 48

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Abbreviatures

A4F – Algae for Future

DGGE – Direção Geral de Geologia e Energia LNEG – Laboratório Nacional de Energia e Geologia OD – Optical density

PNPB – Nacional Plan for Biorefinery Promotion sp. – Specie

spp. – Species

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1. Introduction

1.1. Microalgae history and use

Within the last few decades, the usage and recognition of microalgae as a potential source of a wide range of metabolites has increased. Microalgae are microscopic unicellular organisms highly spread across different habitats throughout the world, capable of converting solar energy into chemical energy via photosynthesis while producing a high variety of metabolites. Currently microalgae are used in the production of valuable compounds such as proteins, lipids, polyunsaturated fatty acids, vitamins, antioxidants, carbohydrates, carotenoids [1] as well as vitamins used in health and cosmetic industry, as food and feed additives for human and animal consumption, amongst others uses. Recently microalgae also started being used in energy production in the form of biofuels due to their high oil content and quick biomass growth [2]. The use of Microalgae in wastewater treatment and heavy metals biosorption cultures has also increased [3].

Throughout history there have been some records of the use of microalgae by men. One of the earliest examples was 2000 years ago in China where a cyanobacteria Nostoc sp. was used as a food source in times of famine. Microalgae biotechnology only really began to develop in the middle of the 20th _{century, mainly as a food source. In the 1960’s Japan started the first industrial scale production of}

the specie Chlorella for human consumption and, two decades later, in the 80’s large scale production facilities were opened all around Asia, India, Israel and Australia [4].

Since then, the algae industry has experienced an exponential development and growth, mainly due to the recognition of the many advantages of microalgae production [5]:

• Microalgae do not compete for the same terrains as food crops, since they do not need cultivable terrains (they can be grown in barren lands, deserts or salterns);

• The water used in their production can be residual water, saltwater and industrial effluents; • High productivity and fast growth rate associated with a high CO2 mitigation, with possibility of

using pollution sources;

• Easy control and manipulation of culture’s conditions (temperature, salinity, pH, nutrients…) allowing an all around the year production of different species of microalgae;

• Possibility to use all metabolites of microalgae production since all of them have value in different contexts.

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Regarding the use of microalgae as a food source and nutrition, a fast-growing interest on the subject has appeared, mostly due to the many advantages that microalgae have compared to normal plant crops [6].

• Microalgae can produce a much higher amount of biomass than normal crops within the same time, making it a rapidly renewable source of biofuel;

• Microalgae production does not compete for terrain with other crops nor does it contribute to soil erosion;

• Microalgae production uses little to no pesticides or herbicides and requires minimal amounts of energy for its cultivation, overall ending up being more environment friendly than many agriculturally based food crops.

Currently they are the most nutrient-dense food known and, due to its mild flavour, it can be added to commonly used food, such as bread, drinks, snacks, or even nutritional supplements [7].

Within the last years, a growing number of successful cases associated with the use of microalgae has arisen and with that the interest and investment of governments in the microalgae industry has also increased. In 2012, the European Union (EU) adopted a strategy entitled “Innovating for Sustainable Growth: A Bioeconomy for Europe”. Its purpose was to increase the development of sustainable productions and limit the negative impact on the environment by funding innovative and sustainable projects that use renewable resources while continuing in satisfying the demand for food, energy and industrial products [4]. With the microalgae industry falling within the criteria of this program, many units of production within Europe were financed, with major increases in France and Ireland more recently, in 2018, the European Parliament and the European Council adopted a new initiative (Directive (UE) 2018/2001) on the promotion of the use of energy from renewable sources, that stipulated objectives and goal to increase the penetration of alternative renewable energy sources in the transport sector, with a clear objective of a 14% use of renewable energy sources by 2030. In this directive it is stated that the use of algae and microalgae in land, sea or photobioreactors is highly considered due to their high energy content (article 25, no.1 paragraph 1 and 4) [8].

Due to an increase interest and investment on this industry, the versatility of microalgae and the range of the products it yields was further explored with extreme results such as the production of a

Nannochloropsis sp. biorefinery capable of extracting pigments for the food industry and oils for

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1.2. Microalgae in Portugal

The ever-growing interest in microalgae has also reached Portugal. With an ample coastline, mild temperature around all year and ample resources at its disposal, it is an ideal place to cultivate and grow microalgae. The use of microalgae in Portugal has increased greatly in the last decade especially due to the higher interest in the product derived from their production and the increasing investment of both the Portuguese government and the work of different entrepreneur companies that early bet on this technology.

Nowadays, there are many companies operating in Portugal concerning the study, development and production of microalgae, namely major ones such as:

• NECTON S.A, a company invested in the culture of marine cultures and salt production, funded in 1997 and located in Faro [10];

• A4F-Algafuel S.A, a company based in Lisbon dedicated to the laboratory study of microalgae and their large-scale production [11];

• BUGGYPOWER, a company with offices in Lisbon and Funchal with a production unit located in Porto Santo in partnership with Electricity Company of Madeira (EEM) [12];

• ALGA2O, LDA, a company based in Coimbra with more than 45 years of experience on the

use of microalgae in the food, medicine and cosmetic industry, and recently started to explore the biofuel industry [13].

Following the aforementioned European initiative adopted in 2018 (Directive (UE) 2018/2001), Portugal approved a National Plan for Biorefinery Promotion (PNPB) [14] in order to meet the objectives set by the European Commission. Within the PNPB, it is anticipated a further investment in already existing alternative sources of energy, such as biomass production plants, residual treatment stations, biorefineries and, amongst others, microalgae production.

The PNPB would also incentivize and promote the development of new biorefineries, as long as certain conditions were met:

• Near population aggregates of over fifty thousand people;

• Presence of industries nearby that generate CO2 emissions (for use as carbon source for the

algae);

• Climate suitable for microalgae production (high solar radiation, low precipitation throughout the year).

In collaboration with Laboratório Nacional de Energia e Geologia (LNEG), the Direção Geral de Geologia e Energia (DGGE) has already determined more than thirty possible locations for microalgae biorefineries (Figure 1).

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The Portuguese government is eager to apply European Directive (UE) 2018/2001, not only for promoting a greener economy, but also to be less dependent on traditional energy sources that relay on international market fluctuations [14]. To do so, new goals for 2030 are being discussed such as the creation of new biorefineries around the country and the investment/benefits to already established companies to start and/or increase the production of alternative energy sources.

All being said, microalgae production in Portugal is expected to grow and prosper in the next 10 years, becoming an industry worth of investment.

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1.3. Overview of Microalgae Biotechnology

1.3.1. Microalgae general characteristics

Microalgae are a diverse group of unicellular organisms comprising prokaryotic and eukaryotic photosynthetic microorganisms. These organisms are known for their rapid adaptation to different environments, as well as their rapid growth, given their simple unicellular structure [15].

Some of the most noteworthy microalgae are Cyanophyceae (blue-green algae), Chlorophyceae (green algae), Bacillariophyceae (of which the diatoms are part of) and Chrysophyceae (golden algae). Many microalgae species are capable of switching from phototrophic to heterotrophic growth. As heterotrophs, the algae rely on glucose and other carbon sources for metabolism and energy. Some algae can even grow mixotrophically [16].

There are two hundred thousand to eight hundred thousand estimated species of microalgae, of which only fifty thousand have been described by science and less than one hundred have been produced at laboratory or industrial scale [17].

Microalgae reproduction is usually accomplished by cell division or fragmentation, allowing a quick cell cycle as well as a rapid cell growth. Compared with terrestrial plants and macroalgae and despite their similar photosynthetic mechanism, their simpler structure and the capacity to grow in a liquid medium, lead to more efficient exchange of water, carbon dioxide and nutrients, making microalgae one of the most efficient biological systems in converting solar energy to organic compounds [16].

1.3.2. Tetraselmis sp.

One widely use microalgae used in industrial production is the Tetraselmis sp., an eukaryotic green unicellular microorganism which corresponds to the scientific classification presented on Table 1.

Table 1 - Accepted scientific classification for Tetraselmis spp. [18].

Domain Eukaryota Kingdom Plantae Class Chlorodendrophyceae Order Chlorodendrales Family Chlorodendraceae Genus Tetraselmis Species Tetraselmis sp.

It has a curved or elliptical body, with an average diameter of 3.5-25 μm and four flagella. Inside it has one unique and large chloroplast (two on very rare occasions). Tetraselmis spp. reproduce asexually, with each parent cell dividing itself into four daughter cells [19].

Currently there are thirty tree accepted species of Tetraselmis [18]. These species inhabit a wide variety of aquatic habitats, varying from saltwater to freshwater (some species even living in both) with the only big restriction being access to sunlight, thus appearing more at surface level or in shallow

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bodies of water [20]. Many of the most used microalgae in the industry, like Tetraselmis suecia and

Tetraselmis chuii, actually share a very similar phylogeny (Figure 2).

Figure 2-Bayesian inference tree of species of the Chlorodendrophyceae class inferred using 18S rDNA

sequences [21].

Most of the known Tetraselmis species are relatively easy to culture, and several of them, such as

Tetraselmis chuii, Tetraselmis suecica, and Tetraselmis tetrahele, are already widely used as a feed in

aquaculture. More recently, euryhaline strains of Tetraselmis that have the ability to develop over a wide range of salinity conditions have gained interest as potential and sustainable sources of lipids for biofuel production [22].

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1.4. Autotrophic microalgae cultivation parameters and strategies

1.4.1. Cultivation systems

Microalgae are very versatile organisms, capable of being cultivated in different conditions. The main types of systems used for microalgae growth can be divided in two categories: open or close systems [23]. The most often used is the open system, due to its capacity to be used in large-scale production in a rather easy maintenance, low upkeep and long durability. The most common open systems are the raceways where the same medium is in constant movement in a cycle moved by a pump or turbine. The system characteristics, such as size, dept and agitation, depend on the cultivated microalgae, existing weather conditions and materials available [24].

However, not all algae can be sustainably cultivated in open systems, due to its inability to thrive in a selective medium/growth environment (high salinity, pH or temperature) that allows a contamination free monoalgal growth [25]. Open systems have other disadvantages such as the lack of control over their conditions, which can result in occurrence of extreme conditions. As such, close systems are becoming more widely used, as technology progresses and systems’ efficiency increases

Figure 3- Two different of open systems. On the left a conventional raceway pound [26], on the right a recent variation of the raceway, cascade raceway [27].

Closed systems have as a main advantage the possibility to control and manipulate all of its conditions, which is particularly important on the production of high value compounds. The formation rate of the compounds can be enhanced by manipulating the system conditions accordingly.

Currently there are three main closed system reactors categorised as photobioreactors (Figure 4). These are systems used to cultivate microalgae which allow a great part of the culture to be exposed to light (>90%) [28].

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The greenwall photobioreactor is a flat plate type of reactor that can either be a glass container or a plastic bag container. The main advantages include low construction cost, high scale up capacity and high surface to volume ratio. On the other hand, it has several disadvantages such as the formation of biofilms and high difficulty in temperature control, especially in large scale [32].

Column photobioreactor advantages include a low capital cost, high surface area to volume ratio, good heat and mass transfer and a relatively homogenous culture environment. However, due to its shape, the column bioreactors are hard to scale-up, as in order to increase the culture volume while continuing to provide efficient gas transfer rates, the reactor diameter should be relatively high. This will decrease the area-to-volume ratio and consequently lower the photosynthetic efficiency of the reactor [33].

Tubular photobioreactors, similarly to greenwall photobioreactors, make a good use of light when compared to column bioreactors. This type of reactor also allows for a more efficient control over the system (pH and temperature) and easy introduction of nutrients. However, the lack of surface area in contact with air in the tubular photobioreactors raises difficulties in gas exchanges. In order to avoid biofilm formation and optimise nutrients, light and gases homology, mechanical pumping is necessary, leading to higher upkeep costs, when compared to other systems. The use of mechanical pumping can also cause shear stress leading to damage to more fragile microalgae cells species [34].

1.4.2. Culture parameters

When cultivating microalgae, in order to optimize its growth and viability, there are several important parameters that one must consider [16], such as:

• Light source; • Temperature; • Salinity;

• Nutrient concentration; • pH;

• Carbon source (carbon dioxide); • Oxygen source;

• Presence of toxic chemicals;

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Light source

Microalgae growth is closely linked to its access to a light source. The presence or absence of light will determinate if the culture’s metabolism is either photosynthesis or aerobic respiration, respectively. The precise control of light intensity on the algae culture is of the utmost importance. Low light intensity corresponds to low growth rate and metabolic activity. As light intensity increases so will the microalgae activity until an optimal point where the light saturation limit is reached, with growth rate and photosynthetic productivity at its maximal. Beyond this point the radiation starts to become harmful to the cell, decreasing its growth rate and eventually leading to cellular death. The exposition to the light source by all the microalgae in culture can be affected by many factors, being the main one the culture turbulence, which will increase as the culture grows and the cell density increases. Homogeneous access to light source by all the microalgae can be optimized with a proper system design capable of stirring the medium, altering the cells closer to the light and decreasing uneven cell densities throughout the bioreactor [15] [16].

Temperature

The medium temperature is one of the most important factors to control. All organisms have an optimal temperature range where their growth and metabolic activity is optimal. That temperature interval changes form microorganism to microorganism, and in the case of Tetraselmis spp. the favourable range is between 10-30º C, with an optimal growth at 25º C [37]. Many microalgae can survive and thrive in temperatures lower than their optimal ones, up to a difference of 15º C. However, when the temperature reaches higher values, a difference of 2–4º C can be fatal to the culture. This parameter can be challenging to control, especially in large-scale productions, where usually it is used a natural light source, that while it is an easy and low-cost solution, it is also unreliable and uncontrollable. With a cloudy sky and/or low temperatures, productivity will drop. In the opposite spectre, during hotter days, it can be challenging to decrease the temperature and reduce light incidence, which in turn can lead to costly increases in production, in order to ensure the viability of the microalgae [5].

Salinity

Salinity present on a culture medium is an important parameter not only for microalgae growth but also for their cellular composition. Concentrations of chlorine and sodium higher than optimal (specific for each microorganism species) can lead to a reduced absorption of other mineral nutrients, such as potassium and manganese, which will alter the ionic rates on the cell composition. Furthermore, excessive salinity will result in salt stress, changing the membrane permeability and decreasing the efficiency of photosynthesis. In the case of organisms from genus Tetraselmis, the optimal salinity varies from species to species. Some are known to grow in non-saline environments such as Tetraselmis

cordiformis, while others, like Tetraselmis suecica can grow in salinities around 30 g/L [18]. Unlike other

parameters here described, salinity is relatively easy to control, needing only to either add brine or fresh water to the photobioreactor in order to adjust its concentration.

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Nutrients

Microalgae, like all living forms, in order to grow and multiply, need access to a great number of nutrients. Some containing carbon, phosphorus or nitrogen, are needed in greater abundance, while others such as copper, zinc, sulphur, calcium or manganese sources only need to be present in vestigial quantities, despite still being essential for cell growth. After designing and optimizing a nutritive medium for the microalgae production, it is important to maintain it constant throughout all the cultivation cycles. In order to confirm the nutrient concentration of the culture properly an elemental analysis is necessary, but it is expensive, and usually takes around two to three weeks to analyse the samples and obtain the results. As an alternative, a more practical approach can be made by measuring the nitrogen present in the culture and, therefore, determine the overall concentration of the other nutrients in the medium and how much nutritional medium there is or has to be added [38]. This method is inaccurate since depletion or accumulation of some of the elements cannot be taken into consideration. Therefore, it is advisable not only to renew large volumes of the culture periodically, but also to proceed an elemental analysis from time to time.

pH

Like temperature, the culture pH is directly linked with its growth. Microalgae can only survive within a certain range of pH and have an optimal development in an even smaller range within the survival range. The culture pH will also influence the type of metabolism exhibited by the cell [39]. This is of extreme importance when trying to produce specific products in microalgae production, since a small deviation from the pretended pH will render useless the production of the target product. The culture pH can be easily controlled by the influx of carbon dioxide. A higher carbon dioxide flow rate will decrease the pH of the culture, while cutting or reducing the flow rate will result in a pH increase [40]. The use of carbon dioxide has also the advantage to serve as a carbon source for the microalgae [41].

Contaminants

The presence of contaminants in the microalgae culture can cause a drop in production up to a total loss of the target microalgae, depending on the type of contaminant. Certain types of bacteria, other microalgae and/or fungi will compete with cultured microalgae for nutrients, space and light. Others like virus can affect the normal function of the algae, eventually leading to their death. Finally, contaminations by predators, like zooplankton and other microorganisms big enough to engulf or devour the algae cells, can cause the death of the entire culture. One of the main uses of large-scale is actually to use it as feed to zooplankton used in aquaculture [42]. The contamination of a culture is often difficult or almost impossible to avoid, even in closed systems, occurring either by operators failure when collecting samples and adding nutrients, incorrect disinfestation of the photobioreactor, or even by contaminants present in the constituents of the medium (water, brine, nutrient mixture). However, there are strategies and procedures capable of minimizing the presence of contaminants and maximizing the growth of the cultures microalgae in the presence of said contaminants. Examples of this include [38]:

- Changing values of pH, temperature or nutrient concentration to values still optimal for the microalgae cultures but less sustainable for the main contaminants;

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- Proper disinfection of the materials used, the nutrient feed pipe and the sample tubes;

- Disinfecting the culture medium using chlorine solutions (that can later be neutralized before the inoculation with sodium hypochlorite);

The presence of toxic chemicals or heavy metals in the solution can also come to affect negatively the cell growth as well as their metabolism [43]. Metals like mercury or lead are highly toxic to microalgae, disrupting their metabolism and, when in higher concentrations, lead to cellular death [44]. Even metals and other compounds essential to the cell’s growth and metabolism, such as copper [45], zinc [46], manganese or potassium, can be harmful to the microalgae if their concentration surpasses a certain threshold. Most microalgae mass-production industries do not usually have this kind of problems due to the use of fresh medium for renovations. However, when the objective is to re-use culture medium from previous cycles, where the accumulation of these elements will become inevitable, the best way to solve this situation is either going through a restricted number of cycles, or to adjust the nutrients added between each cycle.

1.4.3. Medium recirculation strategy

A medium recirculation strategy consists in the process of reintroducing the medium of a harvested culture into the next cycle of the photobioreactor. This will lead to the use of all the unconsumed nutrients from the previous cycle, while saving massive amounts of water that otherwise would be discarded into the river/sewer together with the nutrients. Such action would result on an economic waste for the production company, not to mention an environmental problem due to the amounts of fresh water and resources wasted with each of the non-recirculated cycle [47].

Recirculation strategies are vital for large-scale production of microalgae, due to the reduction of costs regarding water and nutrient supply, with special relevance in the case of saltwater cultivation systems away from the sea, where a higher investment is required to produce saltwater. However, recirculation has its problems and complications. Different microalgae and different conditions will result in different metabolisms, nutrient consumption rate and metabolite production. This will lead to a different composition in the collected culture medium and consequently a necessity to implement an approach to its treatment and adjustment before reintroducing it into the photobioreactor. In result, before applying medium recirculation in a system, one must carefully study, test and optimize the microalgae in use in order to avoid either nutrient depletion or metabolite build-up.

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1.5. Biomass harvesting

Upon the end of a production cycle the biomass is harvested. Despite the increasing interest on microalgae, this critical step of the process is yet to be fully efficient, resulting in a significant loss in biomass, and consequentially, in economic value [48]. Microalgae cell density in the culture medium does not tend to be high, due to competition for nutrients and sunlight, resulting in very diluted cultures, with densities very close to that of water. Associated with their small size (ranging between 3 to 30 µm), the low cell density issue will result in the need to use specific equipment or techniques. However, they are hard to obtain or use, since it varies not only between different species, but also between different purposes (microalgae used for food or high-value metabolites must have a higher purity than fertilizers or animal feed). Even regarding the purity of the product, due to culture medium recycling, some of the metabolites, unconsumed nutrients and toxic impurities will tend to accumulate. Depending on the client specifications for the product, the harvesting approach has to change in order to lead to conforming specification for moisture, salinity and degree of contamination. Lastly, microalgae are sensitive organisms and certain harvesting methods can lead to cell damage or strains. When the target product are undamaged cells, it is necessary to select between a less violent method, with the downside of letting more water, salt and impurities pass, or resorting to a process that can produce a purer biomass at the cost of a lower yield.

1.5.1. Harvesting methods

From an economic standpoint, one of the most important steps of microalgae production is the harvest, therefore its optimization is very important. Depending on how it is performed, the equipment used and the process efficiency, the harvesting step can turn out a heavy profit loss for the company. Therefore, it is crucial to choose the best method for the cultured microalgae [49].

The most known and used method in the microalgae industry is centrifugation. This is easily understood since this method can be used to harvest most of the microalgae species that are produced. In companies that produce different kinds of microalgae, having a common solution and equipment to harvest all of them can be extremely beneficial and cost efficient. Centrifuges are also easy to clean and sanitize, further allowing the processing of different microalgae species due to a decrease on cross contaminations.

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Nonetheless, centrifugation has its own downfalls, being the most expensive harvesting technique [51]. This can be explained by the fact that it consumes a large amount of energy per volume of culture. The consumption further increases the higher the cell recovery efficiency since it will need longer retention times within the centrifuge, operating at a lower flow-rate. In order to avoid excessive energy cost, the harvested medium can go through a flocculation and filtration step before centrifugation to reduce the volume that will pass by the centrifuge, thus reducing retention time and energy consumption. Another downside to the centrifugation is the shear stress it has on the cell, often leading to cell rupture or structural damage. This is particularly unwanted in the cases where the target product is the undamaged cells [48].

While overall centrifugation is the most known and efficient method, in certain specific cases other methods can be better suited for the task. This is the case for filtration, a method more commonly used in solid-liquid separation. In the microalgae industry, as stated previously, it is mainly used as a concentration step to alleviate some of the time and work in the centrifuge, often applied after flocculation or coagulation. However, this technique can be suitable to use in the recovery of relatively large microalgae or those that tend to form colonies or aggregates [52].

1.6. Framework and Goals

As of now, the main issue with large scale production of microalgae is the sustainability of the production systems. Microalgae production already has the advantage of occupying a smaller area than other crops and being able to use adverse terrains where nothing else would easily grow. However, it still struggles with its inefficient use and valorisation of water and nutrients, making it necessary to find strategies to optimize and economize existing resources.

This problem can only be overcome by carefully studying the existing strategies, cutback and improve where there is waste, and further invest on working strategies. This present study has allowed to understand that the reuse of resources, previously deemed unusable or waste, has great potential for a more efficient microalgae production. Nonetheless, to further improve this solution, a deeper study and understanding of its complexity is essential.

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2. Practical experiment design

In this work the microalgae used was a Tetraselmis spp., which came from a previous cycle and was transferred to the photobioreactors in use. The photobioreactors used were plastic greenwalls, three for the study, and two as control, each with a volume capacity of approximately 800 m3_{. The five}

photobioreactors were placed all next to each other, with approximately the same exposure to sunlight and all with the same weather conditions. This experiment took place between March 14th _{and May 6}th

in a country of temperate climate weather (Portugal). The reactors were stationed outside with natural light source, involving daily light cycles with approximately 12 hours of light per day in the beginning of the experiment and 13 hours towards the end. The culture temperature was affected by the weather conditions, with a temperature regulation system consisting in a sprinkling cooling system in order to keep the culture temperature lower than 30º C.

To keep the culture homogeneous an air-flow system was used, which consisted in multiple pipes with two meters length through the greenwall that pumped filtered air into the system. The air used was enriched with a constant flow of CO2 in order to stabilize the culture pH and serve as a carbon source.

The photobioreactor operated in semi-continuous regime, with a constant salinity value of 30 g/L, being periodically harvested and sub consequential renewed.

2.1. Equipment

During the course of this experiment different equipment and recipients were used. Besides the standard equipment used in a production site (hoses, pipe adaptors, falcon tubes, syringes and buckets) and the equipment integrated in the process itself (pipes, refrigeration system, valves and water metering), the remaining main equipment used are described in Table 2.

Table 2 - List of equipment used.

Equipment Model-Manufacturer

pH meter HI98130 - Hana Instruments Centrifuge DHC500 - Guangzhou Fuyi Liquid

Separation Technology Co., Ltd. Filter LF2AL22 - LIQOFLUX Liquid Deposit (5m3_{) -}

Every time a sample was taken from the photobioreactors, the pH meter was used to confirm the culture pH and temperature, making sure it was well within acceptable parameters.

Both the filter and the centrifuge were used in the harvesting of the microalgae culture. The filter is initially used to concentrate most of the culture, while the centrifuge retrieves the biomass in the pretended concentration and moisture conditions.

The 5 m3 _{container is used to receive the permeate derived from the collected harvesting, where}

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2.2. Reagents and solutions

2.2.1. Culture Medium Composition

The culture medium used in this work to cultivate and grow Tetraselmis spp. is a mixture of nutrients developed and optimized by A4F-Algafuel S.A for investigation of marine microalgae cultivation being one of the different media used in the cultivation of microalgae.

The culture medium is the result of mixing four already available solutions in A4F production site (the ratio of the four solutions used will vary for each microalgae species:

• Artificial saltwater, highly concentrated in NaCl;

• Fresh water from ground water source, for dilution of the medium to its intended conditions; • Nutritive medium rich in nitrates and other nutrients;

• Mineral water highly concentrated in various essential minerals not present in the other components, and essential to the microalgae.

After adding all these components in the specified ratio, the medium is filtered in order to remove any biological contaminant present, especially from the fresh and saltwater. It is posteriorly disinfected with a chlorite solution which can be neutralized with sodium thiosulfate if necessary.

The nutritive medium used to supplement the cultures is an outsourced industrial medium based on a recipe developed by A4F-Algafuel S.A. for laboratory-scale cultivation. While it is mostly rich in nitrogen in the form of ammonia (NH3) and nitrate (NO3), the medium also serves as a source of other

elements described in Table 3.

Table 3 - Main constituents present in the composition of the nutritive medium.

Element (%) Nitrogen 10.0 – 30.00 Phosphorus 2.0 – 15.0 Potassium 50.0 – 80.0 Calcium 1.0 – 5.0 Sulphur 0.5 – 3.0 Iron 0.1 – 1.0

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2.2.2. Other solutions

In order to avoid microbial contaminations as much as possible, all materials used are previously placed in a solution of sodium hypochlorite. Furthermore, any connection that needs to be exposed to open air, like the sampling tube, is disinfected with an alcohol solution to sterilize the connection and reduce the risk of microbial contamination.

Before being placed in a photobioreactor and inoculated with the microalgae, sodium hypochlorite is added to the culture medium at a concentration of 50 ppm. Once the culture medium passes to the photobioreactor, o-Toluidine is used to determine the concentration of chlorine still present in the solution. The remaining chlorine is then neutralized using sodium thiosulfate. A resume of the previous solutions is present in Table 4.

Table 4 – List of reagents and solutions used in disinfection and sterilization.

Reagent Use

Sodium hypochlorite Material and Medium disinfection Alcohol solution (70%) Material disinfection

Sodium thiosulfate Hypochlorite neutralization o-Toluidine Chlorine detection

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2.3. Harvesting and recirculation

Upon the culture’s harvest, an amount of approximately 70% of the total volume is collected from each photobioreactor, concentrated in a system of membranes and finally centrifuged to be collected as a paste and frozen. From the concentration step, the supernatant is collected, analysed for salinity and nitrates and complemented with the necessaries amounts in order to have a similar composition of the initial fresh medium. Just like the fresh medium, the recirculated medium is filtrated and disinfected. Finally, it is returned to the photobioreactors, therefore initiating the next cycle of production. A scheme of the medium recirculation is present in Figure 6.

Figure 6 -Simplified schematic representation of the recirculation method used (original).

The harvesting schedule was not the same throughout the experimental procedure. It depended of at least one of the following:

• Client demand – product needed to be collected and delivered;

• Structural issues – the space occupied by the photobioreactor was needed to fulfil company demands;

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2.4. Analytical Methods

Throughout this work, in order to obtain data for analysis and to study if any difference occurred in the system growth, periodical samples were retrieved. Samples were also taken for elemental analysis of the culture medium, supernatant, fresh water, saltwater, nutritive medium and mineral water.

Two types of samples were collected, each collected in a different way. The samples for dry weight quantification were collected using a syringe incorporated in the photobioreactor. Afterwards, each sample was put in a 50 mL Falcon tube and sent to A4F laboratory. The samples for elemental analysis were collected from the formulation deposit at the end of a cycle and after the readjustment of the culture medium, into a 500 mL flask, frozen and sent to an external laboratory.

2.4.1. Culture concentration

The concentration of the samples was obtained by measuring its optical density (OD). The obtained OD value allows the calculation of the dry weight of the sample (g/L) due to a correlation that it is specific for this microalgae species. This value was experimentally obtained within the A4F Laboratories, comparing different samples with known relation of OD to their dry weight obtained in a moisture analysis at 180° C.

2.4.2. Culture productivity

In order to obtain a more accurate comparison between biomass growth in different cultures or different time points, it is important to realize how fast the cell growth is. The culture productivity translates directly into that very same cell growth quantification.

Culture volumetric productivity was calculated according to equation (1), being expressed in grams per litter per day [g/(L·day)]. This was used to calculate intermediate productivities between samples and total productivity of a cycle, using the first and last sample.

𝐶𝑢𝑙𝑡𝑢𝑟𝑒 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑝𝑟𝑜𝑑𝑢𝑡𝑖𝑣𝑦 = 𝑃𝑟𝑜𝑑𝑢𝑐𝑒𝑑 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (𝑔)

𝑉𝑐𝑢𝑙𝑡𝑢𝑟𝑒(𝐿) ∗ 𝑡𝑐𝑢𝑙𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛(𝑑𝑎𝑦𝑠) (1)

2.4.3. Mean and standard deviation

In order to have more reliable results, at least two or more photobioreactors were operated simultaneously. As such, the results of the different photobioreactors operating simultaneously were analysed as being one using their mean value. However, since these photobioreactors operate independently from one another, small differences are bound to appear. In order to acknowledge and quantify these differences, the standard deviation is calculated.

The mean value (𝑥̅) of a dataset was determined according to equation (2), where x represents the value of each element in the dataset and N represents the number of elements of the dataset.

𝑥̅ =∑ 𝑥

𝑁 (2)

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The standard deviation (stdev) was calculated using the equation (3), where x represents the value of each element in the data set, 𝑥̅ is the mean value described above, and N the number of elements of the dataset.

𝑆𝑡𝑑𝑒𝑣 = √∑(𝑥 − 𝑥̅) 2

(𝑁 − 1) (3)

2.4.4. Percentage of variation

The results presentation for elements consumption and accumulation will be presented as a percentage variation, in order to better quantify and compare different elements with different concentrations.

The percentage variation between two values was determined with the equation (4), where 𝑥0 represents a reference value, and 𝑥1 represents the value that is being compared with the reference.

%𝑣𝑎𝑟𝑖𝑎𝑡𝑖𝑜𝑛 =𝑥0− 𝑥1 𝑥0

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3. Results and Discussion

Throughout this work it was studied and developed a recirculation strategy on the cultivation and production of a Tetraselmis spp. in a large-scale unit, within acceptable values of productivity and contamination. This study was conducted by analysing different culture parameters:

Throughout this study, a recirculation strategy on the cultivation and production of a Tetraselmis spp. in a large-scale unit, within acceptable values of productivity and contamination, was both developed and critically reviewed for its viability as an alternative production strategy. This study was conducted by analysing different culture parameters

• Biomass growth; • Productivity; • Temperature; • Solar radiation;

• Elemental analysis and comparison of the culture medium.

The parameters previously mentioned will be evaluated and its influence on the case study – culture medium recirculation – will be subject of study. However, the culture medium pH and salinity will not be analysed during this work, since the pH was continuously monitored during all production cycle, and the salinity value is kept constant during all recirculation cycles. Also, microorganism contamination was not taken into consideration during result evaluation since it was not a parameter with high priority to be analysed by the company, nor did it have a major impact in other previous cultures. All culture media and equipment are washed and disinfected prior to their use and the working conditions are such to minimize any contamination, which will lower the possibility of the cell growth to be affected by a microorganism contamination.

3.1. Biomass growth and productivity analysis of Tetraselmis spp.

culture

This experiment began with an initial cycle, with all photobioreactors receiving fresh medium. This cycle was followed by three more cycles on a total of four cycles. To notice that each renewal cycle had a different duration, since the decision of each harvest was different due to the reason previously mentioned. Due to harsh weather conditions (high temperature in conjunction with problems with the refrigeration system), the last cycle ended very shortly after its start, with the death of all cultures, resulting in an incapacity to acquire any usable data, thus a deeper analysis of the results will not be performed.

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Cycle Starting date Renewal date Production time (days)

Correspondent cycle interval (days)

1 15/03/2019 28/03/2019 14 0 – 14 2 29/03/2019 08/04/2019 11 15 – 26 3 09/04/2019 29/04/2019 21 27 – 46 4 30/04/2019 06/05/2019 7 47 – 53

Samples of the culture were collected throughout these cycles and sent to the laboratory for OD analysis. The values obtained were used to calculate the dry weight of the culture making it possible to determine and compare the growth of the control and test cultures (Figure 7).

Figure 7 – Control vs test dry weight progression throughout the various cycles.

With the obtained values of dry weight, it is possible to determine the productivity of each cycle. This has an importance when comparing the test results and its control counterpart, since it allows a better understanding of the differences in growth that otherwise would be hard to compare, especially in cases where the initial dry weight is not the same (2nd _{and 3}rd _{cycle as an example). In Table 6, the}

average productivity of both control and test cultures can be observed, as well as the percentual difference between them.

Table 6 – Average productivity for all cycles, along with its standard deviation.

Cycle Control Average Productivity (g/L) Test Average Productivity (g/L) Percentual Difference (%) 1st _{0.17 ± 0.02 0.16 ± 0.01 6.06} 2nd _{0.17 ± 0.01 - -} 3rd _{0.08 ± 0.03 0.06 ± 0.03 28.57} 4th _{0.06 ± 0.04 0.03 ± 0.02 66.67} 0,00 0,50 1,00 1,50 2,00 2,50 3,00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 Dry W e ig h t (g /L ) Days

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Comparing both cultures in the first cycle, in which no recirculation has yet been applied, as it can be observed in Figure 7, there is low variation between them, having a similar growth curve and showing little to no difference in productivity values. Given that both began in equal conditions, this was to be expected.

In the second cycle of production, the test culture had to be collected earlier than the experiment planning, due to company internal issues. While this did not interfere with the experience overall, it did lead to a lack of collected samples, which resulted in insufficient data needed to draw any conclusion or comparison with the control. Nonetheless, it is at least possible to notice that the initial growth curve of the dry weight is similar between control and test.

The third cycle was the longest one and the first where visibly the control and the test results diverge from one another, as observed in Figure 7. While the growth curve between them seemed similar, the further the cycle went on the larger the gap grew. This is further evidenced by the difference in productivity between both cultures.

The fourth and final cycle of this experimental work was marked by the culture’s death, resulting in the shortest cycle of the four. However, as shown in Figure 7, the control culture that started with a lower dry weight value in comparison with the test culture, throughout the duration of the cycle, ended up being higher than the test culture. As observed in Table 6, this is corroborated by a significant drop in productivity in the test culture when compared to the control. Despite the lack of results to further support any major conclusion, it can be theorized that a fourth cycle (and a third medium recycle) will have impact on microalgae growth, even without any harsh weather conditions.

3.2. Impact of weather conditions

In order to better understand the influence of medium recirculation, it is important to determine the impact caused by external conditions. Variations in temperature and radiation can lead to changes in the culture’s growth. Continuous days without sunlight or low temperatures can lead to a decrease in microalgae growth and overall productivity. On the opposite side, harsh weather conditions with high temperatures and intense solar radiation can cause cellular death. In order to evaluate the efficiency of the recirculation strategy it is important to differentiate if a culture’s change is associated with the weather or a consequence of the recirculation strategy itself.

In Figure 8 it can be observed the average daily solar radiation to which the photobioreactors were exposed, as well as the range of daily solar radiation.

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By looking at the radiation values for all cycles, it is notable that overall they have been constant with little variation over time. However, from day 29 to 34, which not only the average of the daily radiation is lower than the remaining results, but also the range of radiation that the microalgae were exposed had a significant decrease. These low solar radiation values correspond to the third cycle’s drop on productivity, both on the control and test cultures (Table 7). Therefore, it can be concluded that the lower radiation registered on those days resulted on a drop in microalgae culture growth.

Table 7 – 3rd _{cycle (from day 29 to 46) intermediate productivity results.}

Production Day Test productivity (g/L·day) Control productivity (g/L·day) 29 0.139 0.193 34 0.024 0.026 41 0.060 0.062 46 0.010 0.088

Unlike solar radiation, temperature represents a culture parameter that can be controlled to a certain extent using a cooling sprinkler system. However, this cooling method has its limitations since the water used needs to cross a large section of tubes before reaching the bioreactors. As a result, in higher temperature days, the higher demand for water by all photobioreactors will consequently lead to a decrease on the water flow rate that reaches each photoreactors.

In Figure 9 it is represented the average atmospheric temperature registered by the local weather station during the period of the experiment, as well as the maximum and minimum atmospheric temperature registered on each day.

0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00 100,00 110,00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 So la r Rad ia ti o n (M J /m 3) Days

Solar Radiation Range Avg Solar Radiation

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variation.

▪ - Average temperature

As previously stated, Tetraselmis spp. can thrive between 10–30º C, hitting its optimal growth at 20º C [37]. According to the values represented in Figure 9, the range of temperatures measured was never above the maximum (30º C). It can be also observed that in some occasions the registered temperatures were below 10º C, however that never affected the culture growth since these minimum values were reached just briefly during the night.

On average the temperatures remained at a value between 10–22º C, which is not the optimal growth temperature, it is still within an acceptable range for the culture. Thus, it can be assumed that any negative variation of the culture’s growth should not be associated to the effect of the temperature.

3.3. Elemental analysis of fresh and recycled culture medium

Analysing the impact of external conditions like the weather might provide some insight into the evolution of the culture’s growth but it will not grant a complete analysis. Given the nature of recirculation strategies, where lack of nutrients or the accumulation of toxic substances will tend to occur, the best way to study what is affecting this strategy performance is through an elemental analysis.

Throughout this experimental work, samples for elemental analysis were collected from the first and third cycles. The elemental analysis was only performed on these cycles since this type of analysis is very expensive and time consuming. The first cycle was chosen since it was the starting point, were no recirculation was performed. The third cycle, being the longest one, and so the most representative towards the full microalgae growth cycle, was chosen to study the cumulative effect of the recirculation on culture growth. 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 T e m p e ra tu re (º C) Days

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3.3.1. 1

_{cycle analysis}

For the elemental analysis in the first cycle, the permeate collected was a mixture of all photobioreactors. This decision was based on the fact that no recirculation was performed and the cultures had grown in the same conditions. The permeate collected was then compared to the initial culture medium composition. Initially, the consumption rate of the elements present in the nutritive medium was analysed in order to understand if any of the intended nutrients reached a concentration lower than the necessary or if any of them exceeded it. Table 8 features only elements with a consumption higher than 80% (elements near depletion) or lower than 50% (elements in excess). Elements with low concentration (<0,05%) were also excluded due to analytic uncertainty (concentration below measurable limit).

Table 8 - Nutritive medium element consumption.

Compounds Element Consumption (%) NH3 (as N source) 90.8 NO3- (as N source) 81.2 Cu 94.1 Fe 97.6 K 41.5 Mg 48.5 Mn 98.8 S 44.8 Zn 95.8 Ba 96.9

As expected, all the elements essential for the microalgae growth got partially consumed. None of the components is completely depleted, however some like cooper (Cu), iron (Fe), zinc (Z), manganese (Mn) or barium (Ba) were almost entirely consumed, while others such as potassium (K), magnesium (Mg) and sulphur (S) had a consumption lower than 50%. Taking the above in account, a change in the nutritive medium formulation could be proposed, with either lowering or increasing the concentration of the elements. However, changing the composition of the nutritive medium cannot be done without taking in consideration the different microalgae cultivations in A4F.

After analysing the consumption of elements present in the nutritive medium, the next step was to analyse the elements that started to accumulate, with special attention to those with potentially negative effects on microalgae (Table 9).

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Compounds Element accumulation (%)

Ag

100.0

W

80.0

Al

50.0

Hg

0.0

Be

0.0

As expected from a fresh cycle, there was no major accumulation of any element. The higher values of accumulation registered for silver (Ag) and tungsten (W) were below the measurable scale of the elemental analysis, resulting in uncertainty in their comparison. Furthermore, the measured concentration values for this elements are lower than their toxic concentration value [53].

In the case of aluminium (Al), its concentration was within the measurable range of the elemental analysis, but with a final concentration more than a hundred times lower than the inhibition limit [54]. The amount of aluminium present in the culture medium is low, but since its main source is the nutritive medium (see section 2.2), it may indicate that the nutritive medium requires a future reformulation.

The remaining two notable elements of Table 9, mercury (Hg) and beryllium (Be), are worth mentioning despite their lack of accumulation. These elements are mainly present in the freshwater and saltwater used in the formulation of the medium and tend to be toxic to all life organisms, either microalgae or any possible final consumer (livestock, plants or human). While the present concentration is lower than the toxic limit [55], the fact that these elements easily deposit and will continue to be added to the culture trough their presence in the freshwater and saltwater can be concerning for future recirculation cycles.

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3.3.2. 3rd cycle analysis

In the third cycle elemental analysis was divided in two different parts: • Analysing both control and test consumptions;

• Comparing the difference in composition of the test culture mediums with the first cycle culture medium.

The permeate from the control and test bioreactors was collected and, together with the correspondent culture medium samples, sent for elemental analysis. Afterwards, the results were compared in order to determine what components were being depleted or accumulated in this cycle.

After receiving the results from the external laboratory, all the elements present in the culture permeate were analysed and quantified. However, the variation of some results was within normal or expected range while others had concentration bellow detection limits. The elements with a concentration variation with a potential impact on the culture growth are represented in Table 10.

Table 10 – Elemental consumption on both 3rd _{cycle control and test cultures.}

Element Control Element Consumption (%) Test Element Consumption (%) NH3 54.5 99.3 B 71.1 29.7 Ca 49.9 65.0 Cu 97.1 91.7 Fe 80.6 89.0 K 34.0 12.5 Mg 62.5 -35.9 Mn 97.7 98.7 S 52.7 -7.7 Zn 92.0 93.2 Hg -333.3 -12.5 Al -250.0 -13020.0

Analysing Table 10, it can be observed some discrepancies between in the consumptions of the control and test cultures.

Regarding nitrate consumption, it is shown that the test culture had a much higher nitrate consumption than the control culture, ending up consuming most of the available nitrogen sources. The effects of nitrogen depletion can be directly correlated to the difference on the dry weight slope on Figure 7 at the end of the third cycle (between days 40 and 46).

Regarding elements such as copper, manganese and zinc, while their consumption was almost complete, since this happen both in control and test cultures, the depletion of these elements did not constitute a differentiation factor on microalgae growth.

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Calcium and iron had a consumption rate very similar both on control and test, being between 50% and 90%, which can be considered acceptable. On other hand, the consumption of boron, potassium, magnesium and sulphur was much lower in the test culture than in the control, with the elements magnesium and sulphur suffer an increase of concentration since the beginning of the cycle. While the concentration of any of these elements never reached a toxic level, the fact that these elements were not consumed by the microalgae could demonstrate either a lower metabolic activity or an overfed of those elements, resulting in their accumulation.

The noticeable accumulation of mercury and aluminium in the third cycle is an alarming situation, as both can lead to microalgae growth inhibition. This is of particular concern especially when the microalgae are introduced in the food chain, since mercury and aluminium can be prejudicial and toxic for animal and human beings. Regarding the accumulation of aluminium, while it reaches a staggering high value in the test culture, it is still just 50% below the inhibition limit of Tetraselmis spp. [54]. On the other hand, the value of mercury reached in both cultures, is above 0.02 mg/L. Observing Figure 10, it can be concluded that in both cultures of the third cycle, the accumulation of mercury falls within the growth inhibition values between 20% and 30%.

Figure 10 – Table regarding response of Tetraselmis spp. to the present of mercury in the medium. Table originally published in [44].

Given the tendency for accumulation characteristic of mercury [56] and its presence throughout the third cycle, it is possible to associate the lower productivity seen in Figure 7, when comparing it to the first cycle. The mercury source is both the freshwater and saltwater used for culture medium formulation.