Prospects of the BFT development for utilization of heterotrophic microbiome as a feed raw material source and maintenance of optimal parameters of the cultivation environmentAbstract
The need of the aquaculture development intensification is related with the growing demand of the world community for quality food products produced with minimal environmental impact. The trend of implementing industries that contribute to the achievement of sustainable development of mankind implies the development of new environmentally friendly technologies. The introduction of the biofloc system in aquaculture implies not only an increase in the production profitability due to effective water treatment in cultivation pools, but also the possibility to use the formed activated sludge flocs as raw material for feed additives. However, the formation of the BFT system microbiome is accompanied by significant changes in the species composition of the microbial community. The technology efficiency is largely related to the possibility of microbiome community management, so the study of species composition, quantitative characteristics and biological features of the BFT system components is of great interest. The dynamics of microbiome development, its influence on marketable qualities and morphophysiological characteristics of hydrobionts, as well as the biochemical features of floc raw materials, are investigated comprehensively.
Keywords:
аctivated sludge; amino-acid composition; Clarias gariepinus; hydrobiont; microbial community; microbial protein; probiotic; recirculating aquaculture system
Resumo
A necessidade de intensificar o desenvolvimento da aquicultura está diretamente associada à crescente demanda global por produtos alimentícios de alta qualidade, produzidos com o mínimo impacto ambiental. A tendência de implementar indústrias que contribuam para o desenvolvimento humano sustentável envolve o aprimoramento e a aplicação de novas tecnologias ecologicamente responsáveis. A adoção do sistema de bioflocos (BFT) na aquicultura não implica apenas o aumento da lucratividade da produção, em função da purificação eficiente da água nos tanques de cultivo, mas também possibilita o aproveitamento dos flocos de lodo ativado resultantes como matéria-prima para aditivos alimentares. Entretanto, a formação do microbioma no sistema BFT é acompanhada por mudanças significativas na composição de espécies da comunidade microbiana. A eficácia dessa tecnologia está intimamente relacionada à capacidade de de gestão da comunidade do microbioma, o que torna o estudo de sua composição de espécies, das características quantitativas e das propriedades biológicas de seus componentes um tema de grande relevância. A dinâmica do desenvolvimento do microbioma, sua influência nas qualidades comerciais e nas características morfofisiológicas dos hidrobiontes, bem como as características bioquímicas dos flocos como matérias-primas, são investigadas de forma abrangente.
Palavras-chave:
Lodo ativado; composição de aminoácidos; Clarias gariepinus; hidrobionte; comunidade microbiana; proteína microbiana; probiótico; sistema de aquicultura de recirculação
1. Introduction
One of the main challenges addressed by the Food and Agriculture Organization (FAO) is to ensure healthy diets and food security for all people. Given that the exploitation of terrestrial ecosystems is generally simpler technologically, but is associated with limited space and significant degradation of natural cenoses. Therefore, growing hydrobionts both in natural hydrocenoses and in isolated recirculating aquaculture systems (RAS) is a way to obtain valuable food products with minimal anthropogenic load on the biosphere (Heimann and Delzeit, 2024). The increase in fish production by 2030 is expected to be 12.8%, about 201 million tons. Fish and fishery products will account for 31% of food commodity turnover, with a 33% increase in fishery exports by 2030 (FAO, 2024).
Any method of hydrobiont cultivation implies either exploitation of autotrophic and heterotrophic elements of aquatic ecosystems or external introduction of the whole set of organic substances and trace elements necessary for efficient metabolism as food raw materials. Complex ways of organisms' interaction in ecosystems provide accumulation and transfer of substances and energy. In artificially created systems for growing hydrobionts, the system of substance transportation becomes even more complex, and it is important that technologies provide for the safety of growing objects for the environment and the consumer (Bieg et al., 2022).
Since the process of food digestion of hydrobionts and growth and development processes are the main link in the technological process of cultivation, increasing the efficiency and reducing the cost of food raw materials is an important direction of the fishery intensification (Golovacheva et al., 2024). Researchers have noted that, for example, during shrimp farming, only 15-30% of the introduced feed is utilized, while the rest becomes a substrate for heterotrophic degradation (Rajkumar et al., 2016).
For cultivation of marketable volumes of fish and hydrobionts it is necessary to provide conditions which are optimally close to natural habitat, because changes in conditions reduce marketable qualities of the cultivated object. High environmental friendliness and economy of water utilization in closed water supply systems are compensated by high energy consumption and costs of maintenance of premises occupied by filtration units. Therefore, the BioFloc Technology (BFT) is an effective addition to the existing schemes of recirculating aquaculture systems.
Although the BFT system maturation takes time and requires additional carbon source implementation, which affects the level of costs in industrial applications, there are ways to improve the BFT system efficiency. The intensity of activated sludge floc formation and the quality of protein formed depends on the species composition of the bacteria forming the biofloc system. Several studies (Wu et al., 2022) have used strains with cellulolytic activity to form the BFT system, allowing use of cheaper carbon sources. The efficiency of the BFT system is influenced by the rate of bioflocculant formation, the size characteristics of flocs that determine the rate of ammonia removal, the synthesis of the protein component of flocs and, ultimately, the growth performance of hydrobionts grown in this system (Tkacheva et al., 2020).
Activated sludge flocs, whose growth is stimulated by the released metabolites of hydrobionts, represent a complex of valuable nutrients. The substance of flocs contains carbohydrates, proteins, amino acids, fatty acids and minerals, but their main value, of course, is that it is a rich natural source of protein (Baiduk et al., 2023). In addition, the biofloc system application reduces the importance of the feeding regime as the nutrient substrate is constantly available and in suspension in the pool (Becerril et al., 2017).
Unfortunately, there are few studies devoted to specific descriptions of bacterial genera or species and determining their role in the BFT system. The role of heterotrophic bacteria, the main group of bacteria in the Biofloc system that remove ammonia nitrogen through the nitrification process, has been repeatedly emphasized. In the process of growth and development, these bacteria secrete inorganic compounds that stimulate the role of other floc components and secrete exoenzymes that decompose high molecular weight compounds. The role of Bacillus and Pseudomonas genera in the activity of transformation of toxic nitrogenous compounds has been pointed out (Monroy-Dosta et al., 2015).
A number of studies have searched ways for improving the Biofloc microbiome, for example, by adding rotifers Brachionus plicatilis (Silva et al., 2021). However, it has been marked that the mandatory removal of flocs during the floc biomass growth affects media quality (Khanjani et al., 2023). During sedimentation and removal of settled flocs, the microbial community structure changes and its biodiversity decreases (Zhu et al., 2025). Most researchers, studying BFT systems, believe that the floc microbiome species’ composition and its development as flocs mature and age is the main factor in creation of the comfortable environment for hydrobionts.
The BFT systems’ use as a source of alternative protein in order to enrich fish diets is very promising. Most researchers (Ray et al., 2010) mention the need to periodically remove growing flocs because of the negative effect on the respiratory system of fish. Utilization of flocs protein for feed and feed additive will allow more rational use of energy and feed raw materials, which will be a serious step towards the development of agricultural complex within the framework of sustainable development strategy.
The aim of this study is to compare the impact efficiency of BFT systems and RAS system on the main hydrochemical parameters of the environment, to determine the species composition of the stable ecosystem of the biofloc microbiome. Also, the influence of the BFT system conditions on marketable qualities and morphophysiological characteristics of hydrobionts has been compared. In addition, the characteristics of amino acid composition of microbial protein as a potential raw material for feed production have been investigated.
2. Materials and Methods
2.1. Experimental units and procedures
In order to estimate potential capabilities of the Biofloc systems use which serve as means for maintaining optimal parameters of the environment for hydrobionts’ cultivation and as a protein source for making feed raw materials, the experiment has been planned. It includes stages shown in Figure 1.
At the first stage of the experiment, the BFT system for growing hydrobionts has been created. The RAS system with standard water treatment parameters has been launched simultaneously. The second stage of the experiment involved assessment of the species composition of the formed biofloc system. At the third stage of the experiment, parallel cultivation of hydrobionts in the unit with biofloc medium and in RAS has been carried out. At this stage, the comparative marketable qualities of the growing hydrobionts have been determined. When the hydrobionts reached marketable size, morphophysiological study has been conducted to confirm the favorable effect of the system on the condition of hydrobionts. At the last stage of the experiment, the biological qualities of the extracted dry sludge serving as a feed additive have been investigated (Figure 1).
To create the biofloc medium, which will be effective in terms of treatment of water from biogenic compounds and safe for cultivated hydrobionts, pools with volume 1m3 have been used. The same pools have been used for hydrobionts’ maintenance in the RAS system.
For the BFT system maturation, external nitrogen sources have been used. Previous investigations have shown that achieving the C/N ratio, required for reaching of needed size and qualitative composition of flocs, can be efficiently used in feeds for sturgeon “Sturgeon growth 46/16 VNIRO”. Feed pellets contain 46% of protein, about 16% of lipid compounds and 3% of cellulose.
Molasses with 46.5% of sugar content has been used as the external CO2 source. The share of dry matter in the carbon source is 80.6%, ph – 8.0. The main advantage of the substrate, containing carbon, is its accessibility and low cost.
In each experimental pool with 1 m3 volume, 6 aerators (diffusors), distributed along entire perimeter of the pool. Water temperature has been controlled within the range from 28 to 29 °C. Temperature, oxygen content, pH level has been estimated with the professional water quality measuring instrument WATERLINER WMM-97. The pH level has been maintained within the range from 7.7 to 8.2. Sodium bicarbonate has been used as buffering agent. This acidity level is optimal for nitrification and microbial activity.
Based on previous studies (Baiduk et al., 2023), probiotic strains Bacillus velezensis MT141 and Bacillus velezensis MT142 have been used for the BFT system formation. This combination of strains has shown the effective and rapid flocculus formation. After maturation of biofloc, Clarias gariepinus (Burchell, 1822) (Clariidae) is released into the pool.
2.2. Selection and analysis methods
2.2.1. Water quality analysis
The biofloс system matured for 10 days, during which time the initial concentration of flocks was reached. Water quality parameters including pH, temperature, and oxygen content have been measured using the WATERLINER WMM-97 professional water quality meter. Water samples have been collected in three replicates from each system every 2 days.
Standard analytical protocols (APHA, 2017) have been used to analyze nitrite, nitrate, total nitrogen (TN). Biofloc volume (FV) has been monitored weekly to diagnose maturation of the BFT system. According to traditional methods (Luo et al., 2022), Imhoff cone (1000 mL) was used to determine the floc volume in the pool.
2.2.2. Genetical analysis of the microbiome content
DNA has been isolated according to the method described by Akash Gautam (Gautam, 2022). Sequencing has been performed on a MinION instrument using Rapid Barcoding Sequencing Kit V14 reagents according to the instructions. Quality control of reads has been performed using NanoPlot v1.42.0 program, filtering and rejection of low-quality reads has been performed using Filtlong v0.2.1 program. Taxonomic identification has been performed using GTDB-Tk v4.2.0 (Chaumeil et al., 2022) based on The Genome Taxonomy Database (GTDB) (Parks et al., 2022).
2.2.3. Planting and analyzing of hydrobionts’ condition
At the beginning of rearing, the planting density was 5 eq./l., with a mass of planting material of 16 grams. The water temperature was stable at this stage, 25 ± 0.3 °C, when growing commercial fish.
The first phase of the study lasted for 90 days. Catfish in the experimental pool were fed with sinking fodder in the volume of 2% of fish biomass. Such a feeding rate was chosen in the expectation of the presence of additional feed in the system in the form of activated sludge flocs. Feeding of the object was carried out every 5-6 hours (3 times a day). In the RAS system (control) the feed volume was 3% of fish weight.
At the second stage of the experiment, the study of marketable qualities of catfish of the control and experimental groups was carried out, as well as morphophysiological studies.
The total duration of the experiment was 24 weeks, weighing of objects was carried out every 4 weeks using laboratory scales (A&D, DL (DL-1200), Japan).
2.2.4. Morphophysiological analysis of hydrobionts’ condition
Blood composition and condition were analyzed during the last three weeks.
For hematological studies, blood was taken from the tail artery of catfish from starving fish. Studies of blood biochemical parameters were carried out in the State Budgetary Institution of the Rostov Region “Rostovskaya obl SBBZh s PO” – “Rostovetlaboratoriya”. Statistical processing of the obtained data was carried out by generally accepted methods using Excel 2007 program.
To evaluate the effect of experimental feed with microbial additive, clinical examination, pathologoanatomical autopsy and complete parasitological analysis were used according to generally accepted methods. Fish tissues and organs were examined by compression method using compression (9×12 cm), slide (7.5×2.5 cm) and cover glasses (1.8x1.8 cm). The state of organs and tissues was studied by microscopic method using stereoscopic SIAMS MT-24RF and biological microscopes Saike Digital SK2009H2S4, based on morphometric features of the objects.
2.2.5. Biochemical analysis of microbial sludge composition
For sludge dehydration the domestic convection dehydrator model Libhof PDW-12 with horizontal arrangement of trays and horizontal air movement has been used. The maximum heater power is 800 W, temperature setting discreteness is 5 °C, with a range of 35-90 °C. After drying, the microbial sludge has been analyzed for amino acid composition.
All analyses have been carried out in the testing (scientific and educational) laboratory “Biochemical and Spectral Analysis of Food Products” by method M 04-38-2009. The method is based on decomposition of samples by acid or alkaline hydrolysis (only for tryptophan) with conversion of amino acids into free forms, obtaining phenylisothiocarbamyl derivatives (PIC) derivatives, their further separation and quantification by capillary electrophoresis. Detection is carried out in the UV spectrum region at a wavelength of 254 nm. For tryptophan direct quantitative determination without obtaining PIC derivative with detection at wavelength of 219 nm is provided.
3. Results
3.1. Hydrochemical water indicators
During the start-up phase of the experiment, the fish pools were filled with water, the parameters of which corresponded to the rearing standards meeting the biological needs of Clarias gariepinus.
Oxygen content (DO), pH, temperature and total ammonium nitrogen (TAN) have been measured daily and there has been no deviation from the optimal parameters during the experiment. DO was maintained at the optimum level in both control and experimental pools, temperature and pH were 25.65 ± 0.25 °C and 7.13 ± 0.02, respectively. The dynamics of nutrient nitrogen content is presented in the graphs. In the control study basin (RAS) purification has been carried out by filters, in the basin with the BFT system optimal readings of hydrochemistry have been reached by the time of hydrobionts’ release.
Taking into account the active increase in the total floc volume, the nitrogen and carbon sources introduced at the stage of water treatment were assimilated, and after maturation of the biofloc system, the indicators of the level of biogenic nitrogen compounds in water were maintained in the normal range, which follows from Figures 2, 3, 4 and 5.
3.2. Parameters of the biofloc system
The process of the BFT system formation followed the dynamics of activated sludge flocs formation identified in previous studies. Active floc growth reached its maximum at week 10 of the study, and then excess flocs have been removed daily if the measured floc TSS exceeded 250 mg/L. The parameters of the biofloc system have been relatively stable throughout the experiment.
Stable dynamics of floc formation and effective water purification from biogenic nitrogen indicates the formation of a stable microbial community.
The active process of floc growth indicates the ability of floc bacteria to absorb complex organics. Similar dynamics of hydrochemical parameters indicates the ability of this group of probiotic strains to form a stable microbiome capable of absorbing urea and ammonium compounds, as well as other organics formed in the created artificial biocenosis.
Microbial sludge sequencing has been performed at week 10, when the volume and TSS of flocs reached the maximum level. The cronogram obtained as a result of sequencing is presented in the Figure 6. Quantitative analysis of the obtained data allowed us to identify the dominant microbial genera and associated minor groups in the community.
Among the dominant representatives of the microbiota, 33% belong to the genus Runella Larkin and Williams 1978, a gram-negative, aerobic and immobile genus of bacteria from the family of Spirosomaceae (Figures 6, 7 and 8). A significant number (20%) of the bacteria of the floc community belong to the class Clostridia Rainey 2010. Bacteria of this class, defined as representatives of the family Christensenellaceae, the Christensenellaceae R-7 group, make up 15% of the community. Another 5% is accounted by representatives of the family Peptostreptococcaceae, genus Acetoanaerobium Sleat et al. (1985).
An important component of the phloc community are bacteria of the family Exiguobacteraceae, of the genus Exiguobacterium Collins et al. (1983); the total number of representatives of this genus is 26%. Representatives of the family Bryobacteraceae, the genus Bryobacter Kulichevskaya et al. (2010) account for 11% of the total number of species, the smallest number of dominants were representatives of the genus Diaphorobacter Khan and Hiraishi 2003 (3%).
3.3. Hydrobionts’ condition during the cultivation process
At the end of the 2nd stage of the experiment, African catfish of the genus Clarias Scopoli, 1777 has reached marketable weight by the 6th month of rearing and averaged 960 grams (Table 1). The data in the Table 1 is based on weighing objects every four weeks, for a total of six weighing periods. The rate of weight gain shown by the control group in RAS has been lower, which suggests that, despite the smaller amount of feed, the presence of microbial sludge as a food substrate has determined more comfortable habitat conditions
The growth was recorded from the initial mass (16 g) and was carried out by months growing Clarias gariepinus in the experiment (Figure 9). Obviously, at the initial stage of the experiment in the biofloc system, the growth rate was almost twice as high as the rate of mass gain in RAS.
3.4. Morphophysiological analysis of hydrobionts’ condition
Data on hematological indices of the grown hydrobionts are shown in Table 2. Hematological parameters in the experimental group of fish corresponded to the physiological norm. Leukocytic series of Clarias gariepinus is represented by lymphocytes, neutrophils, monocytes and polymorphonuclear cells.
The morphology of white blood is characterized mainly by lymphocytes, the number of which was in the range of 85.4% – 88.7%, the number of lymphocytes in the experimental group was 3.5% (p <0.1) higher than in the control group. The proportion of monocytes in the blood of catfish in the control group was 5.4%, and in the experimental group 2.3%, confidence intervals were calculated. The low level of significance is chosen for pilot studies or small samples, it should be noted that the sample was below the recommended n>30. Since blood sampling from fish was carried out during life, the sample had to be reduced due to the risk of injury to objects and the impact on the studied marketability. The difference in the average number did not reach statistical significance at the p <0.1 level, high data variability and small sample size could limit the possibility of detecting the effect. Nevertheless, studies on the effect of probiotics on the leukocyte formula of blood should be continued, changing the experimental conditions.
The main hematological criterion is the percentage of different types of leukocytes, this is because they reflect the physiological condition of fish and some aspects of cellular immunity. The result of the analysis of leukocytic profile of the organism of Clarias gariepinus indicates that against the background of application of dry microbial floc in the feeds of fish recorded a reliable increase in the absolute number of leukocytes, while a decrease in the relative number of monocytes. Summarizing, leukocytosis within the physiological norm, with pronounced lymphocytosis, with the presence of an absolute majority in the leukocytic formula and a slight decrease in the proportion of monocytes, states a high degree of development of the cellular link of the immunity system.
Characterizing hematological studies of clarium catfish, it can be stated that the fish was in good physiological condition, indicating favorable feeding and rearing conditions.
The surface of the fish body has not been excessively oily, there have been no ulcers, wounds and other deviations from the physiological norm of the species. Surface mucus was characterized by sufficient transparency, areas of turbidity have been not recorded. The liver was clearly shaped, without fat capsules. Free exudate in the abdominal cavity was absent. According to the results of clinical examination, it has been found that the condition of the examined fish is assessed as satisfactory. No deviations from the physiological norm of the species have been registered.
3.5. Biochemical analysis of the microbial sludge composition
Analysis of the amino acid composition of activated sludge protein showed that a significant percentage of amino acids are valine, glycine and leucine (Figure 10). There is no cysteine in the protein composition, histidine content is less than a percent.
4. Discussion
The use of BFT systems for removal of biogenic pollutants is currently practiced quite widely. In all studies conducted during the series of experiments, hydrochemical and hydrobiological parameters of the environment have remained constant. However, when considering microbiome communities, it is necessary to take into account that fluctuations in temperature, salinity, pH, and oxygen concentration can cause species variability of probiotic complexes. One cannot ignore the possibility of spontaneous spore formation of the main components of the probiotic system, reduction of probiotic impact, emergence of new bacterial species or increase in the concentration of minor elements of the microbial community. Therefore, the development of methods to control the state of microbial communities is highly relevant.
In the present study, the water treatment efficiency of RAS and the BFT systems have been found to be approximately the same, as can be seen from the graphs presented in Section 3 (Results). The satisfactory condition of the reared objects, stable weight gain and absence of physiological and morphological abnormalities indicate the creation of an optimal rearing environment.
Analysis of the species composition of the microbial community by metagenomic sequencing of 16S rRNA genes makes it possible to evaluate dominant and minor genera, trophic interactions arising in the process of flocs maturation and maintaining their stable state. Systemic study of the generic composition of the floc microbiome allows predicting the state of the microbial community and its influence on the qualitative indicators of the artificial hydrocenosis. The microbiome formed in the present study is dominated by members of the genus Runella (33%), a Gram-negative, aerobic and immobile genus of bacteria from the family Spirosomaceae. In a study (Zhao et al., 2024) it has been observed that representatives of this genus have been found as a dominant component in the granular sludge and showed resistance to pollution, in addition, the growth of these bacteria has been stimulated under bright light. Another characteristic feature is the tendency of this genus to grow at temperatures up to +4 and the apparent eurythermal nature of this genus makes it a promising component for water treatment in open water bodies.
The genus Exiguobacterium, representatives of which account for 26% of the total number of floc bacteria, belonging to the Bacillota type, is often found in extreme ecosystems such as glaciers and hot estuaries. A number of strains are highly thermoadaptive and salt tolerant, resistant to ultraviolet light and heavy metals in the environment.
The genus Exiguobacterium, given its broad ecological valence has been found by researchers in the gastrointestinal tracts of marine fish and in microbial biofilms. The researchers note that the specificity of the phospholipid fatty acid set allows for changes in cell membrane function, which determines the ability to adapt to temperature, changes in acidity and other environmental factors. (Dragoni-Rosado et al., 2023) Also in studies it has been revealed that as intestinal symbionts of mealworms strain YT2 of the genus Exiguobacterium allows the worms to process polystyrene (Vishnivetskaya et al., 2009).
Another distinctive ability of representatives of the genus Exiguobacterium is the acquisition of orange pigmentation by the colony, presumably resulting from the biosynthesis of carotenoids C 30. Carotenoids are necessary for the bacterial colony for protection against UV radiation, but their presence in a bacterium that is part of the floc system may provide additional value of the floc substrate as a raw material for feed preparation.
A significant number (20%) of representatives of the family Christensenellaceae, class Clostridia, has also been detected in the studied samples of microbiome flocs. This family has been described relatively recently, but researchers consider representatives of this genus to be a significant factor for maintaining the normal state of human intestinal microflora, and there are assumptions about reducing the risks of metabolic diseases due to a stable level of representatives of the family Christensenellaceae in the intestine. Since some metabolic regulation processes in vertebrates may be similar, additional research is needed on the effects of this floc community on the health status of hydrobionts.
The efficiency of growing Clarias gariepinus in the BFT system is confirmed by higher rates of weight gain. At the same time, it should be noted that the amount of feed received by hydrobionts in the experiment was half the amount of feed introduced into RAS. Obviously, the addition of floc to the catfish diet has been effective enough to meet fish physiological needs. Average growth rates are particularly high in the first months of rearing, and high survival rate of planting material is also shown. Taking into account the economic effect of water treatment using BFT system, providing the fish diet with a complete feed additive makes cultivation of the object even more profitable.
It should be noted that the analysis of morphophysiological condition of Clarias gariepinus has shown no significant differences between the samples grown in RAS and using the BFT system. The analysis of hematological parameters in the experimental group of fish has shown the value of all blood forming elements at the level corresponding to the physiological norm.
Morphology of white blood is characterized mainly by lymphocytes, the number of which has been in the range of 85.4% – 88.7%, the number of lymphocytes in the experimental group has been 3.5% (p < 0.1) more than in the control. The proportion of monocytes in catfish blood in the control group has been 5.4%, and in the experimental group – 2.3%.
The main hematological criterion is the percentage ratio of different types of leukocytes, this is due to the fact that they reflect the physiological state of fish and some aspects of cellular immunity. The result of the leukocytic profile analysis of Clarias gariepinus organism indicates that against the background of the content of the BFT system in fish a reliable increase in the absolute number of leukocytes is recorded, at the same time there is a decrease in the relative number of monocytes. Expressed leukocytosis is not associated with pathological reactions, is within the physiological norm and is accompanied by expressed lymphocytosis. The presence of high values in the leukocytic formula with a slight decrease in the proportion of monocytes, suggests a high level of development of the cellular link of the immunity system. Stable blood formula indices throughout the whole period of growing indicate comfortable growing conditions and high marketable status.
In addition, given that a significant number of studies have found an increase in the activity of nonspecific immunity in fish and arthropods grown in RAS, we can talk about the potential summation of the effect of comfortable growing conditions, valuable feed additive and probiotic effect of microbial community components.
Pathologoanatomical autopsy of Clarias gariepinus has shown a complete normotypic correspondence of the state of organs of fish grown in RAS and BFT systems. The skin of the studied objects is smooth, without damage and signs of infection There is no exudate in the body cavity, serous membranes are clean, without signs of inflammation. Liver of normal color (light brown), without nodules and hemorrhages, heart of normal size and shape, myocardium dense, without visible damage. The condition of the stomach and intestines is normal, the mucous membrane is smooth, free of ulcers and inflammation. The condition of the kidneys has no changes in shape or size, the texture of the organ is homogeneous, with no enlargements or nodules. The gills are pinkish-red in color, clear, with no signs of edema, necrosis or hypertrophy. The condition of lymph nodes shows absence of inflammatory changes.
The conducted studies convincingly testify to the absence of negative influence of BFT systems on the physiological state of Clarias gariepinus, and the ability of floc substrate to replace the lack of food. However, it should be taken into account that food preferences of Clarias gariepinus are very diverse, preferring predation, however, it is omnivorous and can feed even on organic waste, which makes flocs an acceptable food substrate for it (Tkacheva et al., 2020). Most of the hydrobionts grown in RAS are more demanding to food substrates, so the study included the analysis of the biochemical composition of flocs raw material in order to form an additive or systemic component in the feed formulation for different species of hydrobionts.
Protein supplementation for fish feed, obtained as a secondary product during farming, can be very significant, considering that the protein requirement for marine fish reaches 40-55% of the dry weight of feed, for freshwater fish it is slightly lower, 28-40% (Henry et al., 2015). Research in the field of cheapening and improving quality is ongoing (Hasan et al., 2019; Iaconisi et al., 2019), and the amino acid composition of the proteins used, including the ratio of substituted and essential amino acids, is of great importance for the quality of raw materials. Comparing the percentage of essential amino acids in the protein composition of Tubifex tubifex, a popular protein source for fish in aquaculture, it can be observed that the content of some essential amino acids in the meat of naturally reared worm is lower than that of floc protein (Hasan et al., 2019). The content of valine is significantly higher (Figure 11).
Of course, the amino acid composition of animal protein can be managed by changing the composition of feed raw materials (Hasan et al., 2019; Souza et al., 2025), but perhaps managing the species composition of the microbiome could also influence the amino acid ratio. Nevertheless, the study of amino acid composition of floc protein shows that, as a potential feed additive, it contains all essential amino acids.
Research on feed composition and feeding of hydrobionts plays an important role in aquaculture development and sustainable fish production. Nutritional control and the development of balanced diets are fundamental to successful fish farming, making such experiments essential.
5. Conclusions
The studies showed that the environment created by the BFT system corresponded to the required standards and parameters, and positively stimulated the growth of juvenile Clarias gariepinus, the growth rate of which during the first month has been almost twice as high as that of the juveniles reared in RAS, despite the halved amount of feed. The morphophysiological study has not revealed any deviations in blood composition and the state of internal organs, which indicates the absence of toxic effects of microbial community components. Analysis of species composition has shown the presence of genera with potentially high probiotic activity. Studies of floc protein composition have confirmed the presence of a variety of amino acid composition, which meets the requirements for feed raw materials, but it should be emphasized that to recommend the BFT system technology for a diverse range of farmed hydrobionts, additional studies are needed, including the possibility of using flocs as a food substrate for young fish.
Thus, we can speak about the significant potential of BFT systems as a technology in aquaculture and the need for further adaptation of this technology for a wider range of farmed objects.
Acknowledgements
The publication was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-15-2024-528 of 24.04.2024 on the implementation of the large-scale research project within in priority areas of scientific and technological development).
The authors express their gratitude to Victoria Shevchenko, senior researcher at the Research Laboratory “Agrotechnology Center” of DSTU, for the assistance provided during the research and writing of this article, as well as for contributions to previously published material “Influence of the synergetic effect of the biocenosis synthesis of zoogenic colonies of bacteria and protozoa on the sustainable development of aquatic ecosystems”; and to Sofia Tertychnaia, a linguist and translator completed the study program “Translation and Translation Science” of the Faculty of Applied Linguistics at the Don State Technical University (Rostov-on-Don, 344000, Gagarin Sq., 1, Rostov Region, Russian Federation, higher education diploma No. 106131 0835887, issued on July 11, 2025) for the translation of this manuscript to the English language.
Data Availability Statement
The research data is a continuation of the research at the following address: DOI https://www.scielo.br/j/bjb/a/4LkHFRvybw5kw5CCFwHWr5c/ The data presented in the link is publicly available. The scientific data set supporting the results of this research has been published in this article and is not publicly available.
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Edited by
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Editor:
Felipe Polivanov Ottoni
