Headnote
ABSTRACT
Objective: The objective of this work was to evaluate the use of sludge generated during the culture of Arapaima gigas as an alternative medium for Nannochloropsis sp.
Theoretical Framework: Microalgae are photosynthetic micro-organisms with the ability to transform organic waste or effluents into valuable biomass. They are currently of great interest for their use in the food and pharmaceutical industry. Nannochloropsis sp. is a valuable microalga due to its excellent lipid profile.
Method: The sludge used was collected from A. gigas culture ponds at the CITE Acuícola Pesquero Ahuashiyacu (Tarapoto-Peru). An aqueous extract of sludge (EAL) was obtained from the sample by a thermal procedure, which was dosed into the cultures at concentrations of 100, 150 and 200 mL/L. The experimental units followed the traditional batch culture arrangement under laboratory conditions. Guillard F/2 medium was used as control treatment. The characterisation of metals in the medium was performed by coupled plasma induction (ICP) and the determination of ammonium and nitrite by colorimetric tests. In addition, population density, productivity and total lipid content were estimated.
Results and Discussion: Chemical analyses revealed high concentrations of calcium, sodium, magnesium, iron, potassium, phosphorus, ammonium and nitrite (655, 648, 283, 67, 32, 17, 1.5, and 10 ppm, respectively). Likewise, the best growth (3590.00 ± 91.24 x 104 cells/mL) and productivity (0.69 ± 0.01 g/L) was obtained with 200 mL/L of EAL, presenting significant statistical differences (p < 0.05) with respect to the other treatments. In contrast, the highest lipid production (29.67 ± 1.53 %) was observed in the control treatment, far exceeding the EAL cultures; however, no significant statistical difference was observed between EAL treatments.
Research Implications: These results demonstrate the feasibility of using EAL as an alternative medium for the production of Nannochloropsis sp. with a positive environmental and economic impact on the production of A. gigas.
Originality/Value: The use of sludge generated in fish culture, in particular A. Gigas, has not been reported previously. In this study, we demonstrate for the first time the feasibility of using this sludge in the culture of the microalga Nannochloropsis sp. which also has a high potential for use in fish larviculture.
Keywords: Microalgae, Nannochloropsis, Lipids, Sludge, Paiche.
RESUMO
Objetivo: O objetivo deste trabalho foi avaliar o uso do lodo gerado durante o cultivo de Arapaima gigas como meio alternativo para Nannochloropsis sp.
Referencial Teórico: As microalgas são microrganismos fotossintéticos com a capacidade de transformar resíduos orgânicos ou efluentes em biomassa valiosa. Atualmente, são de grande interesse para a sua utilização na indústria alimentar e farmacêutica. A Nannochloropsis sp. é uma microalga valiosa devido ao seu excelente perfil lipídico.
Método: O lodo utilizado foi coletado dos tanques de cultura de A. gigas no CITE Acuícola Pesquero Ahuashiyacu (Tarapoto-Peru). Um extrato aquoso de lodo (EAL) foi obtido da amostra por um procedimento térmico, que foi dosado nas culturas em concentrações de 100, 150 e 200 mL/L. As unidades experimentais seguiram o esquema tradicional de cultura em lote em condições laboratoriais. O meio Guillard F/2 foi utilizado como tratamento de controlo. A caraterização dos metais no meio foi realizada por indução de plasma acoplado (ICP) e a determinação de amónio e nitrito por testes colorimétricos. Além disso, foram estimados a densidade populacional, a produtividade e o teor de lípidos totais.
Resultados e Discussão: As análises químicas revelaram elevadas concentrações de cálcio, sódio, magnésio, ferro, potássio, fósforo, amónio e nitrito (655, 648, 283, 67, 32, 17, 1,5 e 10 ppm, respetivamente). Da mesma forma, o melhor crescimento (3590,00 ± 91,24 x 104 células/mL) e produtividade (0,69 ± 0,01 g/L) foi obtido com 200 mL/L de EAL, apresentando diferenças estatísticas significativas (p < 0,05) em relação aos demais tratamentos. Em contraste, a maior produção de lípidos (29,67 ± 1,53 %) foi observada no tratamento de controlo, excedendo largamente as culturas EAL; no entanto, não foi observada qualquer diferença estatística significativa entre os tratamentos EAL.
Implicações da Pesquisa: Estes resultados demonstram a viabilidade da utilização de EAL como meio alternativo para a produção de Nannochloropsis sp. com um impacto ambiental e económico positivo na produção de A. gigas. Originalidade/Valor: O uso do lodo gerado no cultivo de peixes, em particular A. Gigas, não foi relatado anteriormente. Neste estudo, demonstrámos pela primeira vez a viabilidade de utilização destas lamas na cultura da microalga Nannochloropsis sp. que também tem um elevado potencial de utilização na larvicultura de peixes.
Palavras-chave: Microalgas, Nannochloropsis, Lípidos, Lodo, Paiche..
RESUMEN
Objetivo: El objetivo de este trabajo fue evaluar el uso del lodo generado durante el cultivo de Arapaima gigas como un medio alternativo para Nannochloropsis sp.
Marco Teórico: Las microalgas son microotganismos fotosintéticos con capacidad de transformar residuos orgánicos o efluentes en biomasa de valor. En la actualidad tienen gran interés por su uso en la industria alimentaria y farmacéutica. Nannochloropsis sp. es una microalga de gran valor por su excelente perfil lipídico.
Método: El lodo utilizado se recolectó en los estanques de cultivo de A. gigas en el CITE Acuícola Pesquero Ahuashiyacu (Tarapoto-Perú). Mediante un procedimiento térmico de la muestra se obtuvo un extracto acuoso de lodo (EAL), que se dosificó en los cultivos en concentraciones de 100, 150 y 200 mL/L. Las unidades experimentales siguieron el arreglo tradicional de los cultivos tipo batch, bajo condiciones de laboratorio. Como tratamiento control se empleó medio Guillard F/2. La caracterización de metales en el medio se realizó por inducción de plasma acoplado (ICP) y la determinación amonio y nitritos por pruebas colorimétricas. Además, se estimó la densidad poblacional, productividad y el contenido de lípidos totales.
Resultados y Discusión: Los análisis químicos revelaron altas concentraciones de calcio, sodio, magnesio, hierro, potasio, fosforo, amonio y nitritos (655, 648, 283, 67, 32, 17, 1.5 y 10 ppm, respectivamente). Asimismo, el mejor crecimiento (3590.00 ± 91.24 x 104 cel/mL) y productividad (0.69 ± 0.01 g/L) se obtuvo con 200 mL/L de EAL, presentando diferencias estadísticas significativas (p < 0.05) respecto a los demás tratamientos. En contraste, la mayor producción de lípidos (29.67 ± 1.53 %) se observó en el tratamiento control, superando ampliamente a los cultivos con EAL; sin embargo, no se observó diferencia estadística significativa entre tratamientos con EAL.
Implicaciones de la investigación: Estos resultados demuestran la factibilidad de usar el EAL como medio alternativo para la producción de Nannochloropsis sp., con un impacto positivo en términos ambientales y económicos en la producción de A. gigas.
Originalidad/Valor: El uso de lodos que se generan en el cultivo de peces, en particular de A. Gigas, no ha sido reportado previamente. En este estudio, por primera vez demostramos la factibilidad del uso de estos lodos en el cultivo de la microalga Nannochloropsis sp., la cual además tiene un alto potencial para su uso en la larvicultura de peces.
Palabras clave: Microalgas, Nannochloropsis, Lípidos, Lodos, Paiche.
(ProQuest: ... denotes formulae omitted.)
1 INTRODUCTION
Microalgae, due to their ability to convert organic substances into biomass, offer a promising way to mitigate the environmental impacts caused by the accumulation of organic waste, such as sludge from Arapaima gigas cultivation ponds (Dolganyuk et al., 2020). These wastes, mainly composed of food residues and excrement of the organisms in culture, cause great concern due to their non-treatment and their constant disposition to natural water bodies, which end up being eutrophised since these organic wastes contain the high levels of nitrogen and phosphorus.
The transformation of sludge into useful biomass through the cultivation of microalgae is a viable proposal with a view to reducing the adverse environmental impacts and costs associated with the production of microalgae, since such biomass can also be reused in the cultivation of other organisms. In addition, microalgal technology offers opportunities for the production of substances of economic interest, such as proteins, pigments, lipids and other biocompounds demanded in the aquaculture, industry, energy and food industries (Kandasamy et al., 2022; Roy et al., 2022). It should be noted that microalgae production is still constrained due to high production costs compared to traditional foods (Ahmad & Ashraf, 2023). That is why the development of alternative means for the mass production of microalgae is an attractive option.
The need also arises to address the environmental pollution problems caused by various human activities, such as aquaculture, with the aim of developing more sustainable practices and with a reduced ecological footprint. The cultivation of A. gigas, an important species for the economy and food in the Amazon region, has experienced a notable increase in production in recent years (Gamarra, 2022), but faces little-studied environmental challenges, such as the accumulation of sludge in crop ponds
To address this problem, it is proposed to investigate the effect of the aqueous extract of A. gigas sludge on population growth and lipid content of the microalgae Nannochloropsis sp. In this research, we evaluated different concentrations of this extract to understand its impact on the dynamics of Nannochloropsis sp. culture and lipid production.
2 THEORETICAL FRAMEWORK
Microalgae are photosynthetic microorganisms that grow in marine and continental aquatic environments (Liu & Ruan, 2022). For many years, they have been used in both human and animal diets; however, in recent decades, their cultivation has been oriented towards obtaining nutritional supplements, such as proteins, lipids and antioxidants, in addition to their use to treat organic waste and effluents, as well as in the production of biofertilizer (Roldán, 2022).
493 species of microalgae with high potential for mass production have been described (Segoviano, 2017). It has also been documented that certain species of microalgae represent a highly valuable food resource for humans. Notable examples include Arthrospira flours (Villaró-Cos et al., 2024) and Scenedesmus (Venkataraman et al., 1997), which stand out for their high protein content and food safety. Also, some species of microalgae, such as Chaetoceros and Thalassiosira, have been highlighted for their usefulness as food for larvae of aquatic organisms, while Dunaliella has commercial interest for the production of antioxidants. Finally, certain species of microalgae, such as Gymnodinium, have been linked to episodes of red tides, which can cause the death of both aquatic organisms and man (Medina et al., 2012).
The microalgae Nannochloropsis sp. is considered a promising species for industrial applications for its ability to accumulate high levels of polyunsaturated fatty acids (Assaf, 1989). This microalgae belongs to the class Eustigmatophyceae, which includes the species that exhibit the highest concentration of polyunsaturated fatty acids (PUFAs), among which eicosapentaenoic acid (EPA), arachidonic acid (ARA) and docosahexaenoic acid (DHA) stand out. These fatty acids have a considerable importance in the nutrition of marine organisms, playing a fundamental role in the growth and development of fish larvae, molluscs and crustaceans (Otero et al., 1997; Brown et al., 1999).
On the other hand, A. gigas is one of the five species of tropical freshwater fish that are part of the Arapaimidae family: A. gigas (Schinz, 1822), A. agassizii (Valenciennes, 1847), A. mapae (Valenciennes, 1847), A. arapaima (Valenciennes, 1847) and A. leptosoma (Stewart, 2013) (IIAP, 2017). In Peru, this species inhabits the basins of the Napo, Putumayo, Marañón, Pastaza and Ucayali rivers, with greater abundance in the Pacaya-Samiria National Reserve (Alcántara et al., 2006). Its cultivation is carried out in ponds of 300 to 500 m2, ponds of clay soil (Chu et al., 2017), where after being cultivated large volumes of sludge are generated.
The sludge that comes from these ponds originates from the organic matter that settles at the bottom (food and feces remains), forming layers where anaerobic processes occur. When organic matter increases rapidly in ponds, it settles and decomposes, through a mineralization process that is carried out by fermenting, denitrifying, sulfate-reducing or methane-producing bacteria (Jørgensen, 1982). This results in poor water quality that can harm both growing organisms and the environment. For this reason, it is necessary to carry out periodic removals of the sludge. However, these mineralized elements of the sludge can be reused to generate culture media, as has been described in various works using microalgae cultures.
Microalgae cultures that use organic media, such as sludge, induce modifications in the metabolism of these cells by activating mechanisms related to the synthesis of pigments or lipids (Cañavate, 2011). With regard to the use of sediments derived from fisheries or human activities, Thain (1992), tested Tetraselmis suecica using sludge samples obtaining densities of 25 to 45 x 104 cél/mL on the fifth day and 40 to 160 x 104 cél/mL on the ninth day. The results obtained support the efficacy of using sediments in the production of microalgae.
In addition, it should be considered that the cultivation of microalgae at the laboratory level requires the use of a nutrient medium with an appropriate formulation, usually composed of high purity mineral salts and essential nutrients such as nitrogen, phosphorus, potassium, iron, among others (Paniagua et al., 1986). Guillard's F/2 medium (1975) is best known for being very efficient for many microalgae species (Torrentera & Tacón, 1989). However, this type of commercial culture medium becomes impracticable for its large-scale implementation in the production of microalgae due to its high cost (Martínez, 2003). Furthermore, cultivation media have been developed that can be produced using agricultural fertilizers or by-products such as molasses, organic extracts and biodigested residues, among others. In addition, some media can be prepared from by-products generated by industries, such as sugar, poultry, fisheries or aquaculture (Hernández et al., 2003).
The incorporation of these types of media in the cultivation of microalgae entails a notable decrease in production costs, since their obtaining is accessible and simple (Fernández & Paredes, 2007; Merino & Encomendero, 2012), prior physical, chemical or biological treatment, in order to avoid the presence of unwanted microorganisms. The effectiveness of these culture media is attributed to the versatility of microalgae to take advantage of nutrients, both organic and inorganic, found in such residues. This capacity is due to the ecological plasticity characteristics of the microalgae, which allow them to adapt quickly to these conditions (Fernández & Paredes, 2007).
3 METHODOLOGY
3.1 OBTAINING THE SLUDGE AND PREPARING THE EXPERIMENTAL ENVIRONMENT
The sludge from the A. gigas crop was collected at the CITE Acuícola Pesquero Ahuashiyacu, located in Tarapoto, Peru. Then, it was transported by air to the Laboratory of Evaluation of Aquatic Resources and Auxiliary Species Crops of the National University of Santa Fe (Nuevo Chimbote), in a sealed plastic container hermetically at room temperature. In the laboratory, 1 kg of wet sludge was weighed and 2 L of drinking water was added. This mixture was brought to a boil for 30 minutes, stirring constantly to facilitate the extraction of the nutrients present in the sludge. After this process, the mixture was allowed to rest for a day in refrigeration, in order to allow the separation of the fine particles from the aqueous phase. Finally, the supernatant was separated from the solid phase, which we call aqueous extract of paiche sludge (EALP) and preserved at 4 °C until its use as a culture medium (Figure 1).
3.2 CHEMICAL CHARACTERIZATION OF THE EXPERIMENTAL ENVIRONMENT
An inductively coupled plasma spectroscopy (ICP) analysis of a sample from the experimental medium was carried out. This method was used for the purpose of quantifying chemical elements present in the culture medium. These analyzes were carried out using a sample of 500 mL from the EALP, which was analyzed in the certified laboratories of the company COLECBI SAC. In addition to this, the ammonium and nitrite levels in the medium were quantified using a colorimetric kit of the brand PRO-JBL AQUATEST LAB. This kit has a measurement range of 0 to 2 ppm for ammonium, with a sensitivity of 0.25 ppm, and a measurement range of 0.5 to 220 ppm for nitrates, with a sensitivity of 1.0 ppm.
3.3 EXPERIMENTAL DESIGN
The research was carried out with an experimental design of increasing stimulus (Table 1), evaluating three experimental treatments together with a control treatment. These treatments were selected based on pre-tests performed in the laboratory and worked in triplicate to obtain average results. The objective was to cultivate the microalgae Nannochloropsis sp. using a standard medium (HM) as control, as well as different concentrations of EALP (100, 150 and 200 mL/L).
HM = Heussler-Merino.
3.4 EXPERIMENTAL CROPS
The microalgae Nannochloropsis sp. was supplied by the Laboratory of Auxiliary Species Cultivation of the E.A.P. of Biology in Aquaculture of the National University of Santa Fe. This strain was isolated from the Salaverry wetlands (Trujillo, Peru) and maintained in 2% agar crops on Petri dishes. To start the cultures, the strain was seeded in a liquid medium and scaled as shown in Figure 2. This process was carried out in order to obtain a suitable inoculum size to start the experiments.
During the scaling process in liquid medium, the culture units were kept in constant agitation by aeration provided by a 1 HP blower. In addition, they were exposed to a photoperiod of 24:0, using a 40 watt fluorescent light source that emitted approximately 1500 Lux of light intensity. The culture medium used for both strain maintenance and scaling processes was the HM medium, the components of which are described in Table 2.
The experimental cultures were carried out in glass bottles of 300 mL total volume, with an effective volume of 250 mL. Each culture was inoculated with a ratio of 2 parts inoculum per 10 parts culture medium (v/v). Initial cell density was estimated using a Nikon optical microscope with a Neubauer camera. Constant lighting and shaking was provided as described above.
3.5 DETERMINATION OF POPULATION GROWTH
Crop population growth was determined by daily counts with an optical microscope and a Neubauer camera. To do this, a sample of the algal suspension of each treatment was taken with the help of a Pasteur pipette, then the sample was fixed in the microscope and the counts were made to a 40 X target. Cell density calculations (cell/mL) were performed with the following formula:
... (1)
where:
P is the average number of cells in the counted fields,
D is the dilution and 104 is the constant used to express the volume in mL.
The following formulas were used to determine the population growth rate (µ) and doubling time (TD):
... (2)
... (3)
where:
Nf y Ni representan el número de células final e inicial, respectivamente, y tf y ti representan el tiempo final e inicial, respectivamente.
3.6 DETERMINATION OF TOTAL BIOMASS AND LIPIDS
We used the gravimetric method to determine biomass, which consists of centrifuging a 50 mL sample of the culture at 4000 rpm for 10 minutes in an Eppendorf tube. Afterwards, the supernatant is separated and the resulting pellet is dried in an oven at 70 °C for 5 hours. The biomass is calculated as the difference in weight between the tube with the pellet and the tube alone.
Total lipids were quantified using a one-step extraction procedure (Axelsson & Gentili, 2014). To do this, 15 mL samples were taken from each culture and placed in previously weighed test tubes, then centrifuged at 4000 rpm for 10 minutes. After centrifugation, the liquid phase was removed to obtain the pellet. The pellet was dried in an oven at 55 °C overnight and then weighed to calculate biomass. Next, 3 mL of extraction solution (chloroform and methanol; 2:1 v/v) was added to each sample, shaken vigorously, and allowed to stand for 5 hours. The mixture was centrifuged again, the supernatant was decanted and the pellet was dried again to record the new weight. The lipid content was estimated as a percentage (%) based on the difference in weights before and after the extraction process.
3.7 STATISTICAL ANALYSIS
Statistical analysis was carried out using the average growth and lipid content data to determine possible significant differences between treatments. The normality of the data was determined and a one-way analysis of variance (ANOVA) was applied, followed by Tukey's test as a post-test, with a significance level of 95%. The analysis was performed using SPSS version 20 software.
4 RESULTS AND DISCUSSIONS
4.1 CHEMICAL CHARACTERIZATION OF THE EXPERIMENTAL MEDIUM
The chemical analysis of the experimental medium (EALP) focused on the key nutrients for microalgae cultivation. The results show that calcium and sodium are the predominant elements, with concentrations of 655 ppm and 648 ppm respectively. Magnesium is also present in a significant concentration, at 283 ppm. In addition, significant levels of iron (67 ppm) and phosphorus (17 ppm) were found, essential for crop growth. It is important to note that aluminum and boron should not be carefully considered, as they could have been incorporated by dislodgement from preparation and storage containers. Ammonium and nitrate levels were 1.5 ppm and 10.0 ppm respectively. Other elements, although important, are present in minimal concentrations (Figure 3). These results demonstrate that the EALP medium provides the necessary nutrients for the growth of microalgae.
4.2 POPULATION GROWTH
In our experiment, all experimental treatments demonstrated growth with EALP medium. On the first day, a rapid disparity in population growth was observed, where treatment with 150 mL/L EALP showed the highest growth (151.67 ± 7.64 x 104 cél/mL) compared to the control and other experimental treatments. However, all cultures experienced a lag phase until the third day (Figure 4).
For the fourth day, a marked difference was observed in the growth of the cultures, highlighting the treatment with 200 mL/L of EALP, which exhibited the highest population growth (1705.00 ± 15.00 x 104 cél/mL), surpassing the control and others. experimental treatments. After the fourth day, we observed that the EALP-dosed treatments outperformed the control. The greatest population growth (3590.00 ± 91.24 x 104 cél/mL) was recorded on day 7 with the 200 mL/L treatment, being statistically significant (p < 0.05) compared to the other treatments. Furthermore, treatment with 150 mL showed similar growth (3407.67 ± 17.21 x 104 cél/mL). In contrast, the lowest population growth (2195.00 ± 45.83 x 104 cél/mL) was observed in the control treatment (HM). It is important to highlight that the experimental cultures showed a more intense green pigmentation than the control treatment, this difference being most notable on day 7 (Figure 4).
On the other hand, the highest duplication rate of Nannochloropsis sp. was reached with 200 mL/L of EALP (0.758 ±0.004), while the lowest was recorded in the HM medium (0.676 ±0.003), which coincides with our observations of population density. It is important to highlight that treatment with 150 mL/L of EALP showed a slightly lower performance, although statistically similar to that of 200 mL/L. Furthermore, the shortest doubling times were observed in the EALP treatments (Table 3).
The results show that EALP medium with 200 mL/L favors greater growth of Nannochloropsis sp. compared to the control medium (HM). This is attributed to its superior nutrient content, such as calcium, magnesium, iron, phosphorus and nitrogen. It was observed that the highest growth rate was obtained with 200 mL/L of EALP, while HM medium showed the lowest rate. Treatment with 150 mL/L of EALP was also effective, although slightly statistically lower than that with 200 mL/L. These findings suggest that the optimal concentration of extract for the growth of Nannochloropsis sp. It is between 150-200 mL/L.
In another study, an aqueous extract of mud from El Ferrol Bay has been prepared as a culture medium for the microalgae Spirulina platensis (Menacho & Vera, 2019). With this medium, cultures were dosed at 20, 30 and 40 mL/L, showing that the highest biomass production (0.842 ± 0.055 g/L) was obtained with the treatment of 30 mL/L of EAL. From this work it is inferred that greater growth is associated with a greater concentration of nutrients. These nutrients are limiting for the growth and reproduction of microalgae (AlFadhly et al., 2022).
In contrast, cultures of Nannochloropsis sp. showed a lower biomass production, reaching its maximum value (0.69 ± 0.01 g/L) with 200 mL/L of EALP. This difference is attributed not only to the medium and nutrients, but also to the size of the microalgae, which influences biomass yields (Prieto et al., 2005). Furthermore, it is important to highlight that the size of the microalgae significantly impacts the harvesting process (Roy & Mohanty, 2019).
In another study carried out with D. salina, the effect of the concentration of Argopecten purpuratus sludge extract (EALCA) on the population growth of this microalgae was evaluated. It was found that the culture medium exerts a significant influence on its growth. Higher concentrations of EALCA (40 and 60 mL/L) promoted greater population growth compared to lower concentrations (20 mL/L). These concentrations were also correlated with the levels of nutrients present in the medium, mainly nitrogen (6 ppm), phosphorus (0.002 ppm) and iron
(0.008 ppm), which were lower than the levels supplied in our experiment with EALP. Furthermore, it was observed that the population growth rate of D. salina was higher in cultures with higher concentrations of EALCA (López & Pantoja, 2016).
Likewise, Vereau & Quiroz (2015) investigated the cultivation of S. acutus with aqueous extract of mud from Ferrol Bay, finding that a low concentration of EAL (10 mL/L) increases the percentage of lipids due to the lack of nitrogen, although a lower population density and biomass were also observed. In contrast, a higher concentration of EAL (20 mL/L) results in a lower lipid percentage, but with a higher biomass. In our Nannochloropsis sp. cultures, the same relationship is not observed due to its highly active lipid metabolism. Despite this, we noticed lower concentrations of lipids with the experimental medium, which suggests a higher content of proteins and/or pigments, possibly related to the organic compounds of the paiche waste.
4.3 BIOMASS AND TOTAL LIPIDS
The results of biomass production (g/L) of the crops show significant differences between the treatments with the HM and EALP medium. The lowest amount of biomass (0.51 ±0.01 g/L) was obtained with the HM medium; while, treatments with EALP showed notably higher levels. Specifically, the treatment with 100 mL/L reached a biomass of 0.56 ±0.02 g/L, the 150 mL/L treatment recorded 0.66 ±0.01 g/L, and the 200 mL/L treatment reached the maximum value with a biomass of 0.69 ±0.01 g/L (Figure 5).
Likewise, significant differences were observed in total lipid levels. The HM treatment showed an average lipid content of 29.67 ± 1.53% (g/g dry biomass), while the EALP treatments exhibited lower values: 25.00 ± 4.00%, 24.40 ± 1.64% and 24.67 ± 0.58% for concentrations of 100, 150 and 200 mL/L of EALP, respectively.
Various studies have shown that Nannochloropsis sp. has the ability to accumulate a significant amount of lipids, mainly PUFAS, such as ω-3 and ω-6 (Ma et al., 2016), making it a promising option for the production of biofuels and other lipid-derived products. Rodolfi et al. (2009), showed that Nannochloropsis sp. It has a high lipid production potential under outdoor growing conditions. It was estimated that this microalgae could produce up to 20 tons of lipids per hectare in a Mediterranean climate and more than 30 tons of lipids per hectare in sunny tropical areas. These performances could be improved with the EALP.
Además, Hu et al. (2008), señalaron que muchas microalgas tienen la capacidad de producir cantidades sustanciales de triacilgliceroles (TAG), que son lípidos de almacenamiento, bajo condiciones de estrés fotooxidativo u otras condiciones ambientales adversas. Nannochloropsis sp. es una microalga que, a pesar de tener un genoma corto, posee genes seleccionados para la biosíntesis de lípidos, reportado hasta en 6 especies de este género (Wang et al., 2014); por lo cual, ocasionar estrés, como deficiencia de nitrógeno o fosforo, puede incrementar aún más la acumulación de lípidos.
Regarding the influence of nutrients on the lipid content of Nannochloropsis sp., Wu et al. (2023) found that phosphate concentration affects both cell growth and lipid accumulation, with the limit being 8 mg/L and increasing yields with subsequent phosphate supplementation, with 1, 2 and 3 mg/L starting at day 4; observing a gradual increase from 27.45 to 46.15%, higher than the results due to phosphorus supplementation. In our study, using 100 mL/L EALP, a greater production of total lipids of 25.0 ±4.00% was achieved, however, the control treatment with HM 29.67 ±1.53%, being statistically different (p<0.05) with the experimental treatments. This is because the HM medium provides a high amount of nitrogen, phosphorus, iron and potassium; balanced and effective for the cultivation of microalgae. This microalga shows a high potential for lipid production, making it a promising option for the production of biofuels and other lipid-derived products. However, more research is required to optimize culture conditions and improve lipid productivity of Nannochloropsis sp.
4.4 PHYSICOCHEMICAL PARAMETERS
The crops were monitored and it was observed that there were no notable differences in temperatures, which ranged between 27.65 °C maximum and 26.40 °C minimum. However, it was noted that the pH of the cultures tends to increase as Nannochloropsis sp. grows. This increase was most pronounced in the 200 mL/L EALP treatment, but was observed in all cultures as the microalgae population grew (Figure 6).
Regarding pH, Nannochloropsis sp. shows a high population density at pH=8.5 (Khatoon et al., 2014). Significant variations in this value result in a decrease in population density and lipid production. However, it has been evaluated that the pH-shift effect in N. oculata increases lipid yields by 70-80%, values significantly higher than in other microalgae species (Cavonius et al., 2015). ). The EALP medium, being alkaline, helps to have pH values close to the optimal, compared to the HM medium (control) which decreases the pH. For this reason, it is necessary to carefully control the pH levels of the medium, as this would help maximize the production of Nannochloropsis sp.
5 CONCLUSION
In conclusion, the EALP medium is characterized by containing high concentrations of calcium, sodium, magnesium, iron, phosphorus, nitrogen and zinc, among other micronutrients. Its use has been shown to significantly promote population growth of Nannochloropsis sp. The optimal concentration of EALP to obtain the highest cell density was 200 mL/L, reaching a density of 3590.00 ± 91.24 x 104 cél/mL on day 7. In addition, the EALP medium favors greater biomass production, being the most high (0.69 ± 0.01 g/L) at 200 mL/L, while at 100 mL/L a higher lipid concentration was observed. These findings suggest that the EALP medium provides the necessary nutrients to maintain the culture of Nannochloropsis sp., being a viable alternative for the mass production of this microalgae, with the additional advantage of reusing the sludge from the A. gigas ponds, thus reducing its negative effects on the environment.
ACKNOWLEDGMENT
We thank the teachers of the E.P. of Biology in Aquaculture at the National University of Santa, for their comments during the execution of this work.
References
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