Using BioFloc Technology to Improve Aquaculture Efficiency

Abstract

In the present study, literature information on the functioning of the biofloc technology (BFT) system, its components, the state of the organism of hydrobionts, and water quality is analyzed. It is shown that this technology allows reducing financial costs for water treatment by 30%, increasing the efficiency of protein assimilation in the feed composition by two times, and creating a high-protein substrate, which can be further used as a component of feed for aquaculture. The BFT contains a large number of microorganisms, including photoautotrophic microorganisms (algae), chemoautotrophic microorganisms (nitrifying bacteria), and heterotrophic microorganisms (fungi, infusoria, protozoa, and zooplankton). This technology contributes to the improvement in water quality, aquaculture productivity, and hydrobionts. Despite the higher initial costs, BFT can yield higher economic profits. In this paper, the authors summarize data from many recent studies devoted to BFT. Based on the analysis of a number of studies, it can be concluded that this technology has a high potential for scaling up in industrial aquaculture.

1. Introduction

In recent years, the issue of rational use of water resources in aquaculture has become increasingly acute. The technological process of growing hydrobionts implies the formation of significant volumes of wastewater saturated with various pollutants, which eventually enter into natural aquatic ecosystems, worsening their ecological state [1]. Sustainable aquaculture development, which will provide the world’s population with protein of animal origin, should be associated with the greening of the process of growing hydrobionts without damaging the environment [2,3]. Moreover, sustainable aquaculture development will contribute to reducing hunger and poverty and ensuring food security and economic growth in some countries [4].

The already traditional way of maintaining water quality in aquaculture is the use of a multistage treatment system, through which mechanical and biological water purification is carried out. In the biological filtration stage, the main cleaning agent is nitrifying bacteria that utilize ammonia from the water, which is toxic to fish in high concentrations. However, in recent years, research interest in biofloc technology (BFT) has increased, and many recognize it as a growing system with high potential [5], as well as an environmentally friendly technology [6,7]. BFT has the advantage of targeting the establishment of heterotrophic bacterial communities that utilize ammonium more efficiently compared to nitrifying bacteria [8]. The creation of the required biomass of heterotrophic bacteria is accomplished by adding increased carbon content to the growth systems. When acetate or glycerol is used as a carbon source, 15 g of source per 1 kg of fish daily is required [3].

BFT allows reducing financial costs for water treatment by 30%, increasing the efficiency of protein digestion in the feed composition by almost two times by creating a high-protein substrate (flocculus), which can later be used as a component of feed for aquaculture [6,9]. Increasing the efficiency of feed digestion at this time is an important issue in aquaculture because about 75% of the incoming nitrogen is not digested and remains in the water [10], polluting it. In addition, BFT is promising for use in regions with water scarcity [11].

The first study on the development of BFT began in the 1970s, but more extensive research dates back to the 1990s [12]. It is recommended to grow shrimp and tilapia using bioflocs because these species are able to consume the flocculates that are formed as food [5]. Among tilapia, Oreochromis niloticus [13,14] is the most adapted to being reared in the BFT due to their ability to consume in situ produced particles. This ability of tilapias was proved during the experiment on labeling with stable nitrogen isotopes [15].

Despite the obvious advantages of this technology for cultivating commercial aquatic organisms, the spread of BFT in the aquaculture sector is currently localized. This review provides a comprehensive assessment of the BFT performance. The scientific value of the present review is determined by the accumulation in one article of the diverse literature on the implementation, operation, and impact of the BFT in commercial aquaculture. Materials demonstrating the efficiency of aquaculture object cultivation in the BFT (growth rate, survival rate, and feed conversion ratio) are presented. The main disadvantages of the BFT are briefly presented. It allows for the emphasis of timely critical problems in the implementation of the BFT in production.

2. BFT Components

BFT is a revolutionary approach in aquaculture that allows the development of sustainable and highly productive aquatic culture systems. The method is based on the principle of a closed or partially closed system with minimal water exchange and recycling of nutrients to increase the carbon to nitrogen (C/N) ratio. This creates conditions for the active growth of heterotrophic bacteria, algae, and other microorganisms in the BFT [16]. The microbial consortium formed in BFT not only purifies water of nitrogenous waste but also acts as a live feed rich in protein and other essential nutrients for growing species [17].

The composition of BFT is extremely diverse and depends on many factors, including initial water composition, type of organisms cultured, type of feed used, and aeration regime. BFT components are a complex mixture of organic matter, including feed residues, aquatic feces, dead microbial cells, exopolysaccharides (mucus), colloidal particles, and various organic polymers. These components are held together in a flexible matrix predominantly composed of extracellular polysaccharides secreted by bacteria [18]. The mucus secreted by bacteria plays a key role in the formation and stabilization of bioflocs. The presence of filamentous bacteria and electrostatic interactions between different particles further strengthens the structure of bioflocs [19]. The size of bioflocs varies from a few micrometers to several millimeters, which ensures food availability for different size groups of cultured organisms [18,20]. It is important to note that the density of bioflocs is slightly higher than the density of water, which ensures their slow sedimentation (1–3 m h⁻¹), preventing rapid settling and the formation of anaerobic zones on the bottom [21].

Moreover, within BFT, nitrogen compounds are effectively recycled, which reduces the need for water treatment chemicals. However, there are some nuances to consider. The effective operation of BFT requires careful monitoring of water parameters such as pH, dissolved oxygen, temperature, and C/N ratio [22,23]. The condition of the BFT must be constantly monitored to ensure optimal conditions for their development. Improper management can lead to the development of anaerobic conditions, accumulation of toxic substances, and degradation of water quality. Successful application of BFT requires certain knowledge and skills, as well as continuous monitoring and adjustment of system operating parameters. Despite these challenges, BFT represents a promising way to intensify aquaculture, allowing for environmentally friendly and economically viable aquatic rearing systems [17]. These communities also contribute to improved feed efficiency and growth of cultured organisms [18].

The principle of the creation and functioning of the BFT is based on the process of the formation of edible floccules or flakes (single-celled proteins, SCPs) resulting from heterotrophic processes, where the substrate is feed residues, aquaculture excreta, and excess nutrients. The binder for the flocculi/flakes formed is bacterial slime [5]. As defined in the United States Department of Agriculture’s National Agricultural Library Glossary, biofloc is “the use of aggregates of bacteria, algae, or protozoa held together in a matrix along with solid organic matter to improve water quality, waste treatment, and disease prevention in intensive aquaculture systems” [12].

The entire abundance of microorganisms that make up BFT can be categorized into the following three groups: (1) photoautotrophic microorganisms (algae), (2) chemoautotrophic microorganisms (nitrifying bacteria), and (3) heterotrophic microorganisms (fungi, infusoria, and protozoa) and zooplankton [24].

The most important component of a BFT is microorganisms (including probiotic bacteria), which in symbiosis with other components create the basis for effective cultivation of fish and other hydrobionts. Many endogenous BFT components have probiotic properties [24]. Among exogenous probiotics, the introduction of Bacillus was tested in the BFT, resulting in positive results [25].

The bacterial community formed in each specific case of BFT is much wider. Researchers have noted the presence of bacteria of the genus Bacillus (Bacillaceae), namely, B. subtilis, B. amuloliquefaciens, B. licheniformis [26], B. cereus, B. aquamaris, B. tequilensis, and B. niabensis [27], and bacteria of the genus Cellulomonas (Cellulomonadaceae), for example, C. biazotea [26]. The presence of bacteria of the genus Pseudomonas (Pseudomonadaceae), namely, P. stutzeri and P. denitrificans, and bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae), namely, R. palustris, is recorded [26]. Gram-negative nitrite-oxidizing bacteria of the genus Nitrobacter (Nitrobacteraceae), N. winogradskyi, and ammonia-oxidizing bacteria of the genus Nitrosomonas (Nitrosomonadaceae), N. europaea, are recorded in biofloc systems [26]. There are representatives of the genus Exiguobacterium (Bacillaceae), namely, Exiguobacterium sp., E. aurantiacum, E. profundum, E. indicum [27], and others [28]. According to some estimates, up to 2000 bacterial species can function in a BFT [29]. In some cases, up to 90% of the bacterial community may consist of representatives of Vibrio sp. [30]. The qualitative and quantitative composition of the bacterial community in a BFT is influenced by various factors, including water temperature, C/N ratio, alkalinity, salinity, light, and pH [31].

The high abundance of bacterial species in BFT allows the flocculant to be used as biomaterial for the search for probiotic bacteria. Moreover, researchers have already identified probiotics specific to the BFT, among which the B. licheniformis isolate CPQBA 571-12 DRM 07 demonstrated antagonistic activity against a pathogenic strain of Vibrio alginolyticus (BCCM 2068) [32]. Each bacterial group in the BFT plays a different role. For example, one of the properties of bacteria of the genus Bacillus is to have a probiotic effect on the system and utilization of inorganic and organic nitrogenous compounds; proteobacteria and bacteroidetes participate in the degradation of nitrogen compounds; pseudomonads participate in the processes of denitrification and decomposition of hydrocarbons; and bacteria of the family Saprospiraceae participate in the hydrolysis of complex organic substances [33]. Microbial communities in bioflocs effectively compete with pathogens for resources and space, creating a natural barrier against infections. For example, BFT has been shown to help control pathogens that provoke the development of pathologies such as acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei [34] and pathogens in tilapia [35].

Before starting the BFT, a starter solution is prepared, which may include 300 g of molasses, 100 g of probiotic bacteria, and 10 kg of sea salt (not iodized), which is placed in a 20 L tank and aerated for 1–2 days [5]. Other recommendations for starter solutions with a C/N ratio of 15:1 are to use 1 L of molasses (carbon source) and 1 kg of commercial compound feed (nitrogen source) [26]. Glucose, acetate, starch, wheat, and glycerol can also be carbon sources [36]. Carbon in this system is essential for flake formation [37]. Before inoculating the starter solution into the BFT system, it is necessary to verify the presence of microbial flakes, which is performed using a special Imhoff cone (Figure 1).

Water aeration is an important component for the successful operation of a biofloc system and increasing aquaculture productivity because heterotrophic bacteria use large amounts of oxygen to assimilate ammonia [38]. The selection of an optimal aeration unit is an open question [3]. Nevertheless, there are separate studies on this issue in the literature. For example, for culturing the shrimp, Penaeus vannamei, under BFT conditions, the use of diffuse aerators improved bioflocculus formation and shrimp culture efficiency compared to other aerators tested [39].

In addition to bacteria, unicellular algae, such as Chlorella sp., can be components of a BFT. Bacteria and microalgae in such systems coexist in a mutualistic relationship as follows: bacteria remineralize and regenerate inorganic nutrients and carbon dioxide for use by microalgae, and algae produce oxygen needed by heterotrophic bacteria [40]. Chlorella is a source of essential amino acids and highly unsaturated fatty acids for shrimp [41]. In addition, the presence of microalgae in the system provides food resources for zooplankton [42].

In addition to Chlorella, the following microalgae are recorded in BFT systems: Scenedesmus quadicauda, Pediastrum duplex, Coelastrum, Cyclotella, Navicula, Synedra, Fragilaria, Orthoseira, Rhabdonema, Ulothrix, Skeletonema, Cylindrotheca, Hemiaulus, Phymatodocis, Ulothrix, as well as taxa Cyanobacteria, Chlorophyta, Bacillariophyta, Euglenophyta, and Dinophyta [24]. In addition to microalgae and bacteria, the components of the BFT are microscopic fungi, protozoa, rotifers, ringworms, nematodes, and paddlefish [22]. Microscopic fungi are heterotrophic organisms and carry out the utilization of organic contaminants. The 18S rDNA analysis allowed the identification of nine fungal species present in the biofloc system during shrimp cultivation: Aspergillus versicolor, A. niger, A. tamarii, A. flavipes, A. aculeatus, Penicillium citrinum, P. griseofulvum, Trichoderma virens, and Pestalotiopsis microspora [43].

Thus, a complex ecosystem is formed in the BFT, which can be characterized as a mixotrophic system because it contains organisms with autotrophic and heterotrophic types of nutrition [36].

3. Impact of BFT on Aquaculture Productivity

Experimental cultivation of hydrobionts in the BFT has repeatedly shown greater efficiency than traditional cultivation methods, as demonstrated in scientific articles. In the course of scientific research, the following different species of hydrobionts have been tested in the BFT: Penaeus vannamei (Boone, 1931), Penaeus monodon Fabricius, 1798, Farfantepenaeus (Penaeus) brasiliensis Latreille, 1817, Litopenaeus (Penaeus) stylirostris (Stimpson, 1871), Farfantepenaeus (Penaeus) duorarum (Burkenroad, 1939), Macrobrachium rosenbergii [12,44], tilapias [36,45,46,47,48], trout [49], South American catfish Rhamdia quelen (Günther, 1868) [50,51,52], African clarium catfish Clarias gariepinus (Burchell, 1822) [53], channel catfish Ictalurus punctatus (Rafinesque, 1818) [54]; beluga Huso huso (Linnaeus, 1758) [55], Russian sturgeon Acipenser gueldenstaedtii Brandt, 1833 [56], carp Cyprinus carpio Linnaeus, 1758 [57], Pelteobagrus vachelli (Richardson, 1846) [58], and others. The main biotechnical factors that affect the productivity of a BFT are the stocking density of aquaculture objects and the availability of feed resources, which has been demonstrated in shrimp farming [59,60]. Also, the biofloc-based system, due to its closed nature, prevents the escape of cultured individuals and reduces the spread of diseases. The constant presence of microbial flakes is an additional source of nutrients, which reduces the feed conversion ratio and can reduce feed consumption, which improves the growth and survival of hydrobionts [7]. In an efficient BFT, the cost of fish feed is reduced by 30% because each pellet is basically consumed twice, first as a fresh pellet and then as an edible bioflocculant, which contributes to higher productivity and profitability of production [5]. Biofloc materials have high nutritional value, making them an excellent source of staple food for aquaculture. In dry form, biofloc contains 12% to 50% protein, 0.5% to 41% lipids, 14% to 59% carbohydrates, and 3% to 61% ash [5,16]. In addition, biofloc can include essential omega-3 polyunsaturated fatty acids such as docosahexaenoic and eicosapentaenoic acids and promotes their accumulation in live food organisms such as rotifers, which can then be used as live feed for fry and juveniles [5]. Selection of optimal parameters will make BFT the most productive farming system, especially in regions with water scarcity.

A group of Chinese researchers in experiments in RAS and BFT showed that fish weight, total growth, specific growth rate, and feed conversion ratio were more efficient when fish were kept in BFT [61]. Analysis of the scientific literature, which outlines the results of an experimental study on culturing hydrobionts in the biofloc, allows us to conclude that the BFT may have an advantage in growing some types of aquatic organisms compared to more traditional aquaculture methods (

Table 1. Fish biological parameters of different species when reared in BFT.

Species	DE	EC	IBW	FBW	S	SGR	FCR	Reference
Carp Cyprinus carpio	8 weeks	C/N-10	6.91 ± 0.13 a	11.32 ± 0.34 b	93.33 ± 2.89 b	0.88 ± 0.08 b	2.51 ± 0.63 a	[57]
		C/N-15	6.88 ± 0.08 a	11.40 ± 0.48 b	100 a	0.90 ± 0.07 b	1.67 ± 0.45 ab
		C/N-20	6.91 ± 0.09 a	13.58 ± 0.21 a	100 a	1.21 ± 0.02 a	1.27 ± 0.08 b
Nile tilapia Oreochromis niloticus	84 days	35% CP without BFT	99.61 ± 13.74	127.51 ± 28.17 b	100	-	4.97 ± 0.12 a	[62]
		35% CP with BFT	100.69 ± 13.61	140.72 ± 27.26 a	100	-	3.51 ± 0.44 b
		24% CP with BFT	98.45 ± 12.71	138.58 ± 24.99 a	100	-	3.44 ± 0.45 b
African catfish Clarias gariepinus	122 days	Control	-	956 ± 28 a	98 ± 2 a	-	-	[53]
		BFT	-	1077 ± 77 a	100 a	-	-
South American catfish Rhamdia quelen	21 days	T-HET	-	0.066 ± 0.0125 ab	44.0 ± 10.1 a	-	-	[50]
		T200	-	0.0886 ± 0.0084 a	38.1 ± 3.4 a	-	-
		T400–600	-	0.0457 ± 0.0063 b	54.4 ± 2.4 a	-	-
		T800–1000	-	0.0445 ± 0.0044 b	51.1 ± 2.7 a	-	-
		Control	-	0.0649 ± 0.022 ab	10.2 ± 4.5 b	-	-
African catfish Clarias gariepinus	6 weeks	Control	5.13 ± 0.13	22.60 ± 1.58	60.0 ± 2.3 **	3.52 ± 0.20	-	[63]
		Sucrose BFT	5.07 ± 0.07	24.13 ± 1.86	76.3 ± 4.9 **	3.70 ± 0.22	-
		Glycerin BFT	5.07 ± 0.18	22.63 ± 1.62	90.6 ± 2.4 ***	3.55 ± 0.09	-
		rice bran BFT	5.00 ± 0.11	23.46	22.6 ± 22.6 *	3.68	-
Shrimp Penaeus vannamei	120 days	150 Shrimp/m2	0.96 ± 0.28 a	15.6 ± 1.70 a	92.0 ± 2.55 a	-	1.40 ± 0.09 a	[64]
		300 Shrimp/m2	0.96 ± 0.28 a	16.8 ± 0.93 a	81.2 ± 3.09 b	-	1.29 ± 0.05 a
		450 Shrimp/m2	0.96 ± 0.28 a	9.0 ± 1.20 b	75.0 ± 3.74 c	-	2.41 ± 0.55 b
Shrimp Penaeus vannamei	33 days	Blower	4.30 ± 0.93	12.96 ± 2.63 a	86.0 ± 3.0 a	-	1.71 ± 0.15 ab	[39]
		Vertical pump	4.30 ± 0.93	10.93 ± 2.66 b	92.3 ± 5.68 a	-	1.56 ± 0.17 a
		Propeller	4.30 ± 0.93	12.81 ± 2.21 a	55.0 ± 18.3 b	-	1.99 ± 0.21 b
Shrimp Penaeus vannamei	42 days #	Control	3.03 ± 0.12	10.42 ± 0.03	99.2 ± 1.66	2.93 ± 0.18	1.96 ± 0.15	[32]
		Probiotic	3.03 ± 0.12	10.8 ± 0.18	95.8 ± 3.19	2.97 ± 0.11	2.18 ± 0.21
Shrimp Penaeus vannamei	13 weeks	low solids content	-	22.1 ± 0.3 (21.7–22.7) a	49.7 ± 3.1 (43.9–54.5)	-	2.5 ± 0.1 (2.3–2.7)	[65]
		High solids content	-	17.8 ± 0.2 (15.3–19.7) b	49.4 ± 5.9 (41.7–66.5)	-	3.3 ± 0.4 (2.0–4.0)
Freshwater shrimp Macrobrachium rosenbergii	45 days #	Control	0.25 ± 0.01	2.91 ± 0.07	88.72 ± 1.78	-	-	[66]
		Glucose	0.25 ± 0.01	3.15 ± 0.13	93.85 ± 3.08	-	-
		Sucrose	0.25 ± 0.01	3.09 ± 0.16	91.79 ± 4.95	-	-
		Molasses	0.25 ± 0.01	2.97 ± 0.10	92.82 ± 3.20	-	-
Tilapia Oreochromis niloticus	8 weeks	Control	50.25 ± 0.78 a	146.66 ± 0.85 b	100 a	1.92 ± 0.02 b	0.97 ± 0.01 a	[67]
		BFT	50.61 ± 0.91 a	160.54 ± 3.06 a	100 a	2.04 ± 0.01 a	0.83 ± 0.03 b

DE—duration of the experiment; EC—experimental conditions; IBW (g)—initial body weight; FBW (g)—final body weight; S (%)—survival; SGR—specific growth rate; FCR—feed conversion ratio; CP—crude protein; T-HET—absence of suspended solids at the beginning of the experiment (biofloc formation was carried out by daily addition of dextrose); T200—total suspended solids up to 200 mg L⁻¹; T400-600—total suspended solids from 400 to 600 mg L⁻¹; T800-1000—total suspended solids from 800 to 1000 mg L⁻¹. Mean values with different superscript letters in the same column, given in a row of the same experiment, indicate a significant difference (p < 0.05). *, **, and *** indicate significant differences (p < 0.05) from all others in this research; ^#—No statistically significant difference (p > 0.05).

). However, further research is required to obtain a more extensive array of data.

The data in Table 1 show that the highest survival rate (100%) is characteristic of tilapias. Despite the high survival rate, one of the experiments showed a high feed conversion ratio (FCR) (3.44–4.97) [62], which significantly reduces the economic feasibility of growing hydrobionts in the BFT. In another experimental rearing of tilapias for 8 weeks in the BFT system [67], the FCR was much lower and ranged from 0.83 to 0.97. High values of survival rate, judging from the results of these studies, are also characteristic of carp. When carp were reared for 8 weeks in the Biofloc system, survival rates ranged from 99.33% to 100% [57]. Similar rearing results have been observed for different shrimp species. However, when different means of oxygenating the water were tested, the survival rate of Penaeus vannamei was found to decrease up to 55% when an aerator with a propeller aspirator was used. Judging from the results, this equipment is not suitable for BFT with shrimp, as rupture of the bioflocculant was observed. In addition, low abundance was observed with this aeration method.

4. Condition of Hydrobionts Under Cultivation in BFT

BFT can significantly reduce or even eliminate the problems associated with high stocking densities of hydrobionts [68]. Under biofloc conditions, fish and other aquatic organisms derive many benefits, including improved growth performance, increased survival, optimized feed utilization, and enhanced immune systems against various diseases [69].

The application of BFT has a significant impact on the health of cultured organisms such as fish and shrimp larvae [70]. By improving water quality and providing nutritious biofloc, BFT helps to strengthen the immune system of cultured species, increasing their resistance to disease [70,71]. Limited or zero water exchange in BFT drastically reduces the risk of spreading infections, providing high biosecurity [72].

It is important to note that BFT can contain various biologically active compounds such as carotenoids, chlorophyll, polysaccharides, phytosterols, taurine, and fat-soluble vitamins [16,73]. These components not only promote the growth and health of aquatic organisms but can also serve as powerful immunostimulants. A wide range of microorganisms and their cellular components are used as probiotics to improve the innate immunity of aquatic organisms and enhance their antioxidant status. Numerous studies confirm that BFT stimulates the non-specific immune system in both fish and shrimp, making them more resistant to diseases [66,74,75,76] The BFT can also have a positive effect on antioxidant activity in fish. For example, it is reported in the literature that some fish species, such as O. niloticus and C. carpio, showed an increase in antioxidant enzymes superoxide dismutase and catalase, which may indicate a decrease in oxidative stress and improved health of hydrobionts. Increased antioxidant defense was also observed in Opsariichthys kaopingensis and Carassius auratus (Linnaeus, 1758) cultured in BFT, which was manifested in lower levels of lipid peroxidation and more pronounced resistance to free radicals [68].

In various articles devoted to the influence of BFT on the immune system and health of aquatic organisms, the authors focus on different parameters that allow us to assess the level of influence of BFT on the organism. For example, in the review by Kumar et al. [77], it was described that microorganisms present in the BFT produce signaling molecules that activate the host immune response and help protect the host from pathogens. For example, a significant increase in total antioxidant capacity and superoxide dismutase activity, as well as an improvement in the ratio of reduced to oxidized glutathione, has been observed in shrimp grown in such systems. The bacterial cell wall consists of various components such as bacterial peptidoglycan, lipopolysaccharide, and β-1,3-glucans, which may act as immunostimulatory agents to promote non-specific immune activity in shrimp. The BFT can enhance shrimp survival, final weight, and the activity of the prophenoloxidase (proPO) cascade, which is an important element of the innate immune response in crustaceans. This occurs through upregulation of enzymes such as profenoloxidase1 and profenoloxidase2, serine proteinases, and ras-related nuclear proteins. Thus, it can be concluded that the presence of beneficial microbes in the system contributes to improved growth, immune response, and disease resistance in cultured animals [77].

In a study conducted by Panigrahi et al. [78], the role of non-specific immune responses in Penaeus indicus shrimp was analyzed. In this study, BFT was supplemented with nine strains of bacteria of the genus Bacillus, and it was demonstrated that bioaugmentation using selected strains of Bacillus spp. significantly enhanced the innate immunity of shrimp. This was manifested by an increase in phenol oxidase (PO) activity and an increase in total hemocyte count (THC). Also, the study showed an increase in differential hemocyte count (DHC), and increased phagocytosis and lysozyme (LZM) activity was observed [78]. In L. vannamei, BFT treatment significantly increased THC (47.24 × 10⁶ cells ml⁻¹), hyaline cell count (18.21 × 10⁶ cells ml⁻¹), and granular cell count (29.04 × 10⁶ cells ml⁻¹). There was also an increase in serum protein level (82.7 mg mL⁻¹), PO activity (0.73 optical density at 490 nm), and LZM activity (54.54%) [79]. Hemocytes play a key role in the realization of immune response, phagocytosis, encapsulation, and storage and release of profenol oxidase in crustaceans and other invertebrates [80,81]. Lipopolysaccharides (LPSs) and β-1,3-glucans can have a pronounced effect on stimulation of phenol oxidase activity [68]. Due to the fact that shrimp grown in BFT absorb the formed flocculi and microbial aggregates [82,83,84], the increase in the number of hemocytes and phenoloxidase activity may indicate a positive effect of biofloc on the shrimp immune system [85].

Ahmad et al. [86] demonstrated that tapioca (starchy root)-based biofloc stimulated non-specific immunity in Labeo rohita fry. The increase in the activity of key immune indicators such as nitroblue tetrazolium (NBT), myeloperoxidase (MPO), total immunoglobulin (Ig), and LZM indicated a potent immunostimulatory effect of BFT [86]. These results were confirmed by Menaga et al. [87] on GIFT (genetically improved farmed tilapia), O. niloticus tilapia, where BFT led to increased expression of genes related to immune response such as metallothionein, cathepsins, toll-like receptors (TLRs), interleukin 1 (IL-1), and tumor necrosis factor (TNF). Moreover, BFT demonstrated direct antimicrobial activity against Aeromonas hydrophila, a common fish pathogen [87].

The mechanisms of immunostimulation inherent in BFT are complex and multifaceted. BFT contains a number of biologically active substances with immunostimulatory effects. For example, peptidoglycans (components of bacterial cell walls), beta-glucans (polysaccharides from the cell walls of fungi and algae), and LPS, which are powerful immunomodulators capable of activating various parts of the immune system. These substances act as probiotics, improving the gut microbiota and stimulating the immune system. The interaction of various components of BFT, including poly-β-hydroxybutyrate (PHB), a biodegradable polymer produced by some bacteria, contributes significantly to immunomodulation. PHB can stimulate the production of short-chain fatty acids (SCFAs) by biofloc bacteria. Short-chain fatty acids, in turn, have probiotic properties, promoting the growth of beneficial bacteria in the gut and strengthening the immune system [74,84,88].

Ebrahimi et al. [89] found a significant increase in the level of complement system components in fish reared in the BFT system [89]. Complement components such as C₃ and C₄ are important for opsonization (tagging) of pathogens, lysis (destruction) of pathogen cells, and activation of other immune cells [90]. Increased C₃ levels in O. niloticus [90] and C. argus [91] grown under BFT conditions indicate a more effective innate immune response. Experiments with Nile tilapia, Oreochromis niloticus, grown in BFT system showed a significant increase in complement C₃ level (90.42 ng mg⁻¹ protein) compared to the control group (25.32 ng mg⁻¹ protein) [90].

Enhanced immune response may also be associated with increased plasma protein levels in fish, since total plasma protein, including albumin and globulin, is synthesized in the liver and can serve as an important indicator of health and immune function [92]. In gray mullet, Mugil cephalus, an increase in both albumin and globulin levels in plasma was recorded, which is interpreted by the authors as a sign of adequate immune response [93]. A study by Mansour and Esteban [74] confirms these findings, showing a significant increase in total plasma protein and albumin in Nile tilapia reared in BFT [74], which, according to the authors, is due to an improved innate immune response, which is the first line of defense against infections.

Another study conducted by Nageswari et al. [94] showed that Pangasianodon hypophthalmus reared in BFT based on different plant ingredients (tapioca, sorghum, pearl millet, and finger millet) also showed a significant increase in serum albumin, which may indicate a positive effect of the BFT on fish immunity and may be related not only to microbial protein consumption but also to bioactive compounds present in biofloc such as polysaccharides, lipids, enzymes, and other metabolites of microorganisms that have immunomodulatory properties [94]. Moreover, the composition of BFT can vary depending on the feed used, which may influence its biological activity and consequently the immune response of fish [68].

Accordingly, BFT is not only a way to increase aquaculture productivity but also an effective technique to regulate the health of farmed hydrobionts, with particularly significant effects observed in the area of immune response modulation.

5. Water Quality Using BFT Technology

The accumulation of ammonia due to its untimely removal from aquaculture systems is one of the limiting factors for the intensive development of this industry [95]. There are several approaches to water purification in aquaculture as follows: photoautotrophic algal assimilation, chemoautotrophic bacterial nitrification (CBN), and heterotrophic bacterial assimilation (HBA) [3,12], which are also combined in the BFT. According to the information presented in Khanjani, M.H., Mohammadi, A., and Emerenciano, M.G.C. [24], different biocomponents of BFT have different effects on water quality as follows: (1) photoautotrophic microorganisms reduce NH₃ and NO₃, increase O₂, and have no effect on NO₂; (2) chemoautotrophic microorganisms first increase and then decrease the concentration of NH₃ and NO₂, increase NO₃, and decrease the concentration of dissolved oxygen in water; (3) heterotrophic microorganisms reduce NH₃ and O₂, have no effect on NO₂, and NO₃ levels [24].

Bioflocs fulfill a number of critically important functions, some of which are related to water purification [96]. First of all, they provide effective water purification from ammonia, nitrite, and nitrate—products of the metabolism of aquatic organisms. Heterotrophic bacteria oxidize ammonia to nitrite, and nitrifying bacteria oxidize nitrite to nitrate, which can then be assimilated by algae. Heterotrophic bacteria can also fix ammonia to form amino acids. This nitrification-denitrification process is the basis for maintaining good water quality in the BFT. In addition, bioflocs absorb excess phosphorus compounds and other dissolved organic matter, preventing eutrophication and the accumulation of toxic compounds. At the same time, this “live feed” significantly enriches the diet of the reared organisms, reducing the dependence on expensive commercial feeds [17].

Full effective functioning of the biofloc ecosystem requires careful control and maintenance of various parameters, such as temperature, light level, salinity, etc., at certain levels, as detailed in

Table 2. Basic parameters and their optimal ranges for BFT [23].

Parameter	Optimal Range	Main Information	Reference
Temperature	28–30 °C	Temperatures below 20 °C can significantly slow down the development of the microbial community, which in turn will affect the species being grown.	[22]
Dissolved oxygen	>4 mg L−1 ≥ 60% saturated	Aeration needs to be adjusted according to the biomass of the organisms being grown. Respiration rates as high as 6 mg L−1 (CO2) per hour have been observed in some intensive operations, so ensuring good aeration and water mixing is essential.	[97,98]
pH	6.8–8.0 (freshwater) 7.8–8.4 (brackish and marine)	Deviation from the optimum range can cause various biological reactions such as blood acidosis and alkalosis, resulting in animal stress and reduced growth. Chemical effects can also occur, including ammonia toxicity and the toxicity and solubility of metallic compounds.	[22]
Alkalinity	100–150 mg L−1	Autotrophic and heterotrophic bacteria utilize carbonates as a source of inorganic carbon in the BFT. Therefore, it is important to monitor the buffer capacity and adjust it if necessary.	[98,99]
Total ammonia nitrogen	<1 mg L−1	As pH increases, ammonium loses the H⁺ ion and is converted to ammonia, which is much more toxic to aquatic organisms. Therefore, the pH should remain close to neutral values. If the pH rises, it may be necessary to add additional carbohydrates to stimulate microbial growth.	[6]
Nitrite	0	Studies have shown that nitrite causes a variety of physiological problems in aquatic organisms, including ion imbalances in cellular metabolism, oxidation of hemoglobin to methemoglobin, increased heart rate, and increased water retention in the kidneys.	[100]
Nitrate	<20 mg L−1	Nitrate levels below 20 mg L−1 are considered safe for most aquatic organisms. However, some fish species, such as Danio rerio, can also tolerate concentrations up to 200 mg L−1. However, at levels above 400 mg/l, morphological abnormalities and a significant reduction in growth and survival are observed.	[22,101]
Orthophosphate	<20 mg L−1	Phosphate itself is not toxic to the aquatic ecosystem or farm animals, but high concentrations can promote the growth of harmful cyanobacteria. Phosphorus removal from water bodies can occur through microbial back uptake, although this process is slower than nitrogen removal.	[102]
Settling solids and total suspended solids (TSSs)	5–15 mL L−1 100–300 mg L−1 (TSS)	Lack of slurry and agitation in a BFT can lead to the formation of an anaerobic zone in the tank. This in turn will cause a rapid consumption of dissolved oxygen and may result in the release of dangerous gases such as ammonia, hydrogen sulfide, and methane, which are lethally toxic to the fish and shrimp that are reared there.	[22,98,103,104]
Salinity	Variable (depends on the needs of the animal being reared)	Studies have shown that some species, such as the giant freshwater shrimp (Macrobrachium rosenbergii), thrive in brackish water biofloc systems at salinities of 15 ppt. In contrast, other species such as Nile tilapia (Oreochromis niloticus) show little change in salinity conditions from 0 to 12 ppt. However, in the same study, signs of stress were recorded in tilapia at salinities of 16 ppt and above.	[48,105]

N—nitrogen; P—phosphorus; TSS—total suspended solids.

. Neglecting to maintain optimal conditions in the BFT can provoke the death of beneficial microorganisms, change their oxygen demand, and increase the concentration of pathogens [23].

The use of heterotrophic bacteria as part of a BFT can make the system more cost-effective, which was demonstrated by growing tilapia (Oreochromis niloticus) in recirculating aquaculture systems (RASs) and BFT during an 87-day experiment [61]. In addition, it has been shown in situ that water quality improves when hydrobionts are cultured in the BFT system [5].

When tilapia was cultured for 75 days, a significant reduction (p < 0.05) in total ammonia nitrogen (TAN) was demonstrated (p < 0.05) compared to the control. In addition, the concentration of NH₃ and NO₂ decreased [28]. Furthermore, a relationship between the dynamics of the BFT bacterial community and water quality was shown, with higher flake volume being associated with improved water quality and lower inorganic nitrogen. The BFT showed a decrease in nitrogen dioxide concentration, all other things being equal, compared to the control. In pond cultivation, the reduction in NO₂ in BFT is attributed to the activity of the nitrification process by chemoautotrophic bacteria and the removal of TAN by heterotrophic bacteria present in BFT. Thus, bacterial assimilation of nitrogen reduces the load on natural aquatic ecological systems [106].

The bacterial community formed in the BFT affects the pH of the water. It was experimentally shown that pH decreased in tilapia farming compared to the control, which is explained by the authors by the results of the heterotrophic process, which reduces both alkalinity and pH of water [28,107]. A decrease in water alkalinity was also recorded when BFT was applied in pond aquaculture during the cultivation of P. vannamei shrimp. The low alkalinity is attributed to the release of carbon dioxide by the bacteria during respiration [108].

Thus, the analysis of available data indicates the effectiveness of BFT for maintaining optimal water quality in intensive aquaculture.

6. Disadvantages of Biofloc Technology

Despite its clear benefits, the implementation and effective functioning of BFT are associated with a number of challenges that require further research and development [17,109]. One of the main problems is high initial costs due to expensive high-capacity aerators, especially when using the technology on a large scale [109,110], high energy costs to maintain optimal conditions for the normal functioning of all biofloc elements [17], operating costs (constant monitoring of water parameters and feeding bioclasts with carbon sources) [111], and possible errors in system design and lack of qualified personnel (training of qualified personnel is essential to improve efficiency and prevent unexpected failures in BFT, as effective management of bacteria and cultured organisms plays a key role) [109].

The water temperature required for optimal functioning of the BFT microbiota limits the number of hydrobiont species that can be effectively cultured using this technology. These systems function more efficiently at higher temperatures. Therefore, cold-water fish species or fish farms in northern countries may face difficulties when using BFT [112]. BFT culture may not be suitable for oligotrophic species such as salmonids due to the fish not being able to provide the conditions required for optimal biofloc function; however, the technology has been successfully used in the culture of shrimp, Nile tilapia, common carp, and African catfish [112].

Although the operating costs of conventional culture are lower than those of BFT [113], the BFT allows for increased production with smaller cultivation areas and higher stocking densities, thereby increasing profitability. For example, when producing shrimp at a stocking density of 400 shrimp m⁻² and an average final weight of 10.10 g, the net profit was USD 13.81 m² [114]. Increasing stocking density and growth rate by 20% increases profitability by 57% and 45%, respectively [115]. Also, reducing feed costs by 20% can significantly impact profitability [116].

Optimizing carbon sources is a key factor for the success of BFT, as it directly influences the composition and quality of the biofloc [117]. Therefore, research aimed at identifying optimal carbon sources for various fish species and growing conditions is extremely important [118,119]. Monitoring water parameters (dissolved oxygen concentration, pH, ammonia, nitrites, nitrates, and suspended solids) is an integral part of successful BFT management [120]. Currently, various monitoring methods are used, ranging from simple test strips [121] to complex automatic systems with online measurement of parameters [122]. However, there is a need to develop more advanced and affordable monitoring systems that provide continuous and accurate measurement of key parameters in real time [123]. This would allow for prompt responses to changes in the system and prevent the development of undesirable processes. The development of compact and inexpensive sensor systems that can be integrated into existing BFT is an important task for the future advancement of this technology [124].

7. Conclusions

BFT is an innovative approach to aquaculture, which is a closed ecosystem where fish and crustacean wastes are processed by microorganisms into valuable resources. BFT is based on the creation and maintenance of a highly concentrated biofloc—a kind of “living filter” consisting of bacteria, archaea, algae, protozoa, and other microorganisms. In conventional aquaculture, the high stocking densities required to achieve high productivity often result in the accumulation of ammonia and other toxic compounds, which negatively affects the health and growth of farmed organisms and increases the risk of disease and mortality. BFT solves this problem by creating a natural water purification mechanism. Moreover, the composition of BFT includes various beneficial microorganisms with probiotic properties, which strengthen the immunity of fish and crustaceans and increase their resistance to disease. This is especially important in high-density stocking environments where the risk of infections increases significantly. BFT development contributes to more sustainable and environmentally friendly aquaculture systems. No or minimal wastewater discharges reduce the anthropogenic impact on the environment, preserving water resources and biodiversity. Particular attention is paid to studying the interactions between the different components of the BFT and the effects of these interactions on the health and growth of cultured species. Thus, BFT is a promising technology to take aquaculture development to a new level.

Only a comprehensive approach that combines scientific research with the practical implementation of innovative technologies will unlock the full potential of BFT and ensure sustainable development in aquaculture.