Alternative culture medium for Nannochloropsis oculata mass culture

Alternative culture medium for Nannochloropsis oculata mass culture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Alternative culture medium for Nannochloropsis oculata mass culture Fábio de Farias Neves, Rafael de Oliveira Jaime Sales, Isadora Kaniak Ikeda, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4102702/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microalgae of the genus Nannochloropsis are highly valued in aquaculture due to their protein, fatty acid, and carotenoid content, offering potential as additives or substitutes for fish meal or oil. However, large-scale production encounters challenges, particularly concerning the culture medium. Thus, this study aimed to assess the impact of substituting artificial sea salt with common salt and standard medium with agricultural fertilizer in Nannochloropsis oculata production. Initially, the effects of reduced salinity were evaluated at salinities of 30, 10, 5, and 1 ups, followed by an examination of the effects of replacing artificial sea salt with common salt and using agricultural fertilizer on microalgae growth. Salinities of 30 and 10 exhibited the highest growth parameters. The salt source had no significant impact on culture growth, while the agricultural fertilizer enhanced it. Common salt increased the concentration of eicosapentaenoic acid compared to artificial sea salt, although the total lipid concentration was higher in microalgae cultured with artificial sea salt. The developed medium was validated through semi-continuous cultures in 100 L flat vertical bags, showing to be an economically viable alternative without hindering N. oculata growth. microalgae aquaculture salinity fertilizer fatty acids Figures Figure 1 Figure 2 Introduction With the population growth, the demand for food has been increasing, including marine animal derivatives. It is estimated that by 2030, the total global fish production will reach 209 million tons, with 103 million originating from aquaculture (FAO 2018). With the advancement and growth of this sector, it is inevitable that the demand for raw materials for feed production will increase in parallel, including fish meal and oil. It is assumed that about 16% of capture fisheries will be allocated to produce feed ingredients (FAO 2018). Unfortunately, the demand for these ingredients cannot be met. Therefore, there is a need for more sustainable resources that can reduce the use of fish meal and oil in aquaculture (Nishshanka et al. 2022). In this scenario, microalgae emerge as a viable alternative. Microalgae are microscopic photosynthetic organisms capable of growing in different environmental conditions using sunlight, phosphorus, and nitrogen. These microorganisms are considered superfoods as they are sources of vitamins, carotenoids, antioxidants, essential amino acids, lipids, polysaccharides, and other bioactive compounds (Eze et al. 2023). Due to their high nutritional value and richness in bioactive compounds, they not only promote the nutrition of aquatic organisms but also enhance immunity and disease resistance (Nagappan et al. 2021). Additionally, as a primary source of fatty acids, they can improve the lipid profile in aquatic animals, increasing their nutritional value. Thus, microalgae can be used for the feeding of zooplankton, crustaceans, mollusks, shrimp, and fish (Dineshbabu et al. 2019). Microalgae of the genus Nannochloropsis are among the most used and important in aquaculture due to their composition rich in proteins, long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA), and carotenoids (Ferreira et al. 2021). Studies show the potential of this microalga as an alternative to fish-derived components or as a feed additive. The partial replacement of fish oil with Nannochloropsis sp. in the form of pure powder biomass or its lipids in feed has led to improvements in the growth and fatty acid profile of shrimp larvae, post-larvae, and juveniles (Oswald et al. 2019; Oswald et al. 2020). Also, the partial replacement of fish meal with the microalga in turbot feed increased the concentration of essential amino acids in the meat and improved the organism's antioxidant capacity (Qiao et al. 2019). Gbadamosi and Lupatsch (2018) also observed that using N. salina as a total replacement for fish meal did not affect the performance and health of tilapia compared with traditionally fed fish. Used as a feed additive, a preparation of Nannochloropsis spp. has been shown to enhance the immunity and resistance of shrimp subjected to thermal shock (Guimarães et al. 2021). Despite the advantages of using microalgae as feed in aquaculture, a bottleneck in large-scale microalgae production is the high production cost, including costs related to the culture medium (Carvalho et al. 2019). The application of analytical-grade nutrients as used on laboratory scale is impractical. Therefore, more economical alternative nutrient sources are employed, such as commercial fertilizers. However, since these differ from laboratory culture media, the impact of new sources must be assessed (Nayak et al. 2016; Neto et al. 2018; Carvalho et al. 2019). For marine microalgae, another important factor to consider is salinity. Salinity influences the osmotic and ionic balance of cells in relation to the environment and affects energy expenditure. High or very low salinity values can inhibit the growth of some species and induce significant physiological changes, as well as alter the content of lipids, carbohydrates, proteins, and pigments (Cañavate and Fernández‐Díaz, 2022; Haris et al., 2022). In this regard, the salt source must be evaluated by producers. Many researchers use natural seawater as the base for the culture medium, known as semi-defined medium, as they cannot control the water composition at the time of collection. However, the imprecision of the composition, water quality variation, and risks of water contamination are negative aspects of its use, besides being impractical for non-coastal locations. Artificially salinized water, therefore, is an interesting alternative (Berges et al. 2001), and commercial formulations are available. Unfortunately, the high cost of these artificial sea salts limits their use in massive-scale cultivation. In this context, the objective of this study was to develop a financially viable culture medium to produce the microalga Nannochloropsis oculat a . To reduce costs, the use of common table salt (NaCl) and commercial fertilizer was evaluated in three stages: (i) salinity reduction, (ii) substitution of commercial sea salt with common salt, and standard nutrient medium with agricultural fertilizer, and (iii) evaluation of the new culture medium on a pilot scale. Materials And Methods Microalga and culture conditions The microalga Nannochloropsis oculata was obtained from the culture collection of the Laboratory of Algae Cultivation and Biotechnology of the State University of Santa Catarina and maintained in the microalgae cultivation room in F/2 culture medium (Guillard 1975) and artificial sea salt (salinity 30 ups). All presented experiments were conducted in the cultivation room with a controlled temperature of 23ºC, continuous aeration with filtered atmospheric air, and 24-hour illumination with a light intensity of 66.6 μmol photons m -2 s -1 . Physicochemical parameters such as salinity and pH were regularly monitored. Evaluation of Nannochloropsis oculata growth at low salinity Aiming the reduction of the amount of salt in the cultivation medium of N. oculata , the effects of concentrations of salt below the traditional level (30 ups) on culture growth were assessed. Salinities of 30 (control), 10, 5, and 1 ups were applied (represented by Sal 30, Sal 10, Sal 5, and Sal 1, respectively). Prior to the experiment, the microalga was acclimatized to each tested salinity (lower than 30 ups). Every two days, the culture was subcultured with a cultivation medium at a salinity of 0 to decrease by 2 ups until reaching the desired value. For the experiment, commercial artificial sea salt was used, providing 0.3 g g -1 of sodium, 9 mg g -1 of calcium, 27 mg g -1 of magnesium, 9 mg g -1 of potassium, 0.51 g g -1 of chlorides, 0.06 g g -1 of sulfates, 3.15 mg g -1 of carbonates, and trace elements (unspecified). The cultures were supplemented with F/2 medium in Erlenmeyer flasks containing 1.5 L of culture. The experiment lasted for 9 days and was conducted in triplicate. Selection of salt and nutrient sources In order to assess the feasibility of using a more economical cultivation medium, the use of common table salt (CS) was compared to artificial sea salt (AS) in two different culture media: the standard F/2 medium (F2) and a medium supplemented with agricultural fertilizer (AF) and (NH₄)₂SO₄ (with an N:P ratio equal to 16:1). The agricultural fertilizer used was a NPK type, claiming to contain 18% N, 6% P 2 O 5 , and 18% K 2 O. An experimental design with two factors was applied, resulting in four treatments: ASF2, ASAF, CSF2, and CSAF. Each treatment was conducted in triplicate in 1.5 L Erlenmeyer flasks with a salinity of 20. The experiment lasted for 7 days. At the end, the total lipid content was determined using the Bligh and Dyer method (Bligh and Dyer 1959), and the fatty acid profile was analyzed by gas chromatography-mass spectrometry (GC-MS) in the ASAF and CSAF cultures. Validation of the cultivation medium on pilot Scale The results from the laboratory scale experiment were replicated on a pilot scale using the culture medium with adjustments determined in the previous experiments. The microalgal cultivation was conducted in 100 L flat vertical bags (FVAs) with a culture medium containing agricultural fertilizer supplemented (NH₄)₂SO₄ (N:P ratio equal to 16:1), salinized with common table salt (20 ups). The cultivation was carried out in batch mode until the culture reached between 4,000 x 10 4 and 5,000 x 10 4 cells mL -1 , after which harvests of 45% of the volume were performed every 7 days in a semi-continuous system. After harvesting, the supernatant was returned to the culture and fertilized again. Three replicates were conducted, each lasting 20-30 days. Monitoring of microalgal growth For the growth analysis of the cultures, cell density and dry biomass were regularly monitored. Cell density (CD) was determined by cell counting using a Neubauer chamber under an optical microscope. Dry biomass (DB) was estimated using a gravimetric method. A known volume of the culture was filtered through a pre-dried and weighed fiberglass filter. The filter was then dried in oven at 50 ºC until a constant weight was achieved. The dry biomass is determined by the difference between the final and initial weights of the filter. From these data, the maximum cell density (MCD) was determined, and the specific growth rate (µ, d -1 ) and biomass productivity (g L -1 d -1 ) were calculated using the respective formulas: where CDi is the initial cell density, CDf is the final cell density and Δt is the time interval between CDi and CDf. where DBi is the initial dry biomass, DBf is the final dry biomass and Δt is the time interval between DBi and DBf. Statistical analysis For the experiment with different salinities, the data were analyzed through one-way ANOVA, while for the experiment involving different salt and nutrient sources, a factorial ANOVA was applied. In order to identify statistical differences between the treatments, the Tukey post-hoc test was used, considering a significance level of 0.05. The data are presented as mean ± standard deviation. Results Effect of salinity The salinity of each treatment remained constant according to its respective concentration. Sal 30, Sal 10, and Sal 5 maintained an average pH value equal to or above 9.0 (9.0 ± 0.12, 9.5 ± 0.07, and 9.0 ± 0.13, respectively), while Sal 1 remained at 8.8 ± 0.08. The growth of the CD of N. oculata at different salinities is presented in Fig. 1. It can be observed that the growth of the cultures over time was directly proportional to the increase in salinity, with Sal 10 and Sal 30 exhibiting the highest cell densities from day 2 onwards (p < 0.05), concluding the experiment with an average of 1,611.67 x 10 4 ± 385.46 x 10 4 and 2,125.00 x 10 4 ± 354.29 x 10 4 cells mL -1 , respectively. Fig. 1 Cell growth curve of Nannochloropsis oculata culture at different salinities (30, 10, 5 and 1). Data points represent the means and error bars indicate the standard deviation (n = 3) The same trend occurred regarding DB, which showed higher values in Sal 30 (0.29 ± 0.04 g L -1 ) on the 8th day, followed by Sal 10 (0.16 ± 0.02 g L -1 ), Sal 5 (0.14 ± 0.01 g L -1 ), and Sal 1 (0.03 ± 0.00 g L -1 ). Consequently, the productivity of Sal 30 (0.027 ± 0.006 g L -1 d -1 ) was higher than the other treatments (p < 0.05), while the productivity of Sal 10 was similar to Sal 5 (p = 0.885) and higher than Sal 1 (p = 0.006) (Table 1). However, even though Sal 30 showed higher values, the MCD and µ were not statistically different from Sal 10 (p = 0.290 and p = 0.852, respectively), both reaching MCD on the 7th day of cultivation. Cultures cultivated in Sal 5 showed reduced growth, with MCD, productivity, and µ values approximately 40%, 48%, and 52% lower, respectively, compared with the control. Clearly, in Sal 1, there was no growth of the microalga, resulting in a negative µ (-0.07 ± 0.02 d -1 ) and productivity equivalent to zero (Table 1). Table 1 Growth parameters of Nannochloropsis oculata cultivation at different salinities Salinity MCD (x10 4 cells mL -1 ) Biomass gain (g L -1 ) µ (d -1 ) Productivity (g L -1 d -1 ) 30 2,319.17 ± 482.29 a 0.23 ± 0.04 a 0.25 ± 0.03 ab 0.027 ± 0.006 a 10 1,901.67 ± 146.32 a 0.12 ± 0.02 b 0.28 ± 0.05 a 0.017 ± 0.003 b 5 945.83 ± 162.89 b 0.11 ± 0.01 b 0.13 ± 0.07 b 0.013 ± 0.002 b 1 195.00 ± 28.83 c 0.00 ± 0.01 c -0.07 ± 0.02 c 0.000 ± 0.001 c Data are shown as mean ± s.d. (n = 3). Different letters (a, b, and c) represent statistically significant differences among the different salinities (p < 0.05) Evaluation of alternative sources of salt and nutrients The cell growth curves in Fig. 2 demonstrate that, until the last day of cultivation, the treatments showed an increase in their CD, except for CSF2, which reached a plateau on the 3rd day. The averages of ASAF and CSAF at the end of the cultivation reached the highest cell concentrations (8,200.00 x 10 4 ± 1,025.00 x 10 4 and 7,812.50 x 10 4 ± 137.50 x 10 4 cells mL -1 , respectively), followed by ASF2 (6,616.67 x 10 4 ± 1,169.46 x 10 4 cells mL -1 ) and CSF2 (5,075.00 x 10 4 ± 800.00 x 10 4 cells mL -1 ). Fig. 2 Cell growth curve of Nannochloropsis oculata cultivation in different culture media: ASF2, ASAF, CSF2, and CSAF. Data points represent the means and error bars indicate the standard deviation (n = 3) All treatments maintained a salinity of 20 throughout the cultivation. The growth parameters MCD, productivity, and µ showed no significant difference between the type of salt or nutrient source (p > 0.05). However, the final DB showed significant differences between nutrient sources, with AF treatments yielding higher results than those of F2 (p = 0.023) (Table 2). Regarding pH values, cultures with AS (AF: 8.59 ± 0.15; F2: 8.48 ± 0.12) were higher than those with CS (AF: 8.15 ± 0.15; F2: 8.33 ± 0.10) (p = 0.005). Table 2 Growth parameters of Nannochloropsis oculata cultivation in different culture media (ASF2, ASAF, CSF2, and CSAF) Treatment MCD (x10 4 cells mL -1 ) Final DB (g L -1 ) Productivity (g L -1 d -1 ) µ (d -1 ) ASF2 6,616.67 ± 1432.29 Aa 0,51 ± 0,04 Aa 0,06 ± 0,01 Aa 0,21 ± 0,04 Aa ASAF 7,425.00 ± 1688.93 Aa 0,62 ± 0,02 Ab 0,07 ± 0,00 Aa 0,21 ± 0,03 Aa CSF2 5,366.67 ± 488.83 Aa 0,41 ± 0,08 Aa 0,04 ± 0,01 Aa 0,19 ± 0,02 Aa CSAF 7,291.67 ± 912.53 Aa 0,53 ± 0,12 Ab 0,06 ± 0,01 Aa 0,22 ± 0,01 Aa Data are shown as mean ± s.d. (n = 3). Different letters represent significant differences between the factors of the treatments, with uppercase letters (A) referring to the types of salts (AS and CS), and lowercase letters (a and b) referring to the types of nutrients (F2 and AF) In Table 3 it is possible to verify if there is a significant effect of the isolated factors or their interaction on the response variables based on the p-value. A significant effect was observed only for the nutrient medium on the final biomass and for the type of salt on pH, with a variation rate of 62.30% and 78.84% for their respective factors. The other variables were not influenced by the type of salt or nutrient source. Table 3 Factorial ANOVA of the effect of salt type and nutrient medium on growth parameters and pH of Nannochloropsis oculata . Significant differences are represented by the p-value, and the influence of each factor on the responses is indicated by the percentage of variation. Factors Response variable Salt Medium Salt*Medium MCD 9.25% 87.54% 3.21% p=0.657 p=0.313 p=0.313 Productivity 61.83% 37.41% 0.76% p=0.060 p=0.128 p=0.814 µ 3.03% 37.41% 0.76% p=0.576 p=0.118 p=0.697 Final biomass 37.39% 62.30% 0.31% p=0.061 p=0.023 p=0.848 Biomass gain 40.00% 57.99% 2.02% p=0.098 p=0.054 p=0.686 pH 78.84% 1.00% 20.16% p=0.004 p=0.668 p=0.080 Regarding lipids content, the cultivation of ASAF showed 17.8 ± 0.2% (w/v) of total lipids, while CSAF obtained a ratio of 12.4 ± 0.2%. However, the amount of EPA produced by CSAF was higher (p < 0.001) than that obtained by ASAF, with a difference of 11%. Significant differences were also observed in the production of hexadecanoic acid (p = 0.046) and 9-hexadecenoic acid (p = 0.012), with ASAF showing higher quantities (Table 4). Table 4 – Fatty acid profile of Nannochloropsis oculata cultivated in the culture media ASAF and CSAF. Relative quantity (%) Fatty acid Symbology ASAF CSAF Tetradecanoic acid C14:0 6.70 ± 0.33 a 6.05 ± 0.65 a Hexadecanoic acid C16:0 36.72 ± 0.97 a 30.41 ± 2.98 b 9-Hexadecenoic acid C16:1 25.34 ± 0.39 a 18.30 ± 2.24 b Octadecanoic acid C18:0 0.87 ± 0.63 a 0.85 ± 0.66 a 9-Octadecenoic acid C18:1 3.76 ± 0.86 a 8.73 ± 1.81 a 9,12-Octadecadienoic acid C18:2 6.15 ± 0.27 a 6.56 ± 0.08 a 5,8,11,14-Eicosatetraenoic acid C20:4 0.71 ± 0.52 a 1.98 ± 1.93 a 5,8,11,14,17-Eicosapentaenoic acid C20:5 20.05 ± 0.36 a 31.04 ± 0.23 b Different letters (a and b) represent a significant difference between the different culture media (p < 0.05). Validation of the cultivation medium on pilot scale The cultivations carried out in FVAs maintained a salinity of 20 and an average pH of 8.4 ± 0.62. They were initiated with an average initial CD of 2,443.33 x 10 4 ± 109.11 x 10 4 cells mL -1 , and the batch cultivation lasted an average of 9 days, after which harvests and dilutions began. These harvests occurred every 6-8 days, and µ was calculated during these intervals, resulting in 0.12 ± 0.03 d -1 . The cultures were maintained for an average of 23 days, and the MCD achieved was 5,031.67 x 10 4 ± 429.74 x 10 4 cells mL -1 . The average of DB and CD obtained in the harvests were 0.34 ± 0.01 g L -1 and 4,350.00 x 10 4 ± 849.79 x 10 4 cells mL -1 , respectively. Discussion Salinity is one of the crucial factors in the cultivation of marine microalgae, and there are numerous studies evaluating its effects on the growth and biochemical composition of these microorganisms (Gu et al. 2012; Ishika et al. 2018; Pugkaew et al. 2019) . Changes in salinity induce osmotic stress on organisms, requiring them to undergo physiological adjustments to the new environment (Guo et al. 2019). Most studies on this topic assess the effect of high salinities on the growth of Nannochloropsis . However, in the present experiment, the aim was to understand the impact of lower-than-usual salt concentrations, considering that artificial seawater contributes to increased cultivation costs. No difference in the growth of N. oculata was observed between salinities 30 and 10, except for the dry biomass productivity. Similar conclusions were drawn in studies conducted with Nannochloropsis sp., where reducing NaCl concentration from 27 to 13/13.5 g L -1 did not negatively impact cultivation growth (Martínez-Roldán et al. 2014) and even increased cell density after 7 days of cultivation (Pal et al. 2011). The species N. salina also exhibited higher maximum cell density at lower salinity (22 ups) compared to the traditionally used 34 ups (Bartley et al. 2013). Cultures at salinities 5 and 1 exhibited a significant reduction in growth. In this study, the microalga N. oculata had its growth rate halved at salinity 5; however, it was not completely inhibited, as observed at salinity 1. This characteristic is species-specific. For instance, according to Bartley et al. (2013), N. salina does not grow at salinities below 8. In a study conducted by Zulkifli et al. (2018), N. oculata was able to grow in a culture medium with zero salinity comparable to cultures with saline water, but only up to the 8th day. On the other hand, the strain N. oceania CCALA 804 demonstrates efficient growth in a medium with zero salinity, indicating tolerance to drastic osmotic reductions(Pal et al. 2013; Solovchenko et al. 2014). Considering the similarity between the cultures of the treatments with salinities 30 and 10, with a reduction only in productivity (0.027 vs. 0.017 g L -1 d -1 , respectively), an intermediate salinity (20) was chosen for the subsequent experiments. In the cultivation of marine microalgae, seawater is commonly used as the base of the culture medium by researchers and producers. However, there are factors that make its use impractical, such as variations in water quality due to tides, pollution, and climate, and especially the geographic and economic limitations for locations far from the sea (Berges et al. 2001; Venteris et al. 2013). It should be considered that coastal land is of high value and subject to greater climatic fluctuations. Additionally, seawater intake requires the installation and maintenance of equipment on the coast or offshore. Coastal intake is more economical but more susceptible to problems such as boat collisions, waves, and storms. On the other hand, offshore intake involves more expensive installation and maintenance (Huguenin and Colt 2002). An assessment conducted by Venteris et al . (2013) in the United States concluded that the use of saline waters is a costly alternative compared to freshwater. These disadvantages and complications make artificial salinization an attractive option. For this purpose, there are several artificial sea salts in the market that fulfill the function of simulating seawater, providing salinity, trace elements, and buffers that contribute to water quality. However, these products have a high cost, and consequently, their use becomes impractical in large-scale microalgae production. In this context, after reducing the salt concentration in the culture medium of N. oculata , the substitution of artificial sea salt with common table salt was evaluated. The achieved values of maximum cell density, specific growth rate, final biomass, and biomass gain showed no significant difference between the types of salt. Therefore, replacing artificial sea salt with common table salt is not detrimental to the cultivation of N. oculata , maintaining the same levels of cell growth. The only parameter that showed a significant difference was pH, which likely remained higher in artificial sea salt due to the presence of carbonate salts. Nevertheless, it did not influence growth. Therefore, common salt becomes a viable alternative to seawater intake and, especially, a more economical option compared to artificial sea salt in massive production. While common salt costs around US$ 0.16/kg, sea salt is priced at US$ 2.43/kg, 15 times higher. The use of agricultural fertilizer as a nutrient source is a strategy for reducing the costs of the culture medium in large-scale microalgae production, including Nannochloropsis species (Camacho-Rodríguez et al. 2013; Liu and Bangert 2015; Neto et al. 2018). In the present study, only the nutrient source (medium) affected the final biomass, with the highest concentrations obtained with agricultural fertilizer (FA). The maximum cell density averages for FA were also higher than those achieved by F2, although not showing a significant difference. Therefore, the application of agricultural fertilizer is advantageous compared to the standard F2 medium. It is also worth noting that the interaction between salt and nutrient medium showed no effect on any microalgal growth parameter. Regarding the production of lipids and fatty acids, the culture medium with sea salt showed a higher lipid content but a lower amount of EPA compared to the medium with common salt. From the ASAF to the CSAF treatment, there was a reduction of 6.31% and 7.04% in the fatty acids 16:0 and 16:1, respectively, while an 11% increase in EPA (C20:5) was observed. The sea salt applied in this study contains trace elements that possibly enhance lipid accumulation by Nannochloropsis oculata . It has been demonstrated that different levels of iron, zinc, manganese, and molybdenum in the culture medium significantly interfere with lipid storage in some species of microalgae, but their requirements are species-specific (Ghafari et al. 2018). In contrast, the depletion of calcium and magnesium has been shown to increase lipid content in Chlorella vulgaris and Scenedesmus obliquus (Gorain et al. 2013). Similar to the total lipids, micronutrients influence the fatty acid profile. Savvidou et al. (2020) observed that the depletion of iron and manganese decreased lipid content but increased the levels of polyunsaturated fatty acids in Nannochloropsis oceanica, increasing the amount of EPA (C20:5) by 3.63% and 4.91%, while decreasing some saturated fatty acids. These results align with the findings of the present study. Therefore, it is essential to identify the micronutrients that favor the production of desired lipids and fatty acids by Nannochloropsis oculata to subsequently add them strategically to the medium with common salt. When validating the cultivation medium in FVBs, the obtained data were lower than those presented earlier on a smaller scale. It is noticeable that there was a reduction in both the specific growth rate and the achieved dry biomass productivity. This change was expected since the configuration of the 2 L photobioreactor differs from the 100 L flat vertical bags. Changes in shape, aeration, luminosity, among other aspects, influence cultivation productivity. According to (Borowitzka and Vonshak 2017), scaling up microalgae production introduces important hydrodynamic changes, especially regarding the homogeneity of the culture, which is more challenging to achieve on a larger scale. Thus, light and nutrients are not uniformly distributed to all cells, leading to a reduction in productivity. Despite the cultivation reaching lower growth in this stage of the study, other studies with the same genus show similar and even lower results than those found in the present experiment. When using inorganic fertilizers, N. gaditana was able to achieve a higher biomass concentration (0.4 g L -1 ), but with a specific growth rate equal to 0.15 d -1 on the 6th day of cultivation (Riveros et al. 2018), a value close to that found in the present work. Neto et al. (2018) obtained lower biomass concentrations of N. oculata using fertilizers, with a range from 0.12 to 0.20 g L -1 . In contrast, the optimization of the culture medium by the response surface methodology led to the production of up to 0.58 g L -1 of N. oculata UTEX 2164 in 9 days (Mehra and Jutur 2022), a higher value than that found in the present study. However, it is important to note that different scales and nutrient sources make it difficult to compare results. Given the potential demonstrated by the CSAF medium for N. oculata production, some adjustments are necessary for larger-scale cultivation to achieve productivities close to those observed on a smaller scale. Therefore, more in-depth studies on agitation mode, luminosity (width of the flattened bag), cultivation operation, and the addition of micronutrients should be conducted. Conclusion In the present study, it was observed that the marine microalga N. oculata can grow in a cultivation medium with a salinity of 20, which is lower than the typically applied salinity. Additionally, it was possible to replace artificial sea salt and the standard nutrient medium with common salt and agricultural fertilizer without negatively impacting its growth, making the production of N. oculata more economically viable. It is worth noting that studies on the addition of micronutrients should be conducted later to ensure a good lipid composition of the microalga. Adjustments in the configuration of the flat vertical bags photobioreactor of 100 L are also necessary to achieve growth parameters closer to those obtained on a small scale. Declarations Funding This work was supported by National Council for Scientific and Technological Development (CNPq Project 24556/2021-9), also the Santa Catarina State Foundation for Research and Innovation Support (FAPESC 2023TR302). Competing interests The authors report there are no competing interests to declare. Availability of data and material Not applicable. Code availability Not applicable. Authors' contributions Fábio de Farias Neves: Conceptualization, Investigation, Validation, Writing – original draft, Supervision, Project administration. Rafael de Oliveira Jaime Sales: Conceptualization, Methodology, Investigation, Formal analysis, Experimental work, Writing – original draft. Isadora Kaniak Ikeda: Methodology, Investigation, Formal analysis, Experimental work, Writing – original draft. Ana Carolina de Souza Santos: Experimental work. Ana Flavia Celso Duarte: experimental work. Ricardo Camilo Martins: Experimental work. Rosana de Cássia de Souza Schneider: Formal analysis, Review & Editing. Daniel Pedro Willemann: Review & Editing, Project administration. All authors reviewed the manuscript. Acknowledgments Authors thank the National Council for Scientific and Technological Development (CNPq Project 24556/2021-9), also the Santa Catarina State Foundation for Research and Innovation Support (FAPESC 2023TR302). References Bartley ML, Boeing WJ, Corcoran AA, Holguin FO, Schaub T (2013) Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and invading organisms. Biomass Bioenergy 54:83–88. Berges JA, Franklin DJ, Harrison PJ (2001) Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades. J Phycol 37:1138–1145. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. 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Eze CN, Onyejiaka CK, Ihim SA, Ayoka TO, Aduba CC, Ndukwe JK, Nwaiwu O, Onyeaka H (2023) Bioactive compounds by microalgae and potentials for the management of some human disease conditions. AIMS Microbiol 9:55–74. FAO (2018) The State of World Fisheries and Aquaculture 2018 - Meeting the sustainable development goals. Rome Ferreira M, Teixeira C, Abreu H, Silva J, Costas B, Kiron V, Valente LMP (2021) Nutritional value, antimicrobial and antioxidant activities of micro- and macroalgae, single or blended, unravel their potential use for aquafeeds. J Appl Phycol 33:3507–3518. Gbadamosi OK, Lupatsch I (2018) Effects of dietary Nannochloropsis salina on the nutritional performance and fatty acid profile of Nile tilapia, Oreochromis niloticus . Algal Res 33:48–54. Ghafari M, Rashidi B, Haznedaroglu BZ (2018) Effects of macro and micronutrients on neutral lipid accumulation in oleaginous microalgae. Biofuels 9:147–156. Gorain PC, Bagchi SK, Mallick N (2013) Effects of calcium, magnesium and sodium chloride in enhancing lipid accumulation in two green microalgae. Environ Technol 34:1887–1894. Gu N, Lin Q, Li G, Tan Y, Huang L, Lin J (2012) Effect of salinity on growth, biochemical composition, and lipid productivity of Nannochloropsis oculata CS 179. Eng Life Sci 12:631–637. Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of Marine Invertebrate Animals. Springer US, Boston, MA, pp 29–60. Guimarães AM, Guertler C, do Vale Pereira G, da Rosa Coelho J, Costa Rezende P, Nóbrega RO, do Nascimento Vieira F (2021) Nannochloropsis spp. as feed additive for the Pacific white shrimp: Effect on midgut microbiology, thermal shock resistance and immunology. Animals 11:150. Guo L, Liang S, Zhang Z, Liu H, Wang S, Yang G (2019) Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses. J Oceanol Limnol 37:1353–1362. Haris N, Manan H, Jusoh M, Khatoon H, Katayama T, Kasan NA (2022) Effect of different salinity on the growth performance and proximate composition of isolated indigenous microalgae species. Aquac Rep 22:100925. Huguenin JE, Colt J (2002) Seawater sources. In: Huguenin JE, Colt J (eds) Developments in Aquaculture and Fisheries Science, Second. Elsevier, pp 59–71. Ishika T, Bahri PA, Laird DW, Moheimani NR (2018) The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine, halotolerant, and halophilic microalgae. J Appl Phycol 30:1453–1464. Liu J, Bangert K (2015) Effect of nitrogen source in low-cost media on biomass and lipid productivity of Nannochloropsis salina for large-scale biodiesel production. Environ Prog Sustain Energy 34:297–303. Martínez-Roldán AJ, Perales-Vela H V., Cañizares-Villanueva RO, Torzillo G (2014) Physiological response of Nannochloropsis sp. to saline stress in laboratory batch cultures. J Appl Phycol 26:115–121. Mehra A, Jutur PP (2022) Application of response surface methodology (RSM) for optimizing biomass production in Nannochloropsis oculata UTEX 2164. J Appl Phycol 34:1893–1907. Nagappan S, Das P, AbdulQuadir M, Thaher M, Khan S, Mahata C, Al-Jabri H, Vatland AK, Kumar G (2021) Potential of microalgae as a sustainable feed ingredient for aquaculture. J Biotechnol 341:1–20. Nayak M, Thirunavoukkarasu M, Mohanty RC (2016) Cultivation of freshwater microalga Scenedesmus sp. using a low-cost inorganic fertilizer for enhanced biomass and lipid yield. J Gen Appl Microbiol 62:7–13. Neto WAF, Mendes CRB, Abreu PC (2018) Carotenoid production by the marine microalgae Nannochloropsis oculata in different low-cost culture media. Aquac Res 49:2527–2535. Nishshanka GKSH, Anthonio RADP, Nimarshana PHV, Ariyadasa TU, Chang J-S (2022) Marine microalgae as sustainable feedstock for multi-product biorefineries. Biochem Eng J 187:108593. Oswald ATO, Ishikawa M, Koshio S, Yokoyama S, Moss AS, Dossou S (2020) Effects of dietary Nannochloropsis sp. powder and lipids on the growth performance and fatty acid composition of larval and postlarval kuruma shrimp, Marsupenaeus japonicus . Aquac Nutr 26:186–200. Oswald ATO, Ishikawa M, Koshio S, Yokoyama S, Moss AS, Dossou S (2019) Nutritional evaluation of Nannochloropsis powder and lipid as alternative to fish oil for kuruma shrimp, Marsupenaeus japonicus . Aquaculture 504:427–436. Pal D, Khozin-Goldberg I, Cohen Z, Boussiba S (2011) The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Appl Microbiol Biotechnol 90:1429–1441. Pal D, Khozin-Goldberg I, Didi-Cohen S, Solovchenko A, Batushansky A, Kaye Y, Sikron N, Samani T, Fait A, Boussiba S (2013) Growth, lipid production and metabolic adjustments in the euryhaline eustigmatophyte Nannochloropsis oceanica CCALA 804 in response to osmotic downshift. Appl Microbiol Biotechnol 97:8291–8306. Pugkaew W, Meetam M, Yokthongwattana K, Leeratsuwan N, Pokethitiyook P (2019) Effects of salinity changes on growth, photosynthetic activity, biochemical composition, and lipid productivity of marine microalga Tetraselmis suecica . J Appl Phycol 31:969–979. Qiao H, Hu D, Ma J, Wang X, Wu H, Wang J (2019) Feeding effects of the microalga Nannochloropsis sp. on juvenile turbot ( Scophthalmus maximus L.). Algal Res 41:101540. Riveros K, Sepulveda C, Bazaes J, Marticorena P, Riquelme C, Acién G (2018) Overall development of a bioprocess for the outdoor production of Nannochloropsis gaditana for aquaculture. Aquac Res 49:165–176. Savvidou MG, Boli E, Logothetis D, Lymperopoulou T, Ferraro A, Louli V, Mamma D, Kekos D, Magoulas K, Kolisis FN (2020) A study on the effect of macro- and micro- nutrients on Nannochloropsis oceanica growth, fatty acid composition and magnetic harvesting efficiency. Plants 9:660. Solovchenko A, Lukyanov A, Solovchenko O, Didi-Cohen S, Boussiba S, Khozin‐Goldberg I (2014) Interactive effects of salinity, high light, and nitrogen starvation on fatty acid and carotenoid profiles in Nannochloropsis oceanica CCALA 804. Eur J Lipid Sci Technol 116:635–644. Venteris ER, Skaggs RL, Coleman AM, Wigmosta MS (2013) A GIS cost model to assess the availability of freshwater, seawater, and saline groundwater for algal biofuel production in the United States. Environ Sci Technol 47:4840–4849. Zulkifli AF, Ramli A, Lim JW, Lam MK (2018) Effect of NaNO 3 and NaCl concentration on Nannochloropsis oculata cell biomass and FAME composition for biodiesel production. J Phys Conf Ser 1123:012071. 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Data points represent the means and error bars indicate the standard deviation (n = 3)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4102702/v1/eb67f75fd81251800b50253f.png"},{"id":53153896,"identity":"f59b1fe2-929e-4d4c-8930-e98f05fa70bb","added_by":"auto","created_at":"2024-03-21 09:13:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28259,"visible":true,"origin":"","legend":"\u003cp\u003eCell growth curve of \u003cem\u003eNannochloropsis oculata\u003c/em\u003ecultivation in different culture media: ASF2, ASAF, CSF2, and CSAF. Data points represent the means and error bars indicate the standard deviation (n = 3)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4102702/v1/9c1a0f133744a0daa838699f.png"},{"id":54801398,"identity":"952ed2e1-5982-4a46-bd87-4a1c430d1b06","added_by":"auto","created_at":"2024-04-17 02:52:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":645664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4102702/v1/c9680612-0cc0-4673-856c-dcb32a884980.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alternative culture medium for Nannochloropsis oculata mass culture","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith the population growth, the demand for food has been increasing, including marine animal derivatives. It is estimated that by 2030, the total global fish production will reach 209 million tons, with 103 million originating from aquaculture (FAO 2018). With the advancement and growth of this sector, it is inevitable that the demand for raw materials for feed production will increase in parallel, including fish meal and oil. It is assumed that about 16% of capture fisheries will be allocated to produce feed ingredients (FAO 2018). Unfortunately, the demand for these ingredients cannot be met. Therefore, there is a need for more sustainable resources that can reduce the use of fish meal and oil in aquaculture (Nishshanka et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In this scenario, microalgae emerge as a viable alternative. Microalgae are microscopic photosynthetic organisms capable of growing in different environmental conditions using sunlight, phosphorus, and nitrogen. These microorganisms are considered superfoods as they are sources of vitamins, carotenoids, antioxidants, essential amino acids, lipids, polysaccharides, and other bioactive compounds (Eze et al. 2023). Due to their high nutritional value and richness in bioactive compounds, they not only promote the nutrition of aquatic organisms but also enhance immunity and disease resistance (Nagappan et al. 2021). \u0026nbsp;Additionally, as a primary source of fatty acids, they can improve the lipid profile in aquatic animals, increasing their nutritional value. Thus, microalgae can be used for the feeding of zooplankton, crustaceans, mollusks, shrimp, and fish (Dineshbabu et al. 2019).\u003c/p\u003e\n\u003cp\u003eMicroalgae of the genus \u003cem\u003eNannochloropsis\u003c/em\u003e are among the most used and important in aquaculture due to their composition rich in proteins, long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA), and carotenoids (Ferreira et al. 2021). Studies show the potential of this microalga as an alternative to fish-derived components or as a feed additive. The partial replacement of fish oil with \u003cem\u003eNannochloropsis\u003c/em\u003e sp. in the form of pure powder biomass or its lipids in feed has led to improvements in the growth and fatty acid profile of shrimp larvae, post-larvae, and juveniles (Oswald et al. 2019; Oswald et al. 2020). Also, the partial replacement of fish meal with the microalga in turbot feed increased the concentration of essential amino acids in the meat and improved the organism\u0026apos;s antioxidant capacity (Qiao et al. 2019). Gbadamosi and Lupatsch (2018) also observed that using \u003cem\u003eN. salina\u003c/em\u003e as a total replacement for fish meal did not affect the performance and health of tilapia compared with traditionally fed fish. Used as a feed additive, a preparation of \u003cem\u003eNannochloropsis\u003c/em\u003e spp. has been shown to enhance the immunity and resistance of shrimp subjected to thermal shock (Guimar\u0026atilde;es et al. 2021).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Despite the advantages of using microalgae as feed in aquaculture, a bottleneck in large-scale microalgae production is the high production cost, including costs related to the culture medium (Carvalho et al. 2019). The application of analytical-grade nutrients as used on laboratory scale is impractical. Therefore, more economical alternative nutrient sources are employed, such as commercial fertilizers. However, since these differ from laboratory culture media, the impact of new sources must be assessed (Nayak et al. 2016; Neto et al. 2018; Carvalho et al. 2019).\u003c/p\u003e\n\u003cp\u003eFor marine microalgae, another important factor to consider is salinity. Salinity influences the osmotic and ionic balance of cells in relation to the environment and affects energy expenditure. High or very low salinity values can inhibit the growth of some species and induce significant physiological changes, as well as alter the content of lipids, carbohydrates, proteins, and pigments (Ca\u0026ntilde;avate and Fern\u0026aacute;ndez‐D\u0026iacute;az, 2022; Haris et al., 2022). In this regard, the salt source must be evaluated by producers. Many researchers use natural seawater as the base for the culture medium, known as semi-defined medium, as they cannot control the water composition at the time of collection. However, the imprecision of the composition, water quality variation, and risks of water contamination are negative aspects of its use, besides being impractical for non-coastal locations. Artificially salinized water, therefore, is an interesting alternative (Berges et al. 2001), and commercial formulations are available. Unfortunately, the high cost of these artificial sea salts limits their use in massive-scale cultivation.\u003c/p\u003e\n\u003cp\u003eIn this context, the objective of this study was to develop a financially viable culture medium to produce the microalga \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculat\u003c/em\u003e\u003cem\u003ea\u003c/em\u003e. To reduce costs, the use of common table salt (NaCl) and commercial fertilizer was evaluated in three stages: (i) salinity reduction, (ii) substitution of commercial sea salt with common salt, and standard nutrient medium with agricultural fertilizer, and (iii) evaluation of the new culture medium on a pilot scale.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicroalga and culture conditions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microalga \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e was obtained from the culture collection of the Laboratory of Algae Cultivation and Biotechnology of the State University of Santa Catarina and maintained in the microalgae cultivation room in F/2 culture medium (Guillard 1975) and artificial sea salt (salinity 30 ups). All presented experiments were conducted in the cultivation room with a controlled temperature of 23\u0026ordm;C, continuous aeration with filtered atmospheric air, and 24-hour illumination with a light intensity of 66.6 \u0026mu;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e. Physicochemical parameters such as salinity and pH were regularly monitored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEvaluation of Nannochloropsis oculata growth at low salinity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAiming the reduction of the amount of salt in the cultivation medium of \u003cem\u003eN. oculata\u003c/em\u003e, the effects of concentrations of salt below the traditional level (30 ups) on culture growth were assessed. Salinities of 30 (control), 10, 5, and 1 ups were applied (represented by Sal 30, Sal 10, Sal 5, and Sal 1, respectively). Prior to the experiment, the microalga was acclimatized to each tested salinity (lower than 30 ups). Every two days, the culture was subcultured with a cultivation medium at a salinity of 0 to decrease by 2 ups until reaching the desired value. For the experiment, commercial artificial sea salt was used, providing 0.3 g g\u003csup\u003e-1\u003c/sup\u003e of sodium, 9 mg g\u003csup\u003e-1\u003c/sup\u003e of calcium, 27 mg g\u003csup\u003e-1\u003c/sup\u003e of magnesium, 9 mg g\u003csup\u003e-1\u003c/sup\u003e of potassium, 0.51 g g\u003csup\u003e-1\u003c/sup\u003e of chlorides, 0.06 g g\u003csup\u003e-1\u003c/sup\u003e of sulfates, 3.15 mg g\u003csup\u003e-1\u003c/sup\u003e of carbonates, and trace elements (unspecified). The cultures were supplemented with F/2 medium in Erlenmeyer flasks containing 1.5 L of culture. The experiment lasted for 9 days and was conducted in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSelection of salt and nutrient sources\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to assess the feasibility of using a more economical cultivation medium, the use of common table salt (CS) was compared to artificial sea salt (AS) in two different culture media: the standard F/2 medium (F2) and a medium supplemented with agricultural fertilizer (AF) and (NH₄)₂SO₄ (with an N:P ratio equal to 16:1). The agricultural fertilizer used was a NPK type, claiming to contain 18% N, 6% P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e, and 18% K\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\n\u003cp\u003eAn experimental design with two factors was applied, resulting in four treatments: ASF2, ASAF, CSF2, and CSAF. Each treatment was conducted in triplicate in 1.5 L Erlenmeyer flasks with a salinity of 20. The experiment lasted for 7 days. At the end, the total lipid content was determined using the Bligh and Dyer method (Bligh and Dyer 1959), and the fatty acid profile was analyzed by gas chromatography-mass spectrometry (GC-MS) in the ASAF and CSAF cultures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eValidation of the cultivation medium on pilot Scale\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results from the laboratory scale experiment were replicated on a pilot scale using the culture medium with adjustments determined in the previous experiments. The microalgal cultivation was conducted in 100 L flat vertical bags (FVAs) with a culture medium containing agricultural fertilizer supplemented\u0026nbsp;(NH₄)₂SO₄\u0026nbsp;(N:P ratio equal to 16:1), salinized with common table salt (20 ups). The cultivation was carried out in batch mode until the culture reached between 4,000 x 10\u003csup\u003e4\u003c/sup\u003e and 5,000 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e, after which harvests of 45% of the volume were performed every 7 days in a semi-continuous system. After harvesting, the supernatant was returned to the culture and fertilized again. Three replicates were conducted, each lasting 20-30 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMonitoring of microalgal growth\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the growth analysis of the cultures, cell density and dry biomass were regularly monitored. Cell density (CD) was determined by cell counting using a Neubauer chamber under an optical microscope. Dry biomass (DB) was estimated using a gravimetric method. A known volume of the culture was filtered through a pre-dried and weighed fiberglass filter. The filter was then dried in oven at 50 \u0026ordm;C until a constant weight was achieved. The dry biomass is determined by the difference between the final and initial weights of the filter.\u003c/p\u003e\n\u003cp\u003eFrom these data, the maximum cell density (MCD) was determined, and the specific growth rate (\u0026micro;, d\u003csup\u003e-1\u003c/sup\u003e) and biomass productivity (g L\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e) were calculated using the respective formulas:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere CDi is the initial cell density, CDf is the final cell density and \u0026Delta;t is the time interval between CDi and CDf.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere DBi is the initial dry biomass, DBf is the final dry biomass and \u0026Delta;t is the time interval between DBi and DBf.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the experiment with different salinities, the data were analyzed through one-way ANOVA, while for the experiment involving different salt and nutrient sources, a factorial ANOVA was applied. In order to identify statistical differences between the treatments, the Tukey post-hoc test was used, considering a significance level of 0.05. The data are presented as mean \u0026plusmn; standard deviation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEffect of salinity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe salinity of each treatment remained constant according to its respective concentration. Sal 30, Sal 10, and Sal 5 maintained an average pH value equal to or above 9.0 (9.0 \u0026plusmn; 0.12, 9.5 \u0026plusmn; 0.07, and 9.0 \u0026plusmn; 0.13, respectively), while Sal 1 remained at 8.8 \u0026plusmn; 0.08.\u003c/p\u003e\n\u003cp\u003eThe growth of the CD of \u003cem\u003eN. oculata\u003c/em\u003e at different salinities is presented in Fig. 1. It can be observed that the growth of the cultures over time was directly proportional to the increase in salinity, with Sal 10 and Sal 30 exhibiting the highest cell densities from day 2 onwards (p \u0026lt; 0.05), concluding the experiment with an average of 1,611.67 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 385.46 x 10\u003csup\u003e4\u003c/sup\u003e and 2,125.00 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 354.29 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1\u003c/strong\u003e Cell growth curve of \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e culture at different salinities (30, 10, 5 and 1). Data points represent the means and error bars indicate the standard deviation (n = 3)\u003c/p\u003e\n\u003cp\u003eThe same trend occurred regarding DB, which showed higher values in Sal 30 (0.29 \u0026plusmn; 0.04 g L\u003csup\u003e-1\u003c/sup\u003e) on the 8th day, followed by Sal 10 (0.16 \u0026plusmn; 0.02 g L\u003csup\u003e-1\u003c/sup\u003e), Sal 5 (0.14 \u0026plusmn; 0.01 g L\u003csup\u003e-1\u003c/sup\u003e), and Sal 1 (0.03 \u0026plusmn; 0.00 g L\u003csup\u003e-1\u003c/sup\u003e). Consequently, the productivity of Sal 30 (0.027 \u0026plusmn; 0.006 g L\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e) was higher than the other treatments (p \u0026lt; 0.05), while the productivity of Sal 10 was similar to Sal 5 (p = 0.885) and higher than Sal 1 (p = 0.006) (Table 1).\u003c/p\u003e\n\u003cp\u003eHowever, even though Sal 30 showed higher values, the MCD and \u0026micro; were not statistically different from Sal 10 (p = 0.290 and p = 0.852, respectively), both reaching MCD on the 7th day of cultivation. Cultures cultivated in Sal 5 showed reduced growth, with MCD, productivity, and \u0026micro; values approximately 40%, 48%, and 52% lower, respectively, compared with the control. Clearly, in Sal 1, there was no growth of the microalga, resulting in a negative \u0026micro; (-0.07 \u0026plusmn; 0.02 d\u003csup\u003e-1\u003c/sup\u003e) and productivity equivalent to zero (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Growth parameters of \u003cem\u003eNannochloropsis oculata\u003c/em\u003e cultivation at different salinities\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.017064846416382%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSalinity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.426621160409557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMCD (x10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBiomass gain (g L\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026micro; (d\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.98976109215017%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProductivity (g L\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.017064846416382%\" valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.426621160409557%\"\u003e\n \u003cp\u003e2,319.17 \u0026plusmn; 482.29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.23 \u0026plusmn; 0.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.25 \u0026plusmn; 0.03 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.98976109215017%\" valign=\"top\"\u003e\n \u003cp\u003e0.027 \u0026plusmn; 0.006 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.017064846416382%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.426621160409557%\" valign=\"top\"\u003e\n \u003cp\u003e1,901.67 \u0026plusmn; 146.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.02 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.28 \u0026plusmn; 0.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.98976109215017%\" valign=\"top\"\u003e\n \u003cp\u003e0.017 \u0026plusmn; 0.003 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.017064846416382%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.426621160409557%\" valign=\"top\"\u003e\n \u003cp\u003e945.83 \u0026plusmn; 162.89 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.11 \u0026plusmn; 0.01 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.07 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.98976109215017%\" valign=\"top\"\u003e\n \u003cp\u003e0.013 \u0026plusmn; 0.002 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.017064846416382%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.426621160409557%\" valign=\"top\"\u003e\n \u003cp\u003e195.00 \u0026plusmn; 28.83 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.01 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.283276450511945%\" valign=\"top\"\u003e\n \u003cp\u003e-0.07 \u0026plusmn; 0.02 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.98976109215017%\" valign=\"top\"\u003e\n \u003cp\u003e0.000 \u0026plusmn; 0.001 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eData are shown as mean \u0026plusmn; s.d. (n = 3). Different letters (a, b, and c) represent statistically significant differences among the different salinities (p \u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEvaluation of alternative sources of salt and nutrients\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell growth curves in Fig. 2 demonstrate that, until the last day of cultivation, the treatments showed an increase in their CD, except for CSF2, which reached a plateau on the 3rd day. The averages of ASAF and CSAF at the end of the cultivation reached the highest cell concentrations (8,200.00 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 1,025.00 x 10\u003csup\u003e4\u003c/sup\u003e and 7,812.50 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 137.50 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e, respectively), followed by ASF2 (6,616.67 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 1,169.46 x 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003ecells mL\u003csup\u003e-1\u003c/sup\u003e) and CSF2 (5,075.00 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 800.00 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2\u003c/strong\u003e Cell growth curve of \u003cem\u003eNannochloropsis oculata\u003c/em\u003e cultivation in different culture media: ASF2, ASAF, CSF2, and CSAF. Data points represent the means and error bars indicate the standard deviation (n = 3)\u003c/p\u003e\n\u003cp\u003eAll treatments maintained a salinity of 20 throughout the cultivation. The growth parameters MCD, productivity, and \u0026micro; showed no significant difference between the type of salt or nutrient source (p \u0026gt; 0.05). However, the final DB showed significant differences between nutrient sources, with AF treatments yielding higher results than those of F2 (p = 0.023) (Table 2). Regarding pH values, cultures with AS (AF: 8.59 \u0026plusmn; 0.15; F2: 8.48 \u0026plusmn; 0.12) were higher than those with CS (AF: 8.15 \u0026plusmn; 0.15; F2: 8.33 \u0026plusmn; 0.10) (p = 0.005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Growth parameters of \u003cem\u003eNannochloropsis oculata\u003c/em\u003e cultivation in different culture media (ASF2, ASAF, CSF2, and CSAF)\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"610\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.942528735632184%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.108374384236452%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMCD (x10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFinal DB (g L\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProductivity (g L\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.555008210180624%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026micro; (d\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.942528735632184%\"\u003e\n \u003cp\u003eASF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.108374384236452%\"\u003e\n \u003cp\u003e6,616.67 \u0026plusmn; 1432.29 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,51 \u0026plusmn; 0,04 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,06 \u0026plusmn; 0,01 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.555008210180624%\"\u003e\n \u003cp\u003e0,21 \u0026plusmn; 0,04 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.942528735632184%\"\u003e\n \u003cp\u003eASAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.108374384236452%\"\u003e\n \u003cp\u003e7,425.00 \u0026plusmn; 1688.93 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,62 \u0026plusmn; 0,02 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,07 \u0026plusmn; 0,00 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.555008210180624%\"\u003e\n \u003cp\u003e0,21 \u0026plusmn; 0,03 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.942528735632184%\"\u003e\n \u003cp\u003eCSF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.108374384236452%\"\u003e\n \u003cp\u003e5,366.67 \u0026plusmn; 488.83 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,41 \u0026plusmn; 0,08 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,04 \u0026plusmn; 0,01 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.555008210180624%\"\u003e\n \u003cp\u003e0,19 \u0026plusmn; 0,02 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.942528735632184%\"\u003e\n \u003cp\u003eCSAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.108374384236452%\"\u003e\n \u003cp\u003e7,291.67 \u0026plusmn; 912.53 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,53 \u0026plusmn; 0,12 Ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.19704433497537%\"\u003e\n \u003cp\u003e0,06 \u0026plusmn; 0,01 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.555008210180624%\"\u003e\n \u003cp\u003e0,22 \u0026plusmn; 0,01 Aa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are shown as mean \u0026plusmn; s.d. (n = 3). Different letters represent significant differences between the factors of the treatments, with uppercase letters (A) referring to the types of salts (AS and CS), and lowercase letters (a and b) referring to the types of nutrients (F2 and AF)\u003c/p\u003e\n\u003cp\u003eIn Table 3 it is possible to verify if there is a significant effect of the isolated factors or their interaction on the response variables based on the p-value. A significant effect was observed only for the nutrient medium on the final biomass and for the type of salt on pH, with a variation rate of 62.30% and 78.84% for their respective factors. The other variables were not influenced by the type of salt or nutrient source.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Factorial ANOVA of the effect of salt type and nutrient medium on growth parameters and pH of \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e. Significant differences are represented by the p-value, and the influence of each factor on the responses is indicated by the percentage of variation.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"416\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.3012048192771%\" colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eFactors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eResponse variable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSalt\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSalt*Medium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003eMCD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e9.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e87.54%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e3.21%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.313\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003eProductivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e61.83%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e37.41%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.76%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.814\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026micro;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e3.03%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e37.41%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.76%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.576\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.697\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003eFinal biomass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e37.39%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e62.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e0.31%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.848\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003eBiomass gain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e40.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e57.99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e2.02%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.686\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003e78.84%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003e1.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003e20.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.69879518072289%\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.349397590361445%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.795180722891565%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.668\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.156626506024097%\" valign=\"bottom\"\u003e\n \u003cp\u003ep=0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eRegarding lipids content, the cultivation of ASAF showed 17.8 \u0026plusmn; 0.2% (w/v) of total lipids, while CSAF obtained a ratio of 12.4 \u0026plusmn; 0.2%. However, the amount of EPA produced by CSAF was higher (p \u0026lt; 0.001) than that obtained by ASAF, with a difference of 11%. Significant differences were also observed in the production of hexadecanoic acid (p = 0.046) and 9-hexadecenoic acid (p = 0.012), with ASAF showing higher quantities (Table 4).\u003c/p\u003e\n\u003cp\u003eTable 4 \u0026ndash; Fatty acid profile of \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e cultivated in the culture media ASAF and CSAF.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"562\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.188612099644125%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative quantity (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFatty acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSymbology\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e\u003cstrong\u003eASAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCSAF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003eTetradecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC14:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e6.70 \u0026plusmn; 0.33 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e6.05 \u0026plusmn; 0.65 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003eHexadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC16:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e36.72 \u0026plusmn; 0.97 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e30.41 \u0026plusmn; 2.98 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e9-Hexadecenoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC16:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e25.34 \u0026plusmn; 0.39 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e18.30 \u0026plusmn; 2.24 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC18:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e0.87 \u0026plusmn; 0.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e0.85 \u0026plusmn; 0.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e9-Octadecenoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC18:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e3.76 \u0026plusmn; 0.86 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e8.73 \u0026plusmn; 1.81 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e9,12-Octadecadienoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC18:2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e6.15 \u0026plusmn; 0.27 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e6.56 \u0026plusmn; 0.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e5,8,11,14-Eicosatetraenoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC20:4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e0.71 \u0026plusmn; 0.52 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e1.98 \u0026plusmn; 1.93 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.15302491103203%\"\u003e\n \u003cp\u003e5,8,11,14,17-Eicosapentaenoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.658362989323843%\"\u003e\n \u003cp\u003eC20:5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.683274021352315%\"\u003e\n \u003cp\u003e20.05 \u0026plusmn; 0.36 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.505338078291814%\"\u003e\n \u003cp\u003e31.04 \u0026plusmn; 0.23 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDifferent letters (a and b) represent a significant difference between the different culture media (p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eValidation of the cultivation medium on pilot scale\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cultivations carried out in FVAs maintained a salinity of 20 and an average pH of 8.4 \u0026plusmn; 0.62. They were initiated with an average initial CD of 2,443.33 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 109.11 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e, and the batch cultivation lasted an average of 9 days, after which harvests and dilutions began. These harvests occurred every 6-8 days, and \u0026micro; was calculated during these intervals, resulting in 0.12 \u0026plusmn; 0.03 d\u003csup\u003e-1\u003c/sup\u003e. The cultures were maintained for an average of 23 days, and the MCD achieved was 5,031.67 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 429.74 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e. The average of DB and CD obtained in the harvests were 0.34 \u0026plusmn; 0.01 g L\u003csup\u003e-1\u003c/sup\u003e and 4,350.00 x 10\u003csup\u003e4\u003c/sup\u003e \u0026plusmn; 849.79 x 10\u003csup\u003e4\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e, respectively.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSalinity is one of the crucial factors in the cultivation of marine microalgae, and there are numerous studies evaluating its effects on the growth and biochemical composition of these microorganisms (Gu et al. 2012; Ishika et al. 2018; Pugkaew et al. 2019) . Changes in salinity induce osmotic stress on organisms, requiring them to undergo physiological adjustments to the new environment (Guo et al. 2019).\u003c/p\u003e\n\u003cp\u003eMost studies on this topic assess the effect of high salinities on the growth of \u003cem\u003eNannochloropsis\u003c/em\u003e. However, in the present experiment, the aim was to understand the impact of lower-than-usual salt concentrations, considering that artificial seawater contributes to increased cultivation costs.\u003c/p\u003e\n\u003cp\u003eNo difference in the growth of \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e was observed between salinities 30 and 10, except for the dry biomass productivity. Similar conclusions were drawn in studies conducted with \u003cem\u003eNannochloropsis\u003c/em\u003e sp., where reducing NaCl concentration from 27 to 13/13.5 g L\u003csup\u003e-1\u003c/sup\u003e did not negatively impact cultivation growth (Mart\u0026iacute;nez-Rold\u0026aacute;n et al. 2014) and even increased cell density after 7 days of cultivation (Pal et al. 2011). The species \u003cem\u003eN.\u003c/em\u003e \u003cem\u003esalina\u003c/em\u003e also exhibited higher maximum cell density at lower salinity (22 ups) compared to the traditionally used 34 ups (Bartley et al. 2013).\u003c/p\u003e\n\u003cp\u003eCultures at salinities 5 and 1 exhibited a significant reduction in growth. In this study, the microalga \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e had its growth rate halved at salinity 5; however, it was not completely inhibited, as observed at salinity 1. This characteristic is species-specific. For instance, according to Bartley et al. (2013), \u003cem\u003eN. salina\u0026nbsp;\u003c/em\u003edoes not grow at salinities below 8. In a study conducted by Zulkifli et al. (2018), \u003cem\u003eN. oculata\u0026nbsp;\u003c/em\u003ewas able to grow in a culture medium with zero salinity comparable to cultures with saline water, but only up to the 8th day. On the other hand, the strain \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoceania\u003c/em\u003e CCALA 804 demonstrates efficient growth in a medium with zero salinity, indicating tolerance to drastic osmotic reductions(Pal et al. 2013; Solovchenko et al. 2014).\u003c/p\u003e\n\u003cp\u003eConsidering the similarity between the cultures of the treatments with salinities 30 and 10, with a reduction only in productivity (0.027 vs. 0.017 g L\u003csup\u003e-1\u003c/sup\u003e d\u003csup\u003e-1\u003c/sup\u003e, respectively), an intermediate salinity (20) was chosen for the subsequent experiments.\u003c/p\u003e\n\u003cp\u003eIn the cultivation of marine microalgae, seawater is commonly used as the base of the culture medium by researchers and producers. However, there are factors that make its use impractical, such as variations in water quality due to tides, pollution, and climate, and especially the geographic and economic limitations for locations far from the sea (Berges et al. 2001; Venteris et al. 2013). It should be considered that coastal land is of high value and subject to greater climatic fluctuations. Additionally, seawater intake requires the installation and maintenance of equipment on the coast or offshore. Coastal intake is more economical but more susceptible to problems such as boat collisions, waves, and storms. On the other hand, offshore intake involves more expensive installation and maintenance (Huguenin and Colt 2002). An assessment conducted by Venteris \u003cem\u003eet al\u003c/em\u003e. (2013) in the United States concluded that the use of saline waters is a costly alternative compared to freshwater. These disadvantages and complications make artificial salinization an attractive option.\u003c/p\u003e\n\u003cp\u003eFor this purpose, there are several artificial sea salts in the market that fulfill the function of simulating seawater, providing salinity, trace elements, and buffers that contribute to water quality. However, these products have a high cost, and consequently, their use becomes impractical in large-scale microalgae production. In this context, after reducing the salt concentration in the culture medium of \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e, the substitution of artificial sea salt with common table salt was evaluated.\u003c/p\u003e\n\u003cp\u003eThe achieved values of maximum cell density, specific growth rate, final biomass, and biomass gain showed no significant difference between the types of salt. Therefore, replacing artificial sea salt with common table salt is not detrimental to the cultivation of \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e, maintaining the same levels of cell growth. The only parameter that showed a significant difference was pH, which likely remained higher in artificial sea salt due to the presence of carbonate salts. Nevertheless, it did not influence growth. Therefore, common salt becomes a viable alternative to seawater intake and, especially, a more economical option compared to artificial sea salt in massive production. While common salt costs around US$ 0.16/kg, sea salt is priced at US$ 2.43/kg, 15 times higher.\u003c/p\u003e\n\u003cp\u003eThe use of agricultural fertilizer as a nutrient source is a strategy for reducing the costs of the culture medium in large-scale microalgae production, including \u003cem\u003eNannochloropsis\u003c/em\u003e species (Camacho-Rodr\u0026iacute;guez et al. 2013; Liu and Bangert 2015; Neto et al. 2018). In the present study, only the nutrient source (medium) affected the final biomass, with the highest concentrations obtained with agricultural fertilizer (FA). The maximum cell density averages for FA were also higher than those achieved by F2, although not showing a significant difference. Therefore, the application of agricultural fertilizer is advantageous compared to the standard F2 medium. It is also worth noting that the interaction between salt and nutrient medium showed no effect on any microalgal growth parameter.\u003c/p\u003e\n\u003cp\u003eRegarding the production of lipids and fatty acids, the culture medium with sea salt showed a higher lipid content but a lower amount of EPA compared to the medium with common salt. From the ASAF to the CSAF treatment, there was a reduction of 6.31% and 7.04% in the fatty acids 16:0 and 16:1, respectively, while an 11% increase in EPA (C20:5) was observed. The sea salt applied in this study contains trace elements that possibly enhance lipid accumulation by \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e. It has been demonstrated that different levels of iron, zinc, manganese, and molybdenum in the culture medium significantly interfere with lipid storage in some species of microalgae, but their requirements are species-specific (Ghafari et al. 2018). In contrast, the depletion of calcium and magnesium has been shown to increase lipid content in \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eScenedesmus obliquus\u003c/em\u003e (Gorain et al. 2013).\u003c/p\u003e\n\u003cp\u003eSimilar to the total lipids, micronutrients influence the fatty acid profile. Savvidou et al. (2020) observed that the depletion of iron and manganese decreased lipid content but increased the levels of polyunsaturated fatty acids in \u003cem\u003eNannochloropsis\u003c/em\u003e oceanica, increasing the amount of EPA (C20:5) by 3.63% and 4.91%, while decreasing some saturated fatty acids. These results align with the findings of the present study. Therefore, it is essential to identify the micronutrients that favor the production of desired lipids and fatty acids by \u003cem\u003eNannochloropsis\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e to subsequently add them strategically to the medium with common salt.\u003c/p\u003e\n\u003cp\u003eWhen validating the cultivation medium in FVBs, the obtained data were lower than those presented earlier on a smaller scale. It is noticeable that there was a reduction in both the specific growth rate and the achieved dry biomass productivity. This change was expected since the configuration of the 2 L photobioreactor differs from the 100 L flat vertical bags. Changes in shape, aeration, luminosity, among other aspects, influence cultivation productivity. According to (Borowitzka and Vonshak 2017), scaling up microalgae production introduces important hydrodynamic changes, especially regarding the homogeneity of the culture, which is more challenging to achieve on a larger scale. Thus, light and nutrients are not uniformly distributed to all cells, leading to a reduction in productivity.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Despite the cultivation reaching lower growth in this stage of the study, other studies with the same genus show similar and even lower results than those found in the present experiment. When using inorganic fertilizers, \u003cem\u003eN.\u003c/em\u003e \u003cem\u003egaditana\u003c/em\u003e was able to achieve a higher biomass concentration (0.4 g L\u003csup\u003e-1\u003c/sup\u003e), but with a specific growth rate equal to 0.15 d\u003csup\u003e-1\u003c/sup\u003e on the 6th day of cultivation (Riveros et al. 2018), a value close to that found in the present work. Neto et al. (2018) obtained lower biomass concentrations of \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e using fertilizers, with a range from 0.12 to 0.20 g L\u003csup\u003e-1\u003c/sup\u003e. In contrast, the optimization of the culture medium by the response surface methodology led to the production of up to 0.58 g L\u003csup\u003e-1\u003c/sup\u003e of \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e UTEX 2164 in 9 days (Mehra and Jutur 2022), a higher value than that found in the present study. However, it is important to note that different scales and nutrient sources make it difficult to compare results.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Given the potential demonstrated by the CSAF medium for \u003cem\u003eN.\u003c/em\u003e \u003cem\u003eoculata\u003c/em\u003e production, some adjustments are necessary for larger-scale cultivation to achieve productivities close to those observed on a smaller scale. Therefore, more in-depth studies on agitation mode, luminosity (width of the flattened bag), cultivation operation, and the addition of micronutrients should be conducted.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, it was observed that the marine microalga \u003cem\u003eN.\u003c/em\u003e\u003cem\u003eoculata\u003c/em\u003e can grow in a cultivation medium with a salinity of 20, which is lower than the typically applied salinity. Additionally, it was possible to replace artificial sea salt and the standard nutrient medium with common salt and agricultural fertilizer without negatively impacting its growth, making the production of \u003cem\u003eN.\u003c/em\u003e\u003cem\u003eoculata\u003c/em\u003e more economically viable. It is worth noting that studies on the addition of micronutrients should be conducted later to ensure a good lipid composition of the microalga. Adjustments in the configuration of the flat vertical bags photobioreactor of 100 L are also necessary to achieve growth parameters closer to those obtained on a small scale.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;National Council for Scientific and Technological Development (CNPq Project 24556/2021-9), also the Santa Catarina State Foundation for Research and Innovation Support (FAPESC 2023TR302).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report there are no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u0026aacute;bio de Farias Neves:\u003c/strong\u003e Conceptualization, Investigation, Validation, Writing \u0026ndash; original draft, Supervision, Project administration. \u003cstrong\u003eRafael de Oliveira Jaime Sales:\u003c/strong\u003e Conceptualization, Methodology, Investigation, Formal analysis, Experimental work, Writing \u0026ndash; original draft. \u003cstrong\u003eIsadora Kaniak Ikeda:\u003c/strong\u003e Methodology, Investigation, Formal analysis, Experimental work, Writing \u0026ndash; original draft. \u0026nbsp;\u003cstrong\u003eAna Carolina de Souza Santos:\u003c/strong\u003e Experimental work. \u003cstrong\u003eAna Flavia Celso Duarte:\u003c/strong\u003e experimental work. \u003cstrong\u003eRicardo Camilo Martins:\u003c/strong\u003e Experimental work. \u003cstrong\u003eRosana de C\u0026aacute;ssia de Souza Schneider:\u003c/strong\u003e Formal analysis, Review \u0026amp; Editing. \u0026nbsp;\u003cstrong\u003eDaniel Pedro Willemann:\u003c/strong\u003e Review \u0026amp; Editing, Project administration. All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank the National Council for Scientific and Technological Development (CNPq Project 24556/2021-9), also the Santa Catarina State Foundation for Research and Innovation Support (FAPESC 2023TR302).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBartley ML, Boeing WJ, Corcoran AA, Holguin FO, Schaub T (2013) Effects of salinity on growth and lipid accumulation of biofuel microalga \u003cem\u003eNannochloropsis salina\u003c/em\u003e and invading organisms. Biomass Bioenergy 54:83\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerges JA, Franklin DJ, Harrison PJ (2001) Evolution of an artificial seawater medium: improvements in enriched seawater, artificial water over the last two decades. 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Eur J Lipid Sci Technol 116:635\u0026ndash;644.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVenteris ER, Skaggs RL, Coleman AM, Wigmosta MS (2013) A GIS cost model to assess the availability of freshwater, seawater, and saline groundwater for algal biofuel production in the United States. Environ Sci Technol 47:4840\u0026ndash;4849.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZulkifli AF, Ramli A, Lim JW, Lam MK (2018) Effect of NaNO\u003csub\u003e3\u003c/sub\u003e and NaCl concentration on \u003cem\u003eNannochloropsis oculata\u003c/em\u003e cell biomass and FAME composition for biodiesel production. J Phys Conf Ser 1123:012071.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"microalgae, aquaculture, salinity, fertilizer, fatty acids","lastPublishedDoi":"10.21203/rs.3.rs-4102702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4102702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroalgae of the genus \u003cem\u003eNannochloropsis\u003c/em\u003e are highly valued in aquaculture due to their protein, fatty acid, and carotenoid content, offering potential as additives or substitutes for fish meal or oil. However, large-scale production encounters challenges, particularly concerning the culture medium. Thus, this study aimed to assess the impact of substituting artificial sea salt with common salt and standard medium with agricultural fertilizer in \u003cem\u003eNannochloropsis oculata\u003c/em\u003e production. Initially, the effects of reduced salinity were evaluated at salinities of 30, 10, 5, and 1 ups, followed by an examination of the effects of replacing artificial sea salt with common salt and using agricultural fertilizer on microalgae growth. Salinities of 30 and 10 exhibited the highest growth parameters. The salt source had no significant impact on culture growth, while the agricultural fertilizer enhanced it. Common salt increased the concentration of eicosapentaenoic acid compared to artificial sea salt, although the total lipid concentration was higher in microalgae cultured with artificial sea salt. The developed medium was validated through semi-continuous cultures in 100 L flat vertical bags, showing to be an economically viable alternative without hindering \u003cem\u003eN. oculata\u003c/em\u003e growth.\u003c/p\u003e","manuscriptTitle":"Alternative culture medium for Nannochloropsis oculata mass culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-21 09:13:40","doi":"10.21203/rs.3.rs-4102702/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"422e0d28-31ad-4ef2-9423-ee627b1d2553","owner":[],"postedDate":"March 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-17T02:44:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-21 09:13:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4102702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4102702","identity":"rs-4102702","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}