NON-TRADITIONAL NUTRIENT MEDIA FOR MICROALGAE CULTIVATION

ABSTRACT

This study is based on the selection of optimal nutrient media in the process of industrial cultivation of microalgae isolated from the territory of Uzbekistan. The green microalgae strains Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-15 were selected as the objects of the study. The growth characteristics, biomass yield and biochemical composition of these strains in nutrient media with different compositions were comparatively analyzed. According to the results of the study, the composition of the nutrient medium has a significant effect on the growth intensity of microalgae and the amount of biomass produced. It was found that the strains produced different amounts of biomass in each nutrient medium, and this process was closely related to the amount of nitrogen, phosphorus and potassium elements in the nutrient medium. The results obtained are of significant scientific and practical importance for the industrial cultivation of local microalgae and the management of their biomass composition in the conditions of Uzbekistan.

АННОТАЦИЯ

Данное исследование основано на подборе оптимальных питательных сред для процесса промышленного культивирования микроводорослей, выделенных на территории Узбекистана. Объектами исследования послужили штаммы зеленых микроводорослей Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 и Tetradesmus obliquus Sur-Uz-15. Проведен сравнительный анализ характеристик роста, выхода биомассы и биохимического состава этих штаммов в питательных средах различного состава. Согласно результатам исследования, состав питательной среды оказывает существенное влияние на интенсивность роста микроводорослей и количество производимой биомассы. Установлено, что штаммы продуцируют различное количество биомассы в каждой питательной среде, и этот процесс тесно связан с содержанием азота, фосфора и калия в среде. Полученные результаты имеют важное научно-практическое значение для промышленного культивирования местных штаммов микроводорослей и управления составом их биомассы в условиях Узбекистана.

Keywords: Tetradesmus obliquus, Planktochlorella nurekis, Chlorella sorokiniana, Tetradesmus obliquus, biomass, nitrogen, phosphorus, potassium.

Ключевые слова: Tetradesmus obliquus, Planktochlorella nurekis, Chlorella sorokiniana, Tetradesmus obliquus, биомасса, азот, фосфор, калий.

Introduction. In recent years, aquaculture and fisheries have developed rapidly, and global demand for fish products has increased dramatically. According to FAO, in 2022, aquaculture accounted for more than 50% of global fish production, with farmed fish exceeding wild fish for the first time [1, 2]. This situation makes the problem of creating an efficient and sustainable feed base in the fisheries sector even more urgent.

Feed is the main economic cost in intensive fish farming systems. Scientific sources indicate that fish feed accounts for 50–70% of total production costs in fish farms, and up to 75–90% in some intensive systems [3]. Fishmeal and fish oil, which are widely used in traditional fish feeds, are limited and non-renewable resources, and their production has a negative impact on marine and ocean ecosystems [4].

In this regard, microalgae are considered a promising, renewable and environmentally friendly source of live feed in fisheries. According to scientific research, the protein content of microalgae biomass can reach 40–60% and lipids up to 15–30%, which is higher than many traditional feed components.

Microalgae are rich in amino acids and fatty acids, which are important for fish larvae and young fish, and can successfully replace part of fishmeal [5, 7]. The green microalgae Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-15 selected in this study are promising species characterized by high biomass productivity, high accumulation of protein and lipids. The use of these microalgae as live feed in fisheries not only increases feeding efficiency, but also improves the ecological condition of fish farming reservoirs.

Microalgae are known to actively absorb excess nitrogen and phosphorus compounds in the aquatic environment. Studies have shown that in biological systems with the participation of microalgae, the content of nitrate and phosphate ions in water can be reduced by 30–60% [8,9]. This process reduces the risk of eutrophication in water bodies, improves the living conditions of fish, and helps prevent the spread of diseases.

However, the high biomass yield of microalgae and their biochemical composition directly depend on the nutrient medium selected for cultivation. The main macro elements in the nutrient medium - nitrogen, phosphorus and potassium - are important factors determining the growth rate of microalgae, the amount of biomass and the ratio of protein and lipids in the biomass [6, 7]. Taking into account the differences in the needs of different green microalgae species for these elements, it is an important scientific and practical task to determine the optimal nutrient medium for local strains isolated from the territory of Uzbekistan.

Therefore, this study aims to develop effective and sustainable live feed sources for the fisheries industry on a scientific basis by cultivating microalgae strains isolated in Uzbek conditions in nutrient media with different compositions, evaluating their biomass yield, and the amount of protein and lipids in the biomass.

Research materials and methods.

Research objects. The objects of the study were selected strains of green microalgae (division Chlorophyta) isolated from natural reservoir in Uzbekistan. The following microalgae were studied in the study: Tetradesmus obliquus, Planktochlorella nurekis, Chlorella sorokiniana, and Scenedesmus almeriensis (Fig.1). These microalgae are considered promising species, characterized by high growth rates, biomass yields, and the ability to accumulate proteins and lipids.

Microalgae cultivation conditions and nutrient medium.

In the research process, nutrient medium with two different compositions were used to cultivate the green microalgae Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-15. The selection of nutrient medium was aimed at satisfying the physiological needs of microalgae, increasing photosynthetic activity and ensuring maximum biomass yield. The macro and microelements contained in these media play an important role in cell division, metabolic processes and the synthesis of biochemical substances [5, 6]. As the first option, Chu–13 nutrient medium was used. This medium is considered one of the classic and balanced media for growing green microalgae in laboratory conditions [10, 6]. The composition of the nutrient medium consisted of the following components (g/L): KNO₃– 0.2; K₂HPO₄– 0.04; MgSO₄×7H₂O– 0.1; CaCl₂×6H₂O– 0.08; iron citrate– 0.01; citric acid – 0.1. Microelements were added in ppm: boron  0.5; MnSO₄×7H₂O – 0.5; CuSO₄×5H₂O – 0.02; CoCl₂×2H₂O – 0.02; Na₂MoO₄×2H₂O– 0.02. The initial pH of the nutrient medium was adjusted to 7.5. The Chu–13 medium is supplied with nitrogen and phosphorus sources, ensuring stable cell growth and active functioning of the photosynthetic apparatus. As a second option, a nutrient medium based on NPK 13–40–13 + microelements (TE) was used. In this medium, nitrogen, phosphorus and potassium elements were supplied in a ratio of 13:40:13, respectively, which serves to activate the processes of cell division and energy metabolism due to the high concentration of phosphorus [7,11]. Microelements were added in the following concentrations (g/L): Fe – 0.02; Mn– 0.01; Zn – 0.01; Cu – 0.01; B – 0.01; Mo – 0.007. The initial pH of the nutrient medium was adjusted to 7.5. This medium is aimed at ensuring rapid growth of microalgae and high biomass yield, allowing modeling of conditions close to industrial. The microalgae cultivation process was carried out in laboratory conditions for 14 days. During the cultivation process, the temperature was maintained in the range of 30–35°C, and the pH value of the medium was maintained in the range of 6.5–7.5. An artificial lighting system was used as a light source, providing a light intensity of 3000–4000 lx.

These conditions serve to increase the photosynthetic activity of microalgae, accelerate cell division, and enhance biomass accumulation. During the experiment, the growth dynamics of microalgae were regularly monitored and biomass yield was determined. Samples were also taken at specified intervals to determine the protein and lipid content of the biomass and prepared for further biochemical analysis.

Sterile preparation of solid nutrient medium (1 L) based on Chu-13 and NPK 13–40–13.

In order to isolate and cultivate green microalgae in the laboratory, 1 liter of solid nutrient media based on Chu-13 and NPK 13–40–13 + microelements (TE) was prepared. The selection of nutrient media was based on their ability to meet the physiological needs of microalgae cells, promote cell division, and support photosynthetic activity. Chu-13 nutrient medium is a balanced medium widely used in the cultivation of microalgae in laboratory conditions, ensuring stable cell growth. In the NPK 13–40–13 nutrient medium, nitrogen, phosphorus and potassium elements were provided in a ratio of 13:40:13, with a high content of phosphorus used to accelerate cell division. Microelements were added to the NPK medium in the following final concentrations (g/L): Fe – 0.02; Mn – 0.01; Zn – 0.01; Cu – 0.01; B – 0.01; Mo – 0.007. The main macro components and agar-agar were completely dissolved in distilled water, and agar-agar was added at a rate of 24 g/L to form a solid medium and clearly separate individual colonies. The pH of the nutrient medium was adjusted to 7.5, as this indicator is considered the optimal physiological environment for green microalgae. The prepared solution was sterilized in an autoclave at a temperature of 121°C for 15–20 minutes, which ensures complete inactivation of microorganisms and spores. Microelements were prepared as a separate solution to prevent precipitation at high temperatures and to preserve their biologically active form, and sterile filtered through a 0.22 µm membrane filter. After autoclaving, the basic nutrient media were cooled to 45–50°C and sterile filtered microelement solutions were added under aseptic conditions. The prepared solid nutrient media were poured into sterile Petri dishes in 15-20 ml portions and stored at room temperature until completely solidified. After solidification, the Petri dishes were placed in an inverted position to prevent condensation and stored at 4°C before inoculation of microalgal strains.

Inoculation of microalgae strains on sterile solid nutrient media.

A sterile solid nutrient medium based on NPK 13–40–13 + microelements (TE) was prepared for sowing by pouring it into Petri dishes and completely solidifying it. The sowing of microalgae strains was carried out under laminar air flow, under strict aseptic conditions. For cultivation, samples were taken from the green microalgae strains Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-15 in the active growth stage using a sterile bacteriological hook. The obtained strains were evenly distributed on the surface of the solid nutrient medium. Each microalgae strain was inoculated into separate Petri dishes, and strict measures were taken to prevent cross-contamination between strains. The inoculated Petri dishes were closed, placed upside down to prevent condensation, and grown under the specified light and temperature conditions for microalgae. During the cultivation, the formation of microalgae colonies, growth intensity, and morphological characteristics were regularly monitored.

RESULTS AND DISCUSSION.

Dynamics of microalgae colony formation in NPK 13–40–13 nutrient medium.

The colony formation rate of the green microalgae Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-15 was evaluated as a function of time under NPK 13–40–13 nutrient medium conditions (Fig.2). On the 10th day of cultivation, colony formation rates were low in all species (≈0.4–0.6 units), which is explained by the lag phase of the cells' adaptation to the nutrient medium. At this stage, Planktochlorella nurekis Far-Uz-37 showed a relatively higher value, demonstrating rapid adaptation to the NPK medium. Within 14–21 days the rate of colony formation increased significantly in all microalgae species. In particular, in Planktochlorella nurekis Far-Uz-37, the rate increased from ≈1.0 to ≈2.0 units, and in Tetradesmus obliquus Sur-Uz-15 from ≈0.8 to ≈1.6 units. This period indicates that the microalgae have entered an exponential growth phase. Stable growth was also noted in the species Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Nam-Uz-23, but their growth rate was relatively lower. During the 28–35 day growth phase maximum colony formation rates were observed. On day 35, Tetradesmus obliquus Sur-Uz-15 (≈3.4 units) and Tetradesmus obliquus Nam-Uz-23 (≈3.1 units) reached high colony densities. Planktochlorella nurekis Far-Uz-37 showed a stable result around ≈2.8 units, maintaining consistent growth dynamics throughout the experiment. The maximum value of Chlorella sorokiniana Sur-Uz-03 was ≈2.5 units. These results clearly demonstrate that the high phosphorus content in the NPK 13–40–13 nutrient medium accelerates cell division and colony formation processes. In particular, the high growth rates of Planktochlorella nurekis Far-Uz-37 and Tetradesmus obliquus Sur-Uz-15 species confirm that this medium is promising for industrial biomass production.

Dynamics of microalgae colony formation in Chu-13 nutrient medium.

The dynamics of colony formation of green microalgae Planktochlorella nurekis Far-Uz-37, Tetradesmus obliquus Nam-Uz-23, Tetradesmus obliquus Sur-Uz-15, and Chlorella sorokiniana Sur-Uz-03 in Chu-13 nutrient medium was evaluated over a period of 10–35 days. The results showed that the growth process of microalgae was gradual and time-dependent (Fig.3). At the initial stage of cultivation (day 10), colony formation rates were low in all species, ranging from 0.4–0.6 units. This is explained by the lag phase of the cells' adaptation to the nutrient medium. At 21 days, the microalgae entered an exponential growth phase and the rate of colony formation increased significantly. At this stage, Planktochlorella nurekis Far-Uz-37 and Tetradesmus obliquus Sur-Uz-15 showed relatively high rates, while Tetradesmus obliquus Nam-Uz-23 and Chlorella sorokiniana Sur-Uz-03 showed stable growth.

The colony formation rate reached maximum values on days 28 and 35. In particular, high colony densities were recorded on day 35 for Chlorella sorokiniana Sur-Uz-03 and Planktochlorella nurekis Far-Uz-37, which indicates that they are well adapted to long-term growth on Chu-13 medium. The colony formation process also continued consistently in Tetradesmus obliquus Nam-Uz-23 and Tetradesmus obliquus Sur-Uz-15, achieving high final values. Overall, the results showed that the Chu-13 nutrient medium provided stable growth and colony formation of microalgae. The growth process in this medium was characterized by a relatively slow start and continued in a consistent and stable manner in the subsequent stages. This scientifically substantiates the fact that the Chu-13 nutrient medium is optimal for long-term cultivation of indigenous microalgae in laboratory conditions.

Comparative discussion of Chu-13 and NPK 13–40–13 nutrient media.

In this study, the effects of Chu-13 and NPK 13–40–13 nutrient media on the growth dynamics, colony formation rate, and biomass accumulation of local green microalgae - Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-15 -were compared.

The results showed that the chemical composition of the nutrient medium is a key factor determining the physiological state and growth strategy of microalgae. In particular, cell division continued consistently and a stable increase in colony formation was ensured during long-term cultivation. The balanced ratio of macro and microelements in this medium led to the stabilization of intracellular metabolic processes.

The growth of microalgae in the Chu-13 nutrient medium was initially slow, and a lag phase of cell adaptation to environmental conditions (lag phase) was clearly observed, and the continuity of the growth process and physiological stability in the Chu-13 medium of Chlorella sorokiniana Sur-Uz-03 species were clearly demonstrated. In the conditions of the NPK 13–40–13 nutrient medium, the growth process of microalgae was significantly accelerated. The high concentration of phosphorus activated cellular energy metabolism, nucleic acid synthesis, and cell division. Therefore, microalgae entered the exponential growth phase early and achieved high colony density in a short time.

This was especially clearly observed in the species Planktochlorella nurekis Far-Uz-37 and Tetradesmus obliquus Sur-Uz-15, and their growth in a phosphorus-rich medium was high. However, during long-term cultivation in NPK 13–40–13 medium, a decrease in growth rate was observed in some species. This is explained by the rapid consumption of nutrients by cells, increased metabolic processes, and increased physiological stress factors. As a result, cells may have activated metabolic mechanisms aimed at maintaining vital activity rather than at the growth process. The results obtained in terms of biomass quantity and quality also showed significant differences. While microalgae grown in NPK 13–40–13 medium dominated in terms of total biomass yield, relatively balanced and stable accumulation of proteins and lipids was observed in biomass grown in Chu-13 medium. This indicates that the normal concentration of nitrogen and phosphorus ensures that intracellular biosynthetic processes proceed in an optimal direction.

The results of the comparative analysis show that the Chu-13 nutrient medium ensures the physiological stability of microalgae and creates favorable conditions for their long-term cultivation. The NPK 13–40–13 nutrient medium is effective for achieving intensive growth and high biomass yield in a short period of time. Therefore, the selection of nutrient medium for industrial cultivation of microalgae should be carried out in accordance with the production goal.

Conclusion. In this study, the growth dynamics and colony formation properties of local green microalgae - Tetradesmus obliquus Nam-Uz-23, Planktochlorella nurekis Far-Uz-37, Chlorella sorokiniana Sur-Uz-03 and Tetradesmus obliquus Sur-Uz-1 were comparatively evaluated in nutrient media with different compositions.

he results confirmed that the chemical composition of the nutrient medium directly affects the growth rate, colony formation intensity and biomass yield of microalgae. According to the results of the study, microalgae grew rapidly in the NPK 13–40–13 nutrient medium, achieving high colony density and biomass yield in a short period of time. Although the growth process was relatively slow in the Chu-13 nutrient medium, stable cell development and long-term growth dynamics were observed. On this basis, it was scientifically proven that the NPK 13–40–13 nutrient medium is an optimal environment for intensive biomass cultivation, and the Chu-13 nutrient medium is an optimal environment for sustainable and long-term cultivation of microalgae.

The results obtained are of significant scientific and practical importance in improving the technologies for industrial cultivation of local microalgae, increasing biomass yield, and managing their biochemical composition.

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