Influence of the synergetic effect of the biocenosis synthesis of zoogenic colonies of bacteria and protozoa on the sustainable development of aquatic ecosystemsAbstract
The production of agricultural products, especially livestock and aquaculture products containing high amounts of protein, has a significant impact on the environment. However, humankind cannot forego the production of such products, since a balanced diet is necessary to maintain a healthy lifestyle. In Russia, where climatic features of most regions are unfavorable for agricultural activities, industrial aquaculture has a negative impact on the environment. In order to achieve the Sustainable Development Goals and develop a cyclic bioeconomy, it is necessary to improve fish keeping technologies in RAS or open ponds using probiotic-enriched Biofloc systems designed to purify water from biogenic nitrogen. When studying the synergistic effect of the interaction of various components of floc-forming systems, the most efficient formation of flocs by individual bacterial strains was revealed. The quality of floc protein allows using activated sludge with Bacillus probiotics to obtain floc biomass, which comprehensively reduces the costs of animal feeding, due to the processing of waste products into fodder raw materials. Also, improvement of the technology will reduce energy costs for water treatment, which will contribute to the achievement of sustainable development goals.
Keywords:
clarias gariepinus; hydrobiont; aquaculture; biofloc; probiotic
Resumo
A fabricação de produtos agrícolas, especialmente os de pecuária e aquicultura ricos em proteínas, impacta significativamente no meio ambiente. No entanto, a humanidade não pode deixar de prodizir tais alimentos, pois para manter um estilo de vida saudável é necessária uma alimentação balanceada. Na Rússia, onde as características climáticas da maioria das regiões são desfavoráveis para atividades agrícolas, a aquicultura industrial afeta negativamente o meio ambiente. Para alcançar os Objetivos de Desenvolvimento Sustentável e a bioeconomia cíclica, é necessário melhorar as tecnologias de criação de peixes em sistemas de recirculação aquícola (RAS) ou em tanques abertos, usando sistemas Biofloc enriquecidos com probióticos, projetados para eliminar o nitrogênio biogênico da água. Ao estudar o efeito sinérgico da interação de vários componentes dos sistemas de floculação, foi revelada a formação mais eficiente de flocos por cepas bacterianas individuais. A qualidade da proteína do floco permite usar lodo ativo com probióticos Bacillus para produzir biomassa de floco, reduzindo de forma abrangente os custos de alimentação animal, convertendo os resíduos em matérias-primas para alimentação animal. Além disso, a melhoria da tecnologia reduzirá os custos de energia para o tratamento de água, contribuindo para os objetivos de desenvolvimento sustentável.
Palavras-chave:
bagre-africano; hidrobionte; aquicultura; biofloco; probiótico
1. Introduction
Discussing human impact on the environment and targeted changes in natural ecosystems, negative consequences of such impact should be noted, for instance, climatic changes. Despite of numerous investigations, mechanisms for reducing anthropogenic impact (Pearson, 2024) are not clearly determined, balanced organizational and technological measures are not taken. However, it should be mentioned that the vast majority of studies, regarded to the search of ways for reducing anthropogenic influence on the environment, refer to the agroindustrial complex. Thus, to integrate Russian economy into the global strategy to achieve the Sustainable Development Goals, it is necessary to develop technologies meeting requirements of the cyclic bioeconomy (Das et al., 2022).
The need to supply humankind with food stuffs evokes drastic changes in the global circuit of substances, the breakdown of established food chains in ecosystems leads to an imbalance of major nutrients (FAO, 2023). The disruption of the C/N/P ratio in natural ecosystems as humans exploit them leads to a decrease in the natural bioproductivity of the ecosystem (Das et al., 2022).
The pressure on natural ecosystems is especially high in the production of protein foods. The need to use feed significantly increases the cost of the food stuffs and causes an increase in the ecological load on the ecosystem. To increase the efficiency of livestock and fish farming it is necessary to improve the feed quality, which affects the cost of the feed used, and thus reduces the profitability of production. The attempt to increase profitability leads to limitation of the use of valuable high-nutrient protein components in feeds for hydrobionts, so the task of the present study in the field of feed production is to replace expensive protein components with more affordable ones.
The solution to the problem can be found in the implementation of the concept of sustainable aquaculture as one of the tactical ways to achieve the Sustainable Development Goals. Aquaculture in Russia is one of the most important sectors of food production, and it should be taken into account that in most regions natural and climatic conditions make it most economically feasible to grow objects in recirculating aquaculture systems (RAS). Recirculating aquaculture systems help to reduce the environmental load compared to pond and pasture aquaculture, where pollutants, especially nitrogenous compounds, are discharged into natural water bodies. However, these systems are costly to operate, so there is a constant search to improve their profitability. In the 1980s, the aquaculture industry developed Biofloc technology to improve system water quality and increase fish production. Numerous studies (Luo et al., 2022) have shown the effectiveness of the Biofloc system.
Our investigations are devoted to finding ways for reducing the cost of aquaculture products and improve the quality of marketable objects. The efficiency of water treatment in the Biofloс system is largely determined by the synergistic interaction of bacteria and protozoa that consume organic compounds. While digesting nitrogen and phosphorus contained in the metabolic products of hydrobionts, bacteria, introduced into the RAS, form communities in which trophic relationships emerge.
The operation principle of BFT-systems is to stimulate the natural growth of macroaggregate flocs, enhancing the self-nitrification of water for cultivation. A necessary condition for the functioning of flocs as an analog of biofilter, along with keeping them in suspension, is the presence of a readily digestible source of organic carbon, since bacterial microorganism cultures at the base of flocs are heterotrophic.
The growth of the bacteria, being the basis of the Biofloc system, is carried out with the formation of flocculi. The mucous sheaths formed by the bacteria have adhesive properties, aggregating the bacteria into flocs. The flocs serve as a substrate for the accumulation of other zoogenic bacteria and microorganisms in them, often entering the environment from the gut of hydrobionts. In turn, probiotic microorganisms can enter the gut of fish through while eating them, exerting positive effects on digestion and immunity with proper strain selection (Daniel and Nageswari, 2017). Thus, it is necessary to investigate the available strains of zoogenic bacteria, studying their flocculating ability and potential to create communities of zoogenic organisms with the most significant synergistic effect of cleaning the environment and positive effects on hydrobionts.
Research on the determination of the toxicity rate of the hydrobionts’ biofloc environment via the biotesting method have shown the absence of the toxic impact on the bioindicator. Addition of one probiotic or a combination of probiotics, which are already known, into the biofloc has improved rates of hydrobionts’ marketability and physiological condition under maintenance of water quality parameters. The stunted growth of pathogenic bacteria has been also pointed out as it improves the sanitary condition of the cultivation environment (Baiduk et al., 2023).
As every other technology, Biofloc has a range of limitations and disadvantages. To improve growth rates, species-candidates should be resistant to the high stocking density (Fry et al., 2016). Presence of flocs offers an advantage to species which are omnivorous or have the ability to digest microbial protein. Use of bioflocs in the industrial aquaculture in the cold regions of Russia is followed by the increase in the energy consumption for the active aeration, CO2 emission, and limitation of the list of cultivated boreal species. Nevertheless, advantages of maintaining hydrobionts in RAS play the significant role in the north regions (Nizyaev et al., 2024). Big amount of activated sludge’ sediment can be applied as a feed additive for hydrobionts in the form of the biofloc meal, functional protein ingredient (Gallardo-Colli et al., 2024).
To sum up all the abovementioned, it is necessary to mention that the objective of the present research is to find the most advanced strains of probiotic organisms and study their comprehensive influence on the maintenance of the sustainable condition of hydropionts’ cultivation environment.
2. Materials and methods
2.1. Experimental units and procedures
To test flocculating capabilities of the new bacteria strain, pools with the volume 1 m3 were used. As a nitrogen source for creation of the initial optimal C/N ratio, sturgeon feed “Sturgeon Growth 46/16 VNIRO” («Осетр рост 46/16 ВНИРО») was used. Protein content in it is 46%. The feed contains fat, not less than 16%, and fiber, not less than 3%.
As a carbon source beet molasses was used, the saccharose weight content is 46%, the dry matter weight content – 80.6, the hydrogen index, pH – 8.0. The carbon source selected is affordable and cheap raw material which is often used in the microbiological production.
There were 6 aerators (diffusers) in each 1 m3 experimental pool. They were distributed along all pool surface. Water temperature was controlled within the range from 28 to 29ºC. Temperature, oxygen content, pH level was evaluated with the professional water quality measuring device WATERLINER WMM-97.
The pH level was maintained within the range from 7.7 to 8.2 which optimized conditions for the nitrification and microbial activity. For the acidity maintenance at the level of 300 mg/L, CaCO, sodium bicarbonate was added. It protected the system from the changing pH.
The experimental project meant the formation of the biofloc system on the basis of strains Bacillus velezensis MT141 and MT142 isolated from samples of sediments collected from the river Don at the depth 0-5 cm, at the running research base of the SSC RAS. The CFU concentration for each represented strain was not lower than 2.4·109 CFU/g. As in the preliminary studies this sets of strains shown the high results in the speed of floc-formation in comparison with other probiotic sets, the genetic research was conducted in order to identify strains. After biofloc maturation, Clarias gariepinus were released into pools.
The extraction of samples was conducted every week. Probes were taken from the middle of the pool. Volume of the sample was equal to 1 L. Faucet water, purified and softened with osmotic filters, was added into the pool.
2.2. Sample collection and analysis methods
2.2.1. Water quality analysis
Water quality parameters, including pH, temperature, and oxygen content, were measured with the professional water quality measuring device WATERLINER WMM-97. Water samples were taken in three replications from each system, every 2 days.
Standard analytic protocols (APHA, 2017) were used for the analysis of nitrite, nitrates, total nitrogen (TN).
The biofloc volume (FV) was determined every week using the Imhoff cone (1000 ml) after 5 minutes sedimentation (Luo et al., 2022). The experiment lasted 20 weeks and included weekly sample collection, which were studied as a microbiome community and potential protein source.
2.2.2. Microbiological and biochemical analysis
After performing sample collection, the processing of the biological material was made. 3 dehydrated samples of the substrate were made from the control once via the method of full drying (Kazeev et al., 2016) with the modified range of temperatures: 50ºC, 55ºC, 60ºC accordingly with the use of the Libhof FDW-12 dehydrator. To preserve properties of the potentially valuable feed raw material, drying of microbial sludge was conducted under the technology of the maintenance of working environment temperature and humidity regimes.
To analyze the microbiological contamination of samples, 1 g of substrate was used, which were ground to homogeneous state with the addition of physiological solution (ratio 1:9) in a porcelain mortar. The resulting suspension was used to make a series of serial decimal dilutions (6 orders of dilutions in each sample). Then 0.1 ml of each of the respective dilutions (Anand et al, 2014) were surface seeded on Petri dishes in 3 repetitions on the following nutrient media with standard compositions: GRM agar (FBSUN SSC PMB, Russia), Endo-GRM agar (FBSUN SSC PMB, Russia), urease agar (by Christensen) (HiMedia Laboratories, India), the Chapek's medium (HiMedia Laboratories, India) and starch-ammonia agar, which was made in the laboratory.
To determine the number of bacteria in spore form, the suspension was pasteurized by bringing it to 100°C (Pokhilenko et al., 2022), then a series of serial decimal dilutions were prepared and surface seeding on GRM agar was performed. Seeding was carried out in the II class laminar box (Belakvilon, Belarus). Cultures were grown in thermostats maintaining a constant temperature of 28°C (Anand et al., 2014).
Colonies in the range from 30 to 300 were counted and expressed as colony forming units (CFU/g) after 7 days of incubation for micromycetes and after 2 days for other microorganisms (Anand et al., 2014).
2.2.3. Genetic studies
The DNA was isolated according to the method described by Akash Gautam (Gautam, 2022). The sequencing was performed on the MinION unit using Rapid Barcoding Sequencing Kit V14 reagents according to the instructions. The quality control of reads was performed using the NanoPlot v1.42.0 program, filtering and rejection of low-quality reads was performed using the Filtlong v0.2.1 program. The taxonomic identification was performed using the GTDB-Tk v4.2.0 (Chaumeil et al., 2022) based on the Genome Taxonomy Database (GTDB) (Parks et al., 2022).
2.2.4. Density and analysis of hydrobionts’ condition
At the beginning of rearing, the seeding was 5 eq./L., with a mass of planting material of 16 grams. The water temperature was stable at this stage, around 25±0.3°C when growing commercial fish. Catfish were fed with sinking feeds in the volume of 1-2% of fish biomass. Such feeding rate was chosen in expectation of the presence of additional feed in the system in the form of activated sludge flakes. Feeding of the object was carried out every 5-6 hours (2-3 times a day).
Cultivation was continued for 120 days, weighing of objects was carried out monthly using laboratory scales (A&D, DL (DL-1200), Japan)
3. Results
3.1. Hydrochemical water rates
Fish-rearing pool was filled with water using a pump. Water met corresponding hydrochemical and physical parameters, approved by the norms of water quality composition in the industry standards using a pump (Table 1).
During the experiment, the water quality in the BTF system was regularly monitored. Data on observations are given in Table 2, from which it is obvious that all parameters of the created artificial system correspond to optimal conditions for biofloc culture maturation.
Given the active increase in total flocs, the nitrogen and carbon sources introduced at the water treatment stage were assimilated, and after maturation of the biofloc system, the water levels of biogenic nitrogen compounds were maintained at normal levels, as shown in Figures 1, 2, 3, 4, 5.
3.2. The BTF system parameters
The dynamics of increasing volume of activated sludge flocs was maintained throughout the experiment. The data on the average floc volume are presented in the Table 3. Throughout the experiment the parameters of the biofloc system were relatively stable.
Sufficiently high dynamics of floc formation indicates a high level of exchange and good growth potential of the studied sample. However, microscopy of sludge flocs samples showed the presence of some microscopic eukaryotic organisms, protozoa and roundworms. Further studies are needed to identify these organisms and their role in the biofloc microbiome.
The dynamics of floc formation is shown in Figure 6.
The isolated strains showed a high level of exoprotase and exoamylase secretion. The strains did not show hemolytic and lecithinase activity, there was no resistance to antibiotics. When probiotic characteristics were analyzed, Bacillus velezensis strains MT141 and MT142 showed rapid and abundant growth, high rate of bacterial biomass formation. They also have the ability to secrete lytic enzymes that can break down complex organic substances to simple ones in water, making them more available for fixation by other members of the microbial community.
The studied strain preparation was made experimentally based on a combination of spore-forming bacterial strains. The content of bacillary cells was 2.4-109 CFU/g.
The results of our studies showed that inoculation into the substrate of the BTF system of an experimental probiotic containing 2 strains of bacteria p. Bacillus velezensis (MT141, MT142), had a positive effect on the contamination of the analyzed samples with respect to the presence of Gram-negative Escherichia coli. Thus, when tested on the Endo medium of substrates with commercial and experimental probiotic, it was found that strains MT141 and MT142 reduced the number of colonies forming units of Escherichia coli by 60.9%.
Bacteria belonging to the Escherichia coli group accounted for about 3% of the microbial community studied. The low relative content of E. coli bacteria indicates that the biofloc microbiota successfully suppresses further development of potentially pathogenic intestinal bacteria.
A similar situation was established for the quantitative values of other E. coli group bacteria in the comparative testing of the samples. In the substrate with the experimental probiotic, the number of CFU/g of E. coli bacteria was 71.17% lower compared to the commercial preparation (Table 4).
Quantitative indicators of contamination of the BTF system substrate samples with Escherichia coli and other E. coli bacteria.
Thus, inoculation of an experimental probiotic preparation containing p. Bacillus velezensis strains (MT141 and MT142) into the Biofloc system at its start-up allows to significantly reduce the contamination of the substrate with Escherichia coli bacteria. Temperature treatment of the substrate allows to achieve 100% purity of samples in terms of the presence of Escherichia coli and other ECBG. The principal presence of ECBG is due to the direct connection of the Biofloc system with hydrobionts, with the feces of which there is a release of representatives of intestinal microbiota. Determination of the ecological groups of the microbial community of the Biofloc system showed that in the control sample there is a high number of bacteria capable of absorbing complex organics.
These bacteria are able to absorb organic matter from water and use some of it to build their cells, divide and grow. The other part of organic matter they decompose into metabolic products, including carbon dioxide and nitrogenous compounds.
In a comparative analysis of quantitative indicators of the control and experimental sample, we can conclude about the favorable effect of experimental probiotics on the structure of the microbiome substrate of the Biofloc system. An increased number of bacteria capable of fixing urea (128.9 times) was observed in the experimental sample compared to the control. Thus, the microbial community of the experimental sample can be characterized as active and capable of fixing both organic compounds, urea and ammonium compounds from water.
3.3. Genetic studies
Genetic analysis allowed us to identify the strains used for floc cultivation as MT141 – Bacillus velezensis, MT142 – Bacillus velezensis. Some authors (Gao at al., 2024) consider this bacterial species as an alternative to antibiotics because it has eight gene clusters for the biosynthesis of secondary metabolites associated with antibacterial compounds. Studies on the effect of probiotic properties on Macrobrachium rosenbergii, showed the effect of increasing the immunity of the hydrobiont grown in medium with strain B. velezensis CPA1-1, which makes it a valuable component of the Biofloc system (Gao at al., 2024).
3.4. Hydrobionts’ condition during the cultivation
The dynamics of mass gain exhibited by the test object shows a significant growth rate of the object (Table 5). Taking into account that the marketable weight of African catfish when grown in closed water supply facilities averages 800-900 grams in 10-12 months, the achievement of a total weight average of 711 grams for the experimental period of 4 months is a good indicator of the quality of the environment in which the growth of the object is carried out.
It should be noted that the active dynamics of catfish weight gain was observed despite the fact that the amount of feed was reduced compared to generally accepted norms, but activated sludge, actively growing at high concentration of exogenous carbon and biogenic nitrogen, serves not only as a biological additive, but also as a good food substrate.
Negative dynamics of activated sludge floc formation at 13 and 14 weeks is explained by the limiting content of activated sludge flocs, they begin to clog the gills of Clarias catfish. To reduce the amount of activated sludge flocs, the sedimentation method is used. Water with activated sludge is drained into the tank using a lift pump. The drained water is then sedimented for 15 minutes, then the top layers of water are returned back to the main pool. Next, purified water passed through a reverse osmosis filter was refilled. The precipitated activated sludge flakes, after sampling, are disposed of as household waste.
4. Discussion
Studies of the interaction between the microbiome formed by probiotic cultures in the flocs of activated sludge and the potential object of cultivation, Clarias catfish in an artificially regulated ecosystem allowed us to suggest that despite the need for external regulation of the system parameters, the addition of an external source of carbon, molasses and nitrogen in the composition of feed, in this system we can observe signs of the presence of a synergistic effect of the interaction of zoogenic components of the microbiome. An important element of maintaining the effective state of the biofloc system is the constant provision of its sufficiently high oxygen content as a result of the action of aerators, which requires constant additional energy consumption (Jiang et al., 2023).
However, the active development of the biofloc system resulted in the accumulation of a significant amount of organic matter of flocs, which must be regularly removed due to their negative impact on the respiratory system of hydrobionts. This suggests that there is a certain potential for bioproductivity in the created closed ecosystem, despite the fact that the system is completely heterotrophic. It can be assumed that the interaction of the selected microbial strains was quite successful, creating comfortable conditions for the maturation and interaction of the microbial community. It was not possible to obtain fully matured flocs and examine their unicellular eukaryotic and multicellular components due to the limited duration of the experiment; nevertheless, preliminary studies show that short food chains can be formed within the biofloc, which expands the potential of the biofloc system and creates prerequisites for the emergence of a stable self-regulating system.
Despite the fact that a well-known example of synergetic interaction of microbial components of aquatic ecosystems was the phenomenon of eutrophication, associated rather with complex negative consequences of changes in ecosystem composition, modern studies show the presence of synergistic effects in the complex reduction of toxic stress for aquatic plants (Hu et al., 2024), and accelerated biodegradation of pollutants.
In our study, we observed the preconditions for the emergence of a system in artificial conditions, in which the processes of microbiome self-organization give a complex positive effect both for the cultivation of quality aquaculture products in isolated conditions of RAS and for reducing the costs of feeding and water treatment. The investigated probiotic strains significantly improve water quality by eliminating nutrient pollution by nitrogen compounds. In addition, probiotics were found to effectively suppress potentially pathogenic microorganisms belonging to the E. coli group, thus providing improved environmental sanitation. Since symbiotic organisms are of zoogenic origin, isolated from the natural ecosystem, when entering the gastrointestinal tract of the object favorably affect the intestinal microflora of the growing object.
In addition to the above-described positive effect on the medium-grown object system, bacterial flocs have a high nutritional value. Taking into account that in the experiment the rate of weight gain by hydrobionts was high and the feeding rate was reduced in comparison with that accepted in aquaculture, flocs represent a valuable source of proteins and lipids that are well digested by hydrobionts.
The prospect of expanding the use of the biofloc systems is associated with the search for the most effective floc-forming strains, the study and selection of more highly organized elements of the microbiome, regulation of the ratio of biogenic elements through the introduction of autotrophic components.
Creation of such a complex and its technological improvement may allow, removing excess organic matter from the mature BFT system, to use it for preparation of feed additives and feeds for hydrobionts.
5. Conclusions
Application of the BFT system in aquaculture allows to reduce expenses on the water purification in natural and enclosed systems for hydrobionts’ cultivation which improves profitability of aquaculture enterprises, hydrobionts’ cultivation conditions and provides high quality of products.
Efficiency of the BFT system is determined by its high productiveness and symbiontic characteristics of the initial probiotic strain. The investigated strains have the property to suppress the pathogenic microflora and, thus, to improve sanitary cultivation conditions.
Productivity of the studied strain is stated through the active growth of biofloc volumes and use of flocs by hydrobionts as a fodder substrate and probiotic with immunostimulating effect.
The synergetic effect arising from the interaction of organisms in the BFT system provides its sustainable development and, due to this, which reduces the need for external regulation of processes, resulting in reduced costs of growing hydrobionts. This is in line with the currently stated goals of sustainable development and contributes to expanding the prospects of green economy.
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 a large-scale research project within the priority areas of scientific and technological development).
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