Instituto Tecnológico y de Estudios Superiores de Monterrey

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Instituto Tecnológico y de Estudios Superiores de Monterrey

Campus Monterrey

School of Engineering and Sciences

Culture of Euglena gracilis in photoautotrophy for paramylon production:

effect of pH and media composition

A thesis presented by

María Martín Roldán

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of

Master of Science In Biotechnology

Monterrey Nuevo León, November 15^th, 2021

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Dedication

To myself, for never giving up. For persisting and thriving. For not being afraid of being bad at something new. For failing but getting up.

To my mum, for being always there and the main pillar of the family. For giving me this autonomy and strength to follow my dreams. All what I am is because of her.

For all the women in my family, for being united and persisting together. For facing all the bad and thriving. Because life is not easy, but it is. For those who have already left but are still in our hearts, sending us strength. These last years were not easy for many of us, many disease and death, but we still carry on.

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Acknowledgements

I would like to express my deepest gratitude to all those who have been side by side with me, along the long, but gratifying time. To my tutors for supporting me and contributing to this work.

Additionally, to the GEPEA laboratory and the Erasmus training scholarship to make it possible.

I would like to thank the Tecnológico of Monterrey and University of Oviedo support on this international master’s degree which made me thrive in my scientific career.

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Culture of Euglena Gracilis in photoautotrophy for paramylon production: effect of pH and media composition

by

María Martín Roldán

Abstract

Microalgae have been widely studied in biotechnology for their application in various areas such as bioremediation, production of biofuels, or use in nutrition. More specifically, certain species are recognized to produce compounds of high commercial value. Euglena is a group of microalgae characterized by the generation of the reserve polysaccharide paramylon, with promising prospects for its application in pharmacology, nutrition, to produce bioplastics, or biodiesel.

Paramylon is accumulated mainly in the presence of organic carbon in the culture medium;

however, this represents a challenge when establishing a large-scale culture due to the risk of biological contamination. In this study, an extensive study of the literature was carried out with respect to the autotrophic culture of Euglena gracilis destined to paramylon production. As a result, we evaluated the effect of culture pH, vitamin supplementation, and nitrogen source in the culture medium in order to reach the maximum biomass productivity. A pH of 7.5 and ammonium as nitrogen source were optimum for the autotrophic culture of E. gracilis, while an improvement in productivity was not observed with vitamin supplementation. Finally, it was possible to scale up the photoautotrophic culture of E. gracilis to a 1-L airlift photobioreactor. In conclusion, there is still much optimization work to achieve the biomass and paramylon productivity reported for the heterotrophic cultures of E. gracilis, but the results of this study reveal its viability.

Keywords: Microalgae – Photobioreactors – Euglena – Paramylon – Autotrophy – Culture

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List of Figures

Figure 1. Effect of vitamin supplement in the growth of E. gracilis under photoautotrophic conditions using nitrate as nitrogen source (BBM medium). Error bars represent the standard deviations of the means (n=3 independent replicates). ... 21 Figure 2. Growth of E. gracilis in airlift photobioreactor using nitrate as nitrogen source (BBM medium). On day 5, a high concentration of vitamin supplement was injected (red arrow).

... 23 Figure 3. Growth of E. gracilis in different pH conditions using nitrate as nitrogen source (BBM medium). Trend curves were obtained for turbidity (OD750) measures. Error bars represent the standard deviations of the means (n=3 independent replicates). ... 24 Figure 4. Growth of E. gracilis in different pH conditions using ammonium as nitrogen source (modified BBM medium). Trend curves were obtained for turbidity (continued lines) measures and pH evolution was monitored (discontinued lines) throughout the cultivations. Error bars represent the standard deviations of the means (n=3 independent replicates). ... 26 Figure 5. Dry weight biomass analysis (A and B), pigments content (C and D), and final carbohydrates content (E) for the photoautotrophic cultures of E. gracilis. Analysis were performed at the end of the cultivation time of 13-14 days, for the cultures in Erlenmeyer flasks using (A and C) BBM medium, or (B, D and E) modified BBM medium. Error bars represent standard deviations of the means (n=3 independent replicates). Statistics ANOVA and Tukey’s range test were performed and different letters above error bars represent significantly different means (α= 0.05). ... 28 Figure 6. Morphological variability of E. gracilis cells cultured in photoautotrophic conditions and different pH value of the modified BBM culture medium. Not significative difference was observed among pH treatments during the cultivation time. ... 29 Figure 7. Growth of E. gracilis in bubble bottle using ammonium as nitrogen source (modified BBM medium). Continued line represents optical density (OD750) and discontinued line pH evolution. Error bars represent standard deviations of the means (n=2 independent replicates). ... 30 Figure 8. Growth of E. gracilis in an airlift photobioreactor using ammonium as nitrogen source (modified BBM medium). (A) Cell growth monitoring, where continued line represents turbidity (OD750) measures and discontinued line pH variation through the cultivation time (days);

(B) Airlift photobioreactor prototype. ... 31

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Figure 9. Flocculation efficiency (FE %) of E. gracilis cells under varying pH treatments.

(A) Cell density measured as optical density at 750 nm (OD750) of the supernatant, and (B) flocculation efficiency (%), for each pH treatment during the experimental time. ... 33 Figure 10. Flocculation efficiency (FE %) of E. gracilis cells for different initial biomass concentrations (g L^-1). (A) Cell density measured as optical density at 750 nm (OD750) of the supernatant, and (B) flocculation efficiency (%), for each cell suspension (g L^-1) during the experimental time. ... 33 Figure 11. Growth curves for the photoautotrophic culture of E. gracilis in Erlenmeyer flasks after an electric treatment for paramylon induction. Cultures were already at stationary phase, using the previously determined optimal conditions. Error bars represent the standard deviations of the means (n=2 independent replicates). ... 35 Figure 12. Paramylon content %(w/w) (A) and dry weight biomass (g/L) (B) at the end of the photoautotrophic cultivation of E. gracilis after an electric treatment for paramylon induction. Error bars represent the standard deviations of the means (n=2 independent replicates). Statistics ANOVA and Tukey’s range test were performed, and not significative differences were observed (α= 0.05). ... 35

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List of Tables

Table 1. Companies worldwide which include Euglena-based bioproducts in their catalog (adapted from Barsanti & Gualtieri, 2018). ... 9 Table 2. Summary of culture conditions and analysis performed for the photoautotrophic culture of Euglena gracilis. ... 14

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1. Introduction

Environmental degradation due to human activity is more noticeable every year.

Problems facing waste management, freshwater sources pollution, chemical contamination, or greenhouse gas emissions, are some examples. The need of a change into a sustainable development is urgency. This requires renewable energy production, development of biobased materials, new therapies and pharmaceuticals, and a change in the food production system and the human consumption behaviour. Many research has been done over the decades to overcome this problematic, such as the use of biodegradable plastic polymers, green energy production, or ecological farming (Raza et al., 2018). However, the over-populated world, inequalities among countries policies and the implemented capitalist system put enormous pressure on natural resources making these changes small and insufficient (González-Chang et al., 2020). One of the biggest impacts is caused by energy production, mainly from non-renewable sources.

Clean energy production has passed through several generations, including the use of plant biomass and, more recently, microalgae biomass (Behera et al., 2015). Microalgae are the main source of oxygen and primary producers underwater, ranging from freshwater ponds to marine environments, contributing to the fixation of atmospheric carbon dioxide (CO2) and solar energy to produce its biomass (Cheah et al., 2015).

Under natural conditions, their photosynthetic efficiency, i.e., the conversion rate of light energy into biomass, was reported to be as high as 3 %, while for plants is about 0.2 % (Barsanti & Gualtieri, 2018). The maximum photosynthetic efficiency described, under optimal growing conditions, was about 10 % (Tredici, 2010). Additionally, microalgae can grow fast and colonize even environments with extreme conditions, promoting biogeochemical cycles (Moreno-Sánchez et al., 2017). Their ability to use waste matter as organic carbon source make them a tool for bioremediation processes, with later application of the microalgae biomass for biorefinery, such as biofuel production. These positive traits of microalgae made them promise to produce biofuels which was supported worldwide. However, its feasibility in large scale production is still not clear, since much energy is needed for the production plants, and sterility in the cultures is hard to maintain (Barsanti & Gualtieri, 2018).

On the other hand, microalgae are also a source of bioactive compounds for pharmaceuticals, nutraceuticals, and cosmetics. For human and livestock nutrition, microalgae are a source of protein, around the 60 % of their composition, which constitutes a more sustainable protein source and a new approach in food production

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(Kottuparambil et al., 2019). Euglena is a genus of aquatic unicellular protist, belonging to the phylum Euglenozoa, which present a specific β-1,3-glucan reserve polysaccharide, called as paramylon. This polymer is accumulated in the cytosol as granules constituting the main product of Euglena, unique in its group (Barsanti & Gualtieri, 2020). Paramylon has been reported for many medical applications with promising perspectives. For instance, as immunostimulant for cytokines immune response was reported from decades ago (Kondo et al., 1992); as nutritional supplement increased the survival of mice against influenza virus infection by higher production of interleukins and inflammatory cytokines (Nakashima et al., 2017). Adding Euglena gracilis to diet reduced the visceral fat accumulation in obese mice and promoted lipolysis metabolism, moreover boosted the development of beneficial bacteria in the gut microbiota (Okouchi et al., 2019). Euglena extract was reported to inhibit the early stage of adipocyte- differentiation in human by down-regulation of gene expression involved in the early stage of adipogenesis (Sugimoto et al., 2018). Authors suggested these results were due to the presence of paramylon, which acts as a dietary fiber capable of preventing the absorption of cholesterol and producing short-chain fatty acids in the intestine. On the other hand, paramylon from Euglena can be modified by addition of acyl groups with different alkyl chain length to generate an alternative to petroleum-based resins.

Paramylon monoesters were suggested as a natural pressure-sensitive adhesive (Shibakami & Sohma, 2018). Lastly, regarding microalgae biomass as third generation of biofuels, complex carbohydrates such as paramylon can be hydrolysed using oxidative glucanase enzymes to generate simple sugars for fermentation processes to the production of bioethanol (Al Abdallah et al., 2016).

Paramylon in euglenoids is synthetized in the presence of an excess in carbon source in the culture medium, and Euglena consumes it when there is a carbon starvation period, allowing algae cells to grow and survive (Rodríguez-Zavala et al., 2010). This is possible since Euglena is an obligated mixotrophic microalgae; hence, when a source of organic carbon is available in the environment, it will be used. Therefore, trophic condition of the culture medium plays the main role for paramylon production by Euglena. As a result, industrial effluents rich in organic carbon, such as potato liquor, domestic wastewater, or lignocellulosic compounds from rice bran agro-waste can be used for growing Euglena under heterotrophy, contributing to bioremediation processes (Kuroda et al., 2018;

Šantek et al., 2012; J. Zhu & Wakisaka, 2020). Nevertheless, the use of non-defined culture media leads to difficulties for optimization and scale-up production and may affect purity and quality of the bioproducts. Additionally, using organic carbon promotes a high risk of contamination by fungi and bacteria. For these reasons, microalgae biomass

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production within photobioreactors are preferred to be performed under autotrophic conditions. Regarding autotrophic culture of Euglena for paramylon production, there are not many studies since this polymer is mainly produced in heterotrophy or mixotrophy.

Consequently, optimization of the culture conditions plays an important role. For instance, supplemented media with vitamins, pH of cultivation, light intensity, temperature, and CO2 inlet. A study by Grimm et al. (2015) reported the autotrophic culture of E. gracilis using a vitamin supplement composed by cyanocobalamin and thiamine hydrochloride to ensure cell growth, achieving a dry weight biomass of 3.4 g L^-1 in Erlenmeyer flask cultures of 100 mL of working volume, at a temperature of 28 °C, light intensity of 100 µmol m⁻²s⁻¹, and a CO2 enriched atmosphere. Kitaya et al. (2005) reported a microdroplets system for a multi-culture of E. gracilis, using a chamber to control concentrations of CO2, O2, and humidity, achieving a generation time of 15 hours for photoautotrophic cultures. Accordingly, photoautotrophic culture of E. gracilis for paramylon production needs still for further research.

In the present study, the effect of some parameters in the culture of E. gracilis under photoautotrophic conditions for paramylon production were determined. Proceeding from the evaluation of the pH value and the media composition, at a laboratory scale. A trial on scaling up the culture was carried out to an airlift photobioreactor of 1 L of working volume. In addition, quantification and biochemical characterisation of the microalgae biomass obtained were accomplished.

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4 General objective

Optimize photoautotrophic growth of E. gracilis for paramylon production at laboratory scale by determining the effect of medium composition and pH, and further scaling-up in a bioreactor under optimal conditions.

Specific objectives

- Determine the effect of vitamin supplementation on cell growth by measuring turbidity during the cultivation.

- Determine the effect of different pH levels (3.5, 5.5, 6, 6.5, and 7.5) on cell growth and carbohydrate content by measuring turbidity during the cultivation, final dry weight biomass, pigments and carbohydrates.

- Determine the effect of two different nitrogen sources (nitrate and ammonium) on cell growth by measuring turbidity during the cultivation, final dry weight biomass, pigments and carbohydrates.

- Scale-up the photoautotrophic culture of E. gracilis under optimal conditions to an airlift photobioreactor of 1 L working volume.

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2. Theoretical Framework

Microalgae biotechnology

Microalgae are a diverse group of microorganisms, prokaryotic or eukaryotic with a diverse trophic spectrum, being able to grow in many different ambiences, including those with extreme conditions. Microalgae have a high efficiency in carbon dioxide sequestration promoting oxygenation of aquatic environments and constituting the primary producers in trophic chains. However, their application in biotechnology was not exploited until the beginning of the development of third generation of biofuels, few decades ago (Behera et al., 2015). Microalgae grown within photobioreactor systems allows to generate high amounts of biomass using small surfaces and reducing land exploitation. This biomass has applications such as human food, animal feed, fibres, fertilizers, or biofuels. In fact, microalgae production can be coupled to bioremediation, increasing the sustainability of the whole process. For instance, microalgae are able to grow in polluted water bodies as domestic wastewater or industrial effluents, rich in organic matter (Yadavalli et al., 2014), due to the variability in their nutritional spectrum, capable of using many different substrates. Moreover, some species tolerate high levels of heavy metals, growing in extreme environments. As an example, Euglena mutabilis was reported to proliferate in acidic mine drainages (Brake et al., 2001). Nevertheless, the destination of the microalgae biomass will determine the form of cultivation since microalgae grown in the presence of contaminants and toxics will not be destined to human consumption, but for biofuel production (Barsanti & Gualtieri, 2018).

On the other hand, microalgae species are selected regarding their molecular composition, growth conditions, nutritional requirements, or their local availability. Some species are unique for producing certain molecules with biological activity, which can be destined to the formulation of pharmaceuticals, cosmetics, or nutraceuticals. For instance, Chlorella, Haematococcus, and Porphyridium species are microalgae rich in carbohydrates with anti-inflammatory, immuno-modulating, or antioxidant activities;

pigments such as carotenoids from Dunaliella or Euglena spp.; or polyunsaturated fatty acids (PUFAs), commonly found in Chlorella vulgaris, Haematococcus pluvialis, or Isochrysis galbana (Koyande et al., 2019). Biotechnological production of microalgae for the purification of these bioactive compounds allows to obtain higher yields than those obtained naturally by plant crops (Gissibl et al., 2019). Therefore, the production of valuable compounds from microalgae is a market niche yet to be exploited, which would contribute to a more sustainable production of pharmaceuticals and nutraceuticals.

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Microalgae Euglena: characteristics and morphology

There are species of microalgae relevant to the others due to their ability to grow under certain conditions, their composition, or the production of a specific valuable compound.

Euglena is a genus of aquatic flagellate unicellular protist, belonging to the phylum Euglenozoa, which possess a complex spectrum of nutritional strategies, being an obligated mixotrophic microalgae. Its primary mode of nutrition is photoautotrophy, although, when a source of organic carbon is available, it will be used. Then, Euglena can grow in autotrophy, heterotrophy, or mixotrophy, assuming this a survival advantage, and, regarding biotechnological production, flexibility in the production mode. This capacity is due to photosynthetic machinery acquired via a secondary endosymbiosis with a prasinophytes, a unicellular green alga. Consequently, chloroplasts in euglenoids are surrounded by three membranes and contain chlorophylls A and B, and carotenoids.

Its nucleus is in the central region of the cell, containing from 42 to 45 condensed chromosomes, suggesting a possible polyploidy (Barsanti & Gualtieri, 2020). Regarding its genome, O’Neill et al. (2015) reported at least four endosymbiotic events involving a red alga and a eukaryotic green alga, while the genetic material of Euglena was derived from an ancestral protozoan. Additionally, Euglena possess epigenetic modifications as typical eukaryotic cells, including DNA methylation and histone acetylation, and contains a hypermodified nucleobase, normally found in the DNA of kinetoplastids, the β-D- glucopyranosyloxymethyluracil, or “base J”. Consequently, DNA sequencing and DNA modifications in Euglena are complex.

Euglena presents mobility through two flagella, dorsal and ventral, with different structure and length, inserted at the bottom of their basal bodies. Inside the membrane of the dorsal flagellum, there is a photoreceptor connected to the axoneme and the paraflagellar rod of the flagella, which is a 3D structure formed by stacked membranes of 2D crystals of photoreceptive proteins. Along with the photoreceptor, there is an eyespot located in front of the photoreceptor, on the dorsal side of the reservoir, which contains carotenoids in its globules (β-carotene, diatoxanthin, and other derivates). In response to a light direction or intensity stimulus, the photoreceptor is screened by the eyespot generating a photoswitch of the photoreceptor proteins and results in a change of the helicoidal swimming of cells, avoiding harmful high irradiances of light. Finally, Euglena has a peculiar and complex cell surface, called pellicle, unrelated to vegetal cell walls, composed by polysaccharides, amino sugars, and glycans, organised in overlapping strips which provides contractibility and flexibility to cells (Barsanti & Gualtieri, 2020).

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7 Biotechnological applications of Euglena

Bioremediation and raw biomass

Due to the broad nutritional spectrum of Euglena, able to grow in mixotrophy, byproducts of agricultural industry such as potato liquor (Šantek et al., 2012), lignocellulosic biomass from rice straw (J. Zhu & Wakisaka, 2020), dairy effluents (Yadavalli et al., 2014), or in domestic wastewater (Kuroda et al., 2018), were reported as substrates for Euglena culture. This contributes to nutrient removal from organic waste matter, with later microalgae biomass biorefinery for fertilizer or biofuel production. On the other hand, Euglena species were reported to develop in acidic streams and polluted rivers from mining areas. A study by Valente & Gomes (2007) described Euglena mutabilis as acidophilic microalga indicator of pollution by acid mine drainages, which survives pH values lower than 3, high metals solubility, presence of iron colloids thus high water turbidity, and inorganic carbon and phosphorus deficiency. Previously, Casiot et al.

(2004) reported E. mutabilis able to oxidize arsenic and bioaccumulate it, either inside the cells or adsorbed at the cell surface. Moreno-Sánchez et al. (2017) mentions the capability of E. gracilis cells, with no-functional chloroplasts, to remove Hg²⁺, Cd²⁺, Cr⁶⁺, and Cr³⁺ ions under heterotrophic conditions, while under photo-heterotrophic conditions, E. gracilis removes Cu²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Tc⁷⁺, and Cr⁶⁺, hence contributing to biomineralization processes and biogeochemical cycles.

Regarding the raw microalgae biomass, it can be destined to soil as fertilizer, returning the photosynthetically fixed carbon into its natural cycle. Nevertheless, since Euglena biomass is rich in protein content, high quality lipids and carbohydrates, and bioactive compounds, it is an ideal candidate for application in aquaculture and animal feed (Chae et al., 2006). Regarding the benefits of Euglena, Das et al. (2009) reported an increase in the immune response of fish when using dietary doses of Euglena viridis or direct paramylon in the fish feed (Skov et al., 2012). For human consumption, microalgae, in general, present high nutritional values, and high quantities of minerals. Particularly, paramylon from Euglena was reported as a dietary supplement capable of minimising fatigue and fatigue sensation in human, likely due to its antioxidant activity which was reported higher in patients with paramylon supplemented diet (Kawano et al., 2020). In fact, this is a further step towards functional nutrition.

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8 Bioactive compounds

As an overview, Euglena is a source of bioactive molecules of interest for industrial production: pigments, unsaturated fatty acids, α-tocopherol, the 20 protein-found amino acids (Rodríguez-Zavala et al., 2010), and paramylon. The highest bioactive isomer of vitamin E, α-tocopherol, is used as dietary supplement to counter stress and pollution harmful effects in human, and it represents the 97 % of tocopherol in Euglena, thus the ideal organism for its bioproduction (Ogbonna, 2009; Wilburn et al., 2008). On the other hand, Euglena is a source of carotenoids with human health applications, usually related to the antioxidant activity. Mainly, diatoxanthin, lutein and β-carotene were found in Euglena (Deli et al., 2014), which may prevent from premature aging, protect against certain cancers, cardiovascular diseases, arthritis, diabetes, macular degeneration, or neurodegeneration (Varela et al., 2015). Carotenoids content in microalgae can be promoted to higher yields by using stressful culture conditions, compared to those from vegetal food. For instance, Suh et al. (2006) reported higher astaxanthin accumulation in H. pluvialis by an excessive irradiation and nitrate starvation culture conditions.

Otherwise, wax ester compounds are produced in the euglenoids mitochondria under anoxic conditions, as a survival strategy through which Euglena can obtain energy by converting paramylon into wax esters (Tucci et al., 2010). Wax esters can be accumulated in cells up to 62 % of total lipid content under anaerobiosis, constituting a source for biofuels production (Matsuda et al., 2011). However, most attempts are focused on biosynthesis pathways to promote paramylon instead of wax esters since paramylon is a unique product from euglenoids, thus the main target on Euglena biotechnology.

Paramylon from euglenoids

As described above, carbon storage in euglenoid cells occurs as β-1,3-glucan reserve polysaccharide, called paramylon, which allows cells to survive light deprivation and carbon starvation periods. Paramylon consists of β-1,3-linked glucose subunits with a molecular weight between 100 and 500 kDa (Barsanti et al., 2011), organised intermolecularly as triple helix forming microfibrils, those turning up into fibres, which are arranged to form granules. These molecules are synthesized in different shapes, like ellipses or rods, depending on the specie. Granules are about 1–6µm long, surrounded by a biomembrane and with an unusual high degree of crystallinity, which makes them different from other carbohydrate storage products found in plants or algae, and unique in Euglenoidea (Malkoff & Buetow, 1964). Additionally, granules may constitute over the 80 % (w/w) of the dry weight biomass (Barsanti et al., 2001), thus an attractive target for

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biotechnology. Table 1 shows companies worldwide which include Euglena products, mainly paramylon, in their catalog. It can be observed that main microalgae production is focused on bioactive compounds since the economic feasibility is favourable for those than for biofuels production that must compete with the very low prices of fossil fuels. In addition, in the last decades much research is focused on novel bioactive compounds for dealing with increasingly complex medical issues. New therapies are required while interest on nature-based products rises. Microalgae are a source of bioactive compounds with applications for pharmaceuticals and nutraceuticals. Regarding it, paramylon as a bioactive polysaccharide, has been reported with immunostimulatory and antitumor effects by activation of the gut immune system via β-glucan receptors, such as dectin-1 (Nakashima et al., 2018); it inhibited the process of liver fibrosis, and decreased inflammation caused by non‐alcoholic fatty liver disease (NAFLD) activity in a mouse model, through oral administration of E. gracilis or paramylon extracts (Nakashima et al., 2019). Regarding wound treatment, paramylon film was reported to generate significantly faster healing than conventional cellulose film in mice; results were related to a decrease in the inflammatory response (Yasuda et al., 2018). More recently, regulation of cholesterol and glycemia, hepatoprotective activity, and treatment of some cancers, such as colorectal or gastric, were reported as well (to see more applications, go to the review article of Gissibl et al., 2019).

Table 1. Companies worldwide which include Euglena-based bioproducts in their catalog (adapted from Barsanti & Gualtieri, 2018).

Location Bioproducts Target market Link

USA Prebiotic β-1,3-glucan Pharma and

Personal Care

www.algaeon- inc.com

Japan Micronutrients/prebiotic/

biofuel

Pharma and

Personal Care Bioenergy

www.euglena.jp

USA Micronutrients / β-1,3-glucan

Pharma and

Personal Care

http://valensa.com/

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10 Market scenario for Euglena bioproducts

In microalgae production, there are levels regarding prices for each kind of bioproduct, being raw biomass, biofuels, and primary metabolites the lowest paid, ranging from 500

€ to 1000 € per ton. These are followed by glycolipids, phospholipids, and chlorophylls, which have prices among 10.000 € and 15.000 €. Secondary metabolites involve lowest yields of production but highest market prices; for example, isoprenoids as antioxidants have a price of about 900.000 €; polysaccharides as immune-stimulant around 200.000

€; polyunsaturated fatty acids (PUFAs), and oxylipins, sold either as micronutrients or anti-inflammatory compounds, have prices from 30.000 € to 75.000 €; all prices expressed in € per ton of microalgae biomass (Barsanti & Gualtieri, 2018). Therefore, pharmaceuticals and nutraceuticals, both based on secondary metabolites, constitute the highest selling prices, making the microalgae biomass production process feasible, in compare to biofuels production, which needs still of high research effort to compete with the low fossil fuel prices. For this reason, in this study the focus will be on paramylon production by E. gracilis as a polyvalent bioactive compound with a wide market outlet.

Nonetheless, industrial production of paramylon has not been achieved yet since the highest yield of production is reported for heterotrophic culture of E. gracilis, which faces a high risk of contamination by other microorganisms, affecting the performance and purity of the product. Consequently, the optimal cultivation conditions to obtain the maximum production yield in autotrophy have yet to be determined. A review of the previous bibliography on the optimization of parameters in the autotrophic culture of E.

gracilis to produce paramylon was carried out.

Autotrophic culture of E. gracilis

Paramylon is accumulated specially when there is a source of organic carbon in the culture medium, either mixotrophy or heterotrophy (Malkoff & Buetow, 1964). However, values around 50 % of paramylon were reported for photoautotrophic cultures (Wang et al., 2018). Parameters such as pH value of the culture medium plays an important role, since photosynthetic efficiency decreases through cultivation time due to an increase in the pH level, thus limiting biomass production (Spilling et al., 2011). A low pH value was reported as optimal for the autotrophic culture of E. gracilis since euglenoids cells can tolerate and proliferate under acidic conditions (Wu et al., 2020). On the other hand, it was described the need of a vitamin supplement in the culture medium, composed by cyanocobalamin and thiamine, for E. gracilis cells to grow under autotrophic conditions (Grimm et al., 2015; Wang et al., 2018; Wu et al., 2020). Gas inlet represents another

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key factor in paramylon production since anaerobic conditions lead to degradation metabolism of paramylon to form wax esters (Yoshioka et al., 2020). Matsuda et al.

(2011) reported a 58 % (w/w) of paramylon in E. gracilis cells growing in aerobic dark conditions, significantly higher than in aerobic light or anaerobic dark conditions, because paramylon biosynthesis was favoured over its conversion into wax esters. Additionally, the air inlet is used for agitation of the cultures, allowing a homogeneous distribution of the nutrients, light irradiance, and avoiding cell sedimentation. The carbon dioxide (CO2) inlet, described as % (v/v), serves as inorganic carbon source and to maintain the pH level within a range.

Finally, there are strategies aimed at boosting the accumulation of the product of interest, based on starvation, growth-limiting, or stressful conditions applied to the microalgae. A study on Chlorella zofingiensis reported an accumulation of carbohydrate up to 66.9 % of the dry weight biomass, from which 66.7 % was starch, under nitrogen starvation conditions (Zhu et al., 2014). Considering paramylon as the carbon storage polysaccharide in euglenoids, a similar strategy could be designed to increase its accumulation. More innovative strategies have been described to increase the paramylon production from Euglena. Based on its previous work on H. pluvialis, Kim et al.

(2021) reported a significative increase of 2.5-fold in paramylon production when an electrical treatment using platinum electrodes at 10 mA was applied to E. gracilis cells.

Moreover, a study by Nezammahalleh et al. (2016) reported a growth stimulation of 51 % in photoautotrophic cultures of C. vulgaris after 50 minutes of electric treatment, using a static electric field of 2 kV, and 0.4 g L^-1 of initial cell suspension. Those, time of exposure and initial cell density, resulted as determining factors. On the other hand, there are procedures for the pre-concentration of cells to reduce the culture volume to be treated.

In this way, a decrease in energy consumption by harvesting processes, such as centrifugation or filtration, is considerable. A pre-concentration test was reported for E.

gracilis in which cell flocculation efficiency was increased to more than 80 % after a treatment of high pH, between 8-9, due to magnesium phosphate, calcium phosphate, and their derivatives precipitation (Wu et al., 2020). This study was based on a previous report on C. vulgaris, one of the reference microalgae in biotechnology (Vandamme et al., 2012).

For these reasons, the culture medium composition, including the consideration of a vitamin supplement, as well as pH value, will be addressed in this study to stablish a photoautotrophic culture of E. gracilis aimed at paramylon production. Furthermore,

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strategies for boosting the microalgae biomass and the percentage of carbohydrate content will be considered and carried out if the opportunity is given.

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3. Materials and Methods

In this section, general experimental procedures, culture media, culture conditions, and biomass analysis are described. Table 2 summarizes in chronological order the different attempts to establish the photoautotrophic culture of E. gracilis from flask to bioreactor scale showing the changes made from one experiment to the other. More detail is given in the following subsections of Materials and Methods..

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Table 2. Summary of culture conditions and analysis performed for the photoautotrophic culture of Euglena gracilis.

Experiment 1 2 2’ 3 4 5 6 7

Culture conditions Replicates 3 1 1 3 3 2 1 2

Culture type Erlenmeyer

flask

Airlift PBR

Erlenmeyer flask

Bubbled bottle

Airlift PBR Erlenmeyer flask

Culture volumen (mL) 100 1000 1000 100 100 1000 1000 400

Initial cell density (OD750)

0.2 0.1 0.2 0.2 0.2 0.2 0.3

Inoculum type HT HT HT HT PT PT PT PT

Temperature (°C) 21 21 21 21 21 21 21 21

Light intensity (µmol m⁻² s⁻¹)

21 100 100 21 21 100 100 21

Culture medium BBM BBM BBM BBM Modified BBM Modified

BBM

Modified BBM

Vitamins supplement + - -/+ + + + + + + -

pH of the culture 6.5 7.5 7.5 3.5 5.5 6.5 3.5 6 7.5 7.5 7.5 7.5

Analysis of biomass

Turbidity (OD750) + + + - + + + + + + + + +

Observed growth ● ● ○ ● ● ● ● ●● ●● ●● ●●● ●●● ●

Dry Weight - - - - + + + + + + - - +

Biochemical analysis

Pigments + + - - + + + + + + - - -

Sugars - - - - - - - + + + - - paramylon

The number of replicates indicates independent replicates. HT refers to heterotrophic cultures; PT refers to photoautotrophic cultures. + indicates that vitamins supplement was added to the cultivation or that the specified analysis was carried out. - indicates that vitamins supplement was not added or that the indicated analysis was not carried out. Dry weight and biochemical analysis were performed at the end of the cultivation. Observed growth indicates a qualitative evaluation depending on cultures density, colour, and cells motility observed under the optical microscope: (○) indicates no growth and even pigments degradation; (●) indicates little growth; (●●) indicates growth; (●●●) indicates high growth and bright green cultures.

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15 Microalgae strain and culture media

Euglena gracilis klebs 1 (collection CCALA 349 https://ccala.butbn.cas.cz/en/euglena- gracilis-klebs-1) was cultured under photoautotrophic conditions, for which the effect of a vitamin supplement, pH value of the culture media, and nitrogen source, were tested.

Culture media composition, vitamin supplement, and pH adjustment are described below.

BBM culture medium (Pruvost et al., 2009) composition (g L^-1) was the following: NaNO3, 1.5; MgSO4·7H2O, 0.225; CaCl2·2H2O 0.025; EDTANa2·2H2O, 0.050; FeSO4·7H2O, 0.014; K2HPO4, 0.150; KH2PO4, 0.123; ZnSO4·7H2O, 0.222; Co(NO3)2·6H2O, 0.044;

CuSO4, 0.079; H3BO3, 2.86; MnCl2·4H2O, 1.81; NaMoO4, 0.22. Modified BBM medium contained (g L^-1) 2.6 of (NH4)2HPO4 as nitrogen source instead of NaNO3, while keeping the same concentrations of the other components. Mixotrophic culture of E. gracilis for inoculum obtention was performed by using 50 % of the heterotrophic commercial Sabouraud dextrose broth (Biokar diagnostics), with a composition (g L^-1) of tryptone, 5;

peptic digest of meat, 5; and glucose, 20; and 50 % of BBM medium. pH of the culture media was adjusted by adding HCl before autoclaving to the values 3.5, 5.5, 6, 6.5, and 7.5, for each case. Vitamins were added by filtration, after autoclaving, at the concentrations (mg L^-1): thiamine hydrochloride, 0.5 and cyanocobalamin, 0.05, accordingly to the treatment or condition to be evaluated. Manipulation of the cultures was performed keeping sterility, in a microbiological chamber.

Experimental design

Erlenmeyer flask cultures were established from a mixotrophic culture of E. gracilis growing in stationary phase, at an initial cell density of 0.2 of optical density at 750 nm (OD750). For all flask cultivations, the working volume was 100 mL, temperature room was maintained at 21 °C, light intensity of 21 µmol m⁻²s⁻¹ using fluorescent lamps perpendicular to the cultures, and agitation was performed using magnets. Inorganic carbon source was assumed from the natural exchange with the atmosphere by using cotton plugs. Firstly, the effect of the vitamin supplement was evaluated, using BBM medium with a pH of 6.5. Three independent replicates were performed both for the supplemented cultures and for the control. Secondly, the effect of the pH value was assessed for vitamin supplemented cultures using BBM medium. Three independent replicates were performed for pH 3.5, 5.5, and 6.5 treatments. Finally, the effect of the pH was studied using a different nitrogen source, i.e., the modified BBM medium which contained ammonium instead of nitrate, at the pH values of 3.5, 6, and 7.5.

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16 Culture in 1 L bubble bottle

In order to obtain higher biomass of Euglena gracilis previously to the establishment of the 1-L photobioreactor, an inoculum of E. gracilis was transferred to an autoclaved bubble bottle with 1 L of modified BBM culture medium, supplemented with vitamins, for an initial cell density of 0.2 OD750. A source of sterile air with not defined % (v/v) of CO2

was injected constantly within the culture, using an air filter connected by a sterile tube.

Light intensity was stablished at 100 µmol m⁻²s⁻¹, temperature room at 21 °C, and agitation was assumed by the air bubbling.

Culture in 1 L airlift photobioreactor

An airlift-type photobioreactor, prototype designed in the laboratory with dimensions 15x3x20 cm and a working volume of 1 L (Figure 8B), was used for scaling up the photoautotrophic culture of E. gracilis. The photobioreactor was equipped with two impellers, a central bottom source of air, aimed at agitation, a source of CO2, destined to control pH, and an air and a sampling outlet. A pH sensor was connected to verify pH and temperature during the culture time. Air input and output were connected using sterile air filters. In fact, sterility was kept during all the cultivation. For the establishment of the culture in the airlift photobioreactor, a previous step of sterilization was performed.

Firstly, the isolation and the absence of leaks were checked, after the entire photobioreactor was assembled. Then, the pH sensor was calibrated using buffer solutions, and placed in its input. Secondly, a solution of 0.005 % (v/v) of peroxyacetic acid at 38 % of purity was introduced using the medium input, a peristaltic pump, to ensure its sterility. The acidic solution was performed for 30 minutes. All inputs and outputs were covered with the acid and air filters were placed before removing it.

Following, two washes with sterile osmotized water were performed by using the peristaltic pump, to remove the acid inside. Finally, the inoculum and the sterilized culture medium were introduced through the peristaltic pump. Air filters, tubes, connectors, osmotized water, and culture media, were previously autoclaved. Light intensity was established at 100 µmol m⁻²s⁻¹, temperature room at 21 °C, and agitation by the air flux and impellers.

A first attempt was performed using BBM medium supplemented by vitamins and a pH range of 6.5-7.5. Later, the photoautotrophic culture was stablished using modified BBM medium supplemented by vitamins, at a pH range of 6.5-7.5. Temperature and light intensity were maintained the same.

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17 Monitoring the algal growth and contamination

All cultivations were monitored for cell density, i.e., OD750 and pH evolution, measured by a UV–visible spectrophotometer and a pH meter, respectively, during the cultivation time. In addition, cell morphology, motility, and contamination were observed occasionally under the optical microscope for every culture, and photos were taken randomly through the cultivations in the different conditions, using immersion oil for the maximum resolution of 100x.

Dry weight biomass analysis

Dry weight biomass was measured at the end of the cultivations, of 13-15 days, for which filters inside aluminium capsules were used. Those, previously dried in a stove at 105 °C for 24 hours, were pre-weighted using an analytical balance. A volume of 3 mL of E.

gracilis culture was weighed inside a test tube previously tared, using the analytical balance, and added to the filter using a vacuum pump. Following, two washes of the test tube using osmotized water were performed to ensure the passage of all the cellular biomass to the filter. Three replicates for each sample. Filters with the biomass were dried for 24 hours in the stove at 105 °C and weighted later. All the process was carried out using stainless steel tweezers to avoid touching the capsules and filters. Results were expressed as grams of biomass per litre of culture (g L^-1). An analysis of the variance between biomass production through the different conditions was performed using the statistic ANOVA and the Tukey’s range test to find the means which were significantly different from each other.

Pigments content analysis

Pigments content was measured at the end of the cultivation time of 13-15 days. For this analysis, an aliquot of 2 mL of culture was centrifuged at maximum velocity (13.4 rpm) for 15 minutes and supernatant was discharged. Pellet was resuspended in 1.5 mL of methanol (analytical quality) and incubated for 45 minutes at 45 °C. Then, absorbance at (i) 480nm, (ii) 652nm, (iii) 665nm, and (iv) 750nm was measured by using a UV–Visible spectrometer and quartz cuvettes. These wavelengths correspond to the characteristic

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wavelengths of (i) carotenoids, (ii) Chlorophyll B, (iii) Chlorophyll A and (iv) turbidity.

Pigments content (µg mL^-1) was calculated using the following equations (Ritchie, 2006):

[Chl − A] = V₂

V₁ (−8.0962 x A652+ 16.5169 x A665) [Chl − B] = V₂

V₁ (27.4405 x A₆₅₂− 12.1688 x A₆₆₅) [Carotenoids] = V2

V₁ (4 x A₄₈₀)

Total carbohydrate content analysis

Total carbohydrate content was measured by the method described by Dubois et al.

(1956). For each culture, an aliquot of 1 mL was centrifuged at maximum velocity (13.4 rpm) for 15 minutes and supernatant was discharged. Pellets were resuspended in 1 mL of osmotized water. At the same time, serial dilutions of 0, 0.025, 0.05, 0.075, and 0.1 (g L^-1) of glucose were made to obtain a standard curve. The same procedure was performed for the culture samples and the glucose solutions: 500 µL of sample were deposited in a test tube, and 500 µL of phenol were added. Following, 2.5 mL of sulfuric acid (>96 %) were added and samples were incubated for 10 minutes at room temperature. Then, 10 seconds of vortex were applied to each tube and 15 minutes of incubation at room temperature. Finally, 30 minutes of incubation were performed in a water bath at 35 °C, then absorbance was measured at 483 nm. Three technical replicates were carried out for each sample.

pH-induced flocculation test

Flocculation test was performed using a mixotrophic culture of E. gracilis growing exponentially after 9 days of cultivation, in a 4 L of working volume bubble bottle, at a pH of 5.1, temperature of 21 °C, and light intensity of 100 µmol m⁻²s⁻¹. Initial inoculum concentration was 4.83 g L^-1, measured by dry weight biomass analysis. Cell suspensions at an initial cell density of 0.9 OD750, in 800 mL of working volume beakers, were prepared using osmotized water. Cell suspensions were adjusted by adding HCl to six different pH values of 5, 6, 7, 8, 9, and 10. Beakers were placed in the flocculation test unit (ORCHIDIS® Flottatest FLOC6) and the experiment was conducted in three-

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time intervals of 30 minutes shaking at 125 rpm. Supernatant samples were taken after each interval of time from the centre of the beakers, and OD750 was measured to evaluate turbidity, indirectly related to cell flocculation. Later, flocculation efficiency (FE) was calculated using the following equation (adapted from Wu et al., 2020):

FE =𝑂𝐷𝑏 − 𝑂𝐷𝑎

𝑂𝐷𝑏 x 100 %

Where ODb is the OD750 of the cell suspension before the pH adjustment, and ODa is the OD750 after the flocculation treatment.

After verifying the optimal pH for cell flocculation, a second experiment was performed for different concentrations of cell suspension: 0.50, 1.30, 2.15, and 3.00 (g L^-1). Similarly, time intervals of shaking at 125 rpm were carried out, these time for 30, 60, 90, and 120 minutes. Supernatant samples were taken between intervals, and OD750 measured. FE was calculated alike.

Correlation between optical density and dry weight

Serial dilutions of a photoautotrophic culture of E. gracilis at an initial OD750 of 3.00 were prepared in a volume of 10 mL. Dry weight analysis was performed for each dilution in a total of three technical replicates, as indicated above. At the same time, 1 mL was used to determine OD750. Results were represented, OD750 versus dry weight biomass (g L^-1), and the linear regression equation was obtained.

Paramylon induction strategy: electric treatment

A pulsed electric field was applied to photoautotrophic Erlenmeyer flasks cultures (200 mL) of E. gracilis, in modified BBM medium, pH value of 7.5, after reaching stationary phase. Three treatments of 50 pulses using 7.5 kV cm^-1, corresponding to a volume specific energy of 74.25 kJ kg^-1 of cell suspension. Cultures were maintained at 21 °C and 50 µmol m⁻²s⁻¹. After 4 days, 200 mL of fresh modified BBM medium were added and cultures were monitored for growth by measuring OD750 for 7 days. At the end of the cultivation time, dry weight biomass and paramylon %(w/w) were analyzed. Paramylon was determined by gravimetry as described in previous literature (Grimm et al., 2015).

Two independent replicates were performed for the control and the treatment.

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20 Statistical analysis

The statistical method used for the data analysis of variance, ANOVA test, was performed using the statistical package Minitab® 1.3 version 19 (State College, USA) for a significance level of α= 0.05. The single-step multiple comparison Tukey’s range test was carried out when means differed significantly, to identify those different from each other.

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4. Results and Discussion

Effect of vitamin supplementation

Experimental cultures were grown in Erlenmeyer flasks using a mixotrophic inoculum of E. gracilis, a working volume of 100 mL, BBM medium, light intensity of 21 µmol m⁻²s⁻¹, pH value of 6.5, and temperature room of 21 °C. Inorganic carbon source was assumed from natural exchange with the atmosphere. Two different conditions were studied:

vitamin supplemented medium and a control without vitamins. Growth was monitored for 13 days as culture turbidity, OD750 (Figure 1).

Figure 1. Effect of vitamin supplement in the growth of E. gracilis under photoautotrophic conditions using nitrate as nitrogen source (BBM medium). Error bars represent the standard deviations of the means (n=3 independent replicates).

Supplementing BBM media with vitamins did not improve largely E. gracilis growth when compared to controls, and it cannot be concluded a difference since error bars overlap.

However, the nil effect of vitamins to cell growth is not evident since the mixotrophic inoculum volume could bring contribution of vitamins from the heterotrophic medium to the cultivations. Moreover, this could explain the high variability within cultures. To further evaluate the effect of the vitamin supplement on cell growth, the inoculum volume must be centrifuged and wash to remove the remains of the culture medium.

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The need of E. gracilis cells for vitamin supplement in the culture medium have been widely reported. Grimm et al. (2015) mentioned concentrations (mg L^-1) of 0.01 of thiamine hydrochloride, and 0.05 of cyanocobalamin; Wang et al. (2018) reported 2.5 of vitamin B1, and 0.01 of vitamin B12 to ensure growth. On the other hand, there are authors using concentrations of 0.01 μg L^-1 of vitamin B1, and 0.5 ng L^-1 of vitamin B12 (Wu et al., 2020). Nevertheless, our results did not confirm the reported importance for the vitamin supplement (Figure 1) and further assays must be accomplished. This factor is important to consider regarding the scale-up for an industrial production of Euglena biomass since the need of high quantities of vitamins involves higher production costs.

Culture in airlift photobioreactor: effect of vitamin supplementation

Considering that cell growth in Erlenmeyer flask is limited by the exchange of CO2 with the atmosphere, a culture of E. gracilis was stablished in a 1 L airlift photobioreactor to obtain higher biomass production. A mixotrophic inoculum of E. gracilis was introduced in an airlift photobioreactor using BBM medium without vitamin supplement, at an initial cell density of 0.1 OD750. The pH was established at a range of 6.5-7.5, controlled using a sterile CO2 inlet. Agitation was performed using impellers and a sterile air inlet. Light intensity was stablished initially at 50 µmol m⁻²s⁻¹. On day 5, a high concentration of vitamin supplement was added to assess the possibility of boosting cell growth, obtaining a temporary increment in cell density which decreased over time. At the end of the cultivation, cell density remained low (Figure 2) and culture turned yellow, indicative of the degradation of cellular pigments, then further analyses were omitted. A second attempt to establish the culture in the airlift photobioreactor under photoautotrophic conditions was carried out, using this time an initial cell density of 0.2 OD750, vitamin supplemented BBM medium, and 100 µmol m⁻²s⁻¹of light intensity, though cell growth was limited and culture turned yellow, as previously (data not shown).

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Figure 2. Growth of E. gracilis in airlift photobioreactor using nitrate as nitrogen source (BBM medium). On day 5, a high concentration of vitamin supplement was injected (red arrow).

Photoautotrophic culture of E. gracilis in photobioreactors has been described by many authors. Wang et al. (2018) reported the effect of different culture conditions to E. gracilis cell composition, including paramylon production, using a 2.5 L stirred tank, and obtaining a dry weight biomass production of 5 g L^-1, with paramylon content of more than 53 %, after 14 days cultivation at 23 °C. However, we failed to obtain the photobioreactor culture, as cell density remained low (Figure 2), probably due to a low initial cell density which was not able to overcome the light intensity, or to generate changes in the culture medium to enable growth. A minimum of 0.2 OD750 was concluded as necessary for starting E. gracilis cultures in photoautotrophy. Previous literature reported a value of 0.3 OD780 for inoculum (Wang et al., 2018); or a cell density of 0.6

·10⁶ cells mL^-1 (Sumida et al., 2007) for the autotrophic culture of E. gracilis. On the other hand, a second attempt to establish the photoautotrophic culture of E. gracilis in the airlift photobioreactor, using a higher initial cell density, and a light intensity of 100 µmol m⁻² s⁻¹, within the range of 100-150 µmol m⁻²s⁻¹ which was reported as optimal for E. gracilis growth (Kitaya et al., 2005; Wang et al., 2018; Wu et al., 2020), to assess if the low light intensity used before was not allowing cell growth in photoautotrophic conditions. Finally, it resulted again in failure (data not shown). Therefore, a lack of some element in the culture medium or not using optimal culture conditions could be affecting cell growth since neither the source of CO2 appeared to be the cause, nor the vitamins supplement boosted growth. We proceeded to evaluate the effect of pH since authors reported different optimal pH values in previous literature (Hurlbert et al.,1971; Grimm et al., 2015;

Wu et al., 2020).

.

0.00 0.05 0.10 0.15 0.20 0.25

0 5 10 15 20 25

OD750

Cultivation time (d)

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Culture in Erlenmeyer flasks: pH effect using BBM medium

Experimental growth conditions in Erlenmeyer flasks were as previously described with the only modification of the pH value. In this case, three different pH values were assessed: 3.5, 5.5, and 6.5. Growth was measured by cell density (OD750) during the cultivation time of 14 days (Figure 3). Dry weight biomass and pigments content were analysed at the end of the cultivation and represented in Figure 5A and 5C, respectively.

Figure 3. Growth of E. gracilis in different pH conditions using nitrate as nitrogen source (BBM medium). Trend curves were obtained for turbidity (OD750) measures.

Error bars represent the standard deviations of the means (n=3 independent replicates).

Differences in cell density among the pH treatments were not significative since error bars overlap and showed high variability within each treatment. Maximum cell density obtained was between 0.4 and 0.6 OD750 at the end of the cultivations. In addition, a statistic ANOVA test was carried out for the dry weight biomass measures, resulting a not significative difference (p-value of 0.154) between the means of each pH treatment (Figure 5A), using a significance level of α= 0.05. Regarding the pigments content, concentrations were low in overall, which meant low health status of cells. Carotenoids represented near the 20 % of pigments, then 30 % of chlorophyll B, and 50 % of chlorophyll A, for every pH treatment since not significative differences were observed due to high values of standard deviations (Figure 5C). High quantity of carotenoids in comparison to chlorophylls is related to cell stress or not optimal culture conditions, such as excessive light exposure or a lack of some essential nutrient in the culture medium.

In fact, variability was high in every analysis performed, which proved not experimental

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error but likely inherent to the cultivation in the assessed conditions. The microscopical surveillance of the cultures did not show differences in cell morphology and motility among the pH treatments, but morphological variability among cells was present in every culture (data not shown). Accordingly, we could not conclude an optimal pH value for the photoautotrophic culture of E. gracilis in the described culture conditions.

Previous literature reported optimal pH values in the culture of E. gracilis, and its capability to grow in low pH levels. For instance, Hurlbert & Bates (1971) reported an initial pH value of 5.4 which shortened the lag phase of autotrophic cultures of E. gracilis.

Wu et al. (2020) described optimal acidic conditions for E. gracilis, unlike many microalgae, and the gradually decrease in the pH through the cultivation time. Many microalgae promote an increase of pH during cell growth, leading to a decrease in photosynthetic efficiency; for this reason, a CO2 bubble source is established to keep pH within an appropriate range (Spilling et al., 2011). On the other hand, acidity conditions may affect the carbon dioxide concentrating mechanism of E. gracilis, or the proteins integrity and enzymatic performance. Some authors reported neutral pH values, between 6.5-7, for the optimal growth of E. gracilis (Grimm et al., 2015; Wang et al., 2018).

Considering this controversy with respect to the optimal pH for E. gracilis culture, we evaluated the effect of pH in cell growth, but we did not obtain a significative difference among treatments (Figure 3). Error bars indicated a high variability, hence inconclusive results. Moreover, cell biomass production was low (Figure 5A), and pigments content indicated a low health status of cells (Figure 5C), even using a vitamin supplemented medium. This assay was carried out using a mixotrophic inoculum of E. gracilis, which could be contributing to variability in results, and the not detected effect of pH on cell growth. Accordingly, to assess the culture medium composition as likely affecting our results, BBM medium components were compared to Cramer & Myers (1952) medium, highly reported for the autotrophic growth of E. gracilis. Minerals composition were similar (Table 1, Annex I) except for the nitrogen source, ammonium phosphate, (NH4)2HPO4. Further research of literature, Richter et al. (2015) reported the incapability of E. gracilis to use nitrate as nitrogen source, explaining the low cell density reached in E. gracilis cultures using BBM medium since nitrogen was apported as sodium nitrate, NaNO3. Therefore, nitrogen was a limiting factor and new assays were performed to assess the effect of pH, using ammonium phosphate in the culture medium, referring to it as modified BBM. On the other hand, a photoautotrophic inoculum was obtained in modified BBM medium to avoid variability in the following assays, likely coming from the mixotrophic inoculum volume.

Instituto Tecnológico y de Estudios Superiores de Monterrey