High-density polyethylene (HDPE)-lined ponds with well-prepared dikes are most preferred for large-scale biofloc fish or shrimp production. HDPE-lined ponds with sufficient elevation and central drainage are used for effective biofloc-based farming. The BFT can be conveniently applied in re-circulatory aquaculture or raceway systems either by in situ inclusion or ex situ floc production through an activated sludge system and by putting the harvested biofloc in the production system. The system is adequately agitated and aerated to keep the microbial floc in suspension. For the development of biofloc, a small nitrogenous source (fish feed or urea) is added to previously filled water tanks. Then, a healthy carbon source like starch, molasses, or wheat flour is uniformly spread on the surface of water. To create a microbial mass, clay is softened and passed through a sieve (53-μm sized particles or less) and then added to the microbial reservoir. In addition, the use of farm wastewater containing nitrogenous wastes is helpful as an inoculum. The addition of 20 g of clay, 10 mg of ammonium sulfate, and 200 mg of carbonaceous organic matter such as molasses stimulates biofloc formation in 1 L of water (19). The primary inoculum in biofloc production cycle improves microbial mass formation. However, care should be taken before using any commercial inoculum for this purpose. Heavy aeration is required for keeping the floc under suspension. The use of clay and water rich in biofloc production cycle as the primary inoculum improves microbial mass formation in the new culture system (20). Heterotrophic bacteria are more active than other bacteria because of the presence of the carbonated organic matter, which assists the removal of carbon and nitrogen from the water, and produces microbial biomass (13). This microbial biomass is attached and fed by other organisms in the water forming biofloc (14). Physical and chemical parameters like temperature, oxygen, pH, alkalinity, total nitrogen, ammonium, nitrite, and nitrate should be measured; subsequently, appropriate responses should be adopted quickly (21, 22). As explained in Figure 1, there are three major approaches for successfully generating biofloc.

Biofloc is defined as a set of organic matters formed usually at high density as suspended particles (33–35), and it comprises 60–70% of organic materials, and combination of fungi, algae, bacteria, protozoa, rotifer, nematodes, and other inorganic substances (36). In a zero-water exchange system, water quality is maintained by two functional bacterial population, viz., heterotrophic ammonia- assimilative and chemoautotrophic-nitrifying bacteria (37, 38). As the culture progresses, the color changes from green to brown, indicating the transition from algal-dominated to bacterial biofloc-dominated system. The number of bacteria in biofloc ponds can be between 10⁶ and 10⁹ ml⁻¹ of floc plugs, which contain between 10 and 30 mg dry matter, making the pond a biotechnological industry (39). Khanjani et al. (14) specified that the maturation of the biofloc can be determined by the number of heterotrophic bacteria per ml, which is 3.36 × 10⁷. The type of carbon source, salinity level, and species to be cultured are major factors determining the different group of organisms present in biofloc (40). The biofloc collected from Litopenaeus vannamei tanks contained 24.6% phytoplankton (dominated by diatoms like Thalassiosira, Chaetoceros, and Navicula), 3% bacterial biomass (two-thirds was Gram-negative and one-third was Gram-positive), a small amount of protozoan community (98% flagellates, 1.5% rotifers, and 0.5% amoeba), and 33.2% detritus; the remaining quantity of 39.25% was ash (16). However, the composition may vary within different culture systems. Of the total organic sludge, it is believed that only 2–20% is living as microbial cells, while the rest is total organic matter (41).

Some of the dominant bacterial species found in the biofloc system are Proteobacterium, Bacillus, and Actinobacterium, and traces of other bacterial species (Roseobacter and cytophaga) are also present in the system (42). For a bacterial species to run a cellular machinery, it needs nitrogen like ammonium ion and a carbon source such as sugar, starch, and cellulose to make it responsive to toxic ammonia from the aquaculture system. The ability to adhere to suspended particles and surfaces as well as the use of organic matter are important physiological properties of bacteria in biofloc. The role of bacteria here is to transform the toxic form of nitrogen to one that is toxic only at high concentrations by the process called nitrification. This bacterial growth via promoted nitrogen uptake decreases ammonium concentration more rapidly than nitrification (38). The rapid occurrence of heterotrophic bacteria is due to the growth rate and microbial biomass yields per unit substrate of heterotrophs being a factor 10 higher than that of nitrifying bacteria (38). The microbial biomass yield per unit substrate of heterotrophic bacteria is about 0.5 g biomass C/g substrate C used (43). The optimum C:N ratio in an aquaculture system can be maintained (C:N ratio 15–20:1) by adding different locally available cheap carbon sources and/or reducing protein percentage in feed. Under optimum C:N ratio, inorganic nitrogen is immobilized into bacterial cell, while organic substrates are metabolized. Bacteria have a nutrient conversion ratio as high as 50% and possess fast multiplication rates converting toxic product in the biofloc system into highly nutritious and useful microbial protein (42). Therefore, the biofloc system could be considered as a microbial ecological sequence model. It is reported that heterotrophic microbial communities like Pseudomonas, Bacillus, Sphingomonas, Micrococcus, Nitrospira, Nitrobacter, and yeast maintain water quality and manages the physiological health of cultured species in a biofloc system (44).

Carbon-nitrogen ratio in biofloc systems plays a vital role in the restriction of toxic inorganic nitrogenous compounds into beneficial bacterial cells acting as a source of food for cultured organisms (10). Immobilization of inorganic nitrogen takes place when the C:N ratio of the organic matter is higher than 10 (45). In a biofloc system (intensive culture), the growth of heterotrophic bacteria is restricted by dissolved organic carbon. The population of the heterotrophic bacteria in the system is stimulated by adding a supplementary carbohydrate source or reducing the feed protein level manipulating the C:N ratio and creating a demand for nitrogen (ammonia). The generated ratio of organic carbon and inorganic nitrogen reflects the requirement and composition of heterotrophic bacterial cells. Substantial development of useful microbial growth and fixation of ammonia in a culture medium are possible by directly or indirectly adding a carbon source in limited discharge systems (changing C:N ratio) (11, 15, 37, 46). Thus, manipulating the C:N ratio results in shift from an autotrophic to a heterotrophic system (4, 10).

Biofloc systems possess a dynamic nutritional value and can be used as a complete aquatic food source and supply bioactive compounds (47). There are many factors that can affect nutritional value such as food preference, floc density in water, and ability of animals to ingest and digest microbial proteins (38). The heterotrophic bacterial population in a biofloc system produces single-cell proteins, which act as a source of food for carps, shrimps, and tilapia (48–50). Qualitatively, biofloc contains crude protein (50%), crude lipid (2.5%), fiber (4%), ash (7%), and 22 kJ g⁻¹ energy (51). It is observed that the nutritional value of biofloc depends on the biochemical compounds present, particle size, and digestibility of cultured organisms. Ekasari (17) found that particle sizes of <100 μm and more than 45 μm are more promising for L. vannamei, Oreochromis niloticus, and Perna viridis because of higher nutritional values. The highest level of proteins and lipids are found in the particle size of >100 μm, whereas the amino acid concentration is found highest in <45 μm. Table 2 presents various studies conducted on different aquatic species by adding different carbon sources and microbes reported.

It is reported that the feed conversion ratio (FCR) values of a fish culture in the biofloc technology can be reduced to 1.2–1.29 from 1.52 in clear water (22). In clear water treatments, feed efficiency was recorded as low as 66.81%, whereas the same was 84.26% in BFT (22). Ballester et al. (55), in his study on the BFT, used wheat bran and molasses as carbohydrate source and revealed the nutritional breakdown of biofloc as 30.4% crude protein, 4.7% crude fat, 8.3% fiber, 39.2% ash, and 29.1% nitrogen-free extract. Khanjani et al. (13) used molasses, starch, wheat flour, and their mixtures as carbon sources and suggested that experimenting with different carbon sources changes the nutritional composition of the biofloc produced. Ray et al. (40) revealed that in super-intensive shrimp culture systems, an environment friendly plant-based diet can produce results similar to those from fish-based feed in the BFT, and that controlling the concentration of particles could improve water quality and shrimp production. Samocha et al. (66), in his study on 120 days of L. vannamei biofloc culture, reported 92, 81, and 75% survival with stocking of 150, 300, and 450 shrimps/m², respectively, and detected no significant changes in FCR when feeding L. vannamei with different percentages of CP diets; he claimed that floc biomass provides a complete source of cellular nutrition to the system. The exact mechanism of growth enhancement by microbial flocs is unknown, but it is believed that continuous consumption of native proteins without previous treatments (56) could possess a growth factor enhancing the growth in the system. Biofloc improves ingestion and digestion of supplied feed and provides cellular nutrition to cultured organisms (67). Emerenciano et al. (57) stated that the presence of biofloc in the brood-stock diet of Litopenaeus vannamei and Penaeus stylirostris improved reproductive performance and egg quality. In his study, L. vannamei females were cultured under biofloc conditions and fed with fresh food, and later showed higher egg production and better spawning rates, and contained higher levels of highly unsaturated fatty acids (HUFAs) in eggs.

In addition to improving target species production, the BFT can help conserve the amount of water required in aquaculture. Biofloc, compared to traditional aquaculture practices, provides a more sustainable approach with minimum water exchange and lower feed intake, transforming it into a low-cost sustainable technology for aquaculture development (5, 15). Traditional aquaculture systems with regular water exchange require up to 80 m³ kg⁻¹ shrimp, but intensive shrimp farming systems with zero exchange require just 1–2.26 m³ kg⁻¹ shrimp (38). When compared to a recirculating aquaculture system (RAS), biofloc systems based on tilapia production utilized about 40% less water (68). Majority of studies using the BFT found that nitrogen and phosphorus waste in this system could be decreased further, supporting the importance of the system in improving aquaculture productivity and minimizing environmental effects from aquaculture production systems (68, 69). Heterotrophic bacteria in a biofloc system assimilate inorganic nitrogen compounds at a faster rate than denitrifying bacteria, resulting in a 10-fold increase in microbial biomass (38). As a result, if there are enough organic carbon sources available, heterotrophic bacteria can immobilize ammonia in biofloc in a matter of hours or days (38). Although heterotrophic bacteria are primary nitrogen conversion agents, the biofloc system also aids in nitrification, phototrophic nitrogen uptake, and denitrification (17). Farm biosecurity and biofloc technology are two key aspects that must be considered for long-term intensive aquaculture sustainability. The BFT improves biosecurity by limiting water exchange, increasing environmental control, creating biological and physical (indoor) barriers to infections, and boosting the immune system.

Wasielesky et al. (70) studied various aspects like endurance, growth, feeding pattern, and feed conversion ratio (FCR) of L. vannamei juveniles in a biofloc system, and positive association was observed between growth of shrimp and protein content, depicting the benefits of the BFT for the culture of white-legged shrimp (L. vannamei). Another study on white-legged shrimp in the BFT (8) showed higher growth rates, improved water quality, and increment in the final body weight of the shrimp. The comparative analysis of Farfantepenaeus brasiliensis post larvae when stocked with and without a biofloc system showed better growth and survival in the biofloc system because of high nutritional environment (34). Pinto et al. (71), in his study on culturing the L. vannamei utilizing artificial seawater in the BFT, concluded that the BFT system was zootechnically and financially viable. Ferreira et al. (30), in his study on the BFT, reported the presence of Bacillus species demonstrating the probiotics nature of the system. Huang et al. (72), in his study on L. vannamei in the BFT, observed an increase in the concentration of Actinobacteria, Alteromonadaceae, and Rhodobacteraceae and a decrease in the number of Cyanobacteria, Mycoplasmataceae, and Vibrio, indicating t the higher C:N ratio in the system responsible for enhancing and promoting beneficial bacteria and ultimately suppressing harmful pathogens. Hence, from the above studies, it can be explained that the BFT could be used as an innovative strategy to develop and grow enriched probiotic microbes for improving health and enhancing growth of cultured species (73).

It is of prime importance to conduct research on species for the BFT, which are commercially important and have a social interest. The only sole member of the freshwater prawn species having published literature associated with the BFT is Macrobrachium rosenbergii, allowing it to be used commercially. Considering the research on shrimps includes species like L. vannamei (74), Penaeus monodon (46), Litopenaeus setiferus (75), Fenneropenaeus merguiensis (76), and Farfantepenaeus paulensis (77). There are many other species of the family to which the BFT has successfully extended such as F. brasiliensis (34), F. duorarum (57), L. stylirostris (34), L. schmitti (78), Marsupenaeus japonicus (42), and Fenneropenaeus indicus (79).

Oreochromis aureus (10), Oreochromis mossambicus (39), and Oreochromis niloticus (51) are among the freshwater fish species studied using the BFT. The technology has also been extended to two major commercially important carps, i.e., Catla cattle (80) and Labeo rohita (50). For the ornamental fish industry, various species have also been studied using this technology like Poecilia reticulata (81), Scatophagus argus (82), Carassius auratus (83), and Pseudotropheus saulosi (84). Some other commercially important species that have been studied using the BFT are shown in Table 3.

To make any aquaculture venture profitable, there is a need to reduce production costs and increase profitability. The key deciding factors for the aquaculture industry to sustain in the long-run are feed cost and environmental protection (21). In this technology, the carbon source used is mainly a by-product that is derived from either the plant or animal food industry and is locally available. Before the stocking of post larvae and during the grow out culture, cheap sources of carbohydrates like molasses and plant meals are applied in the system to provide food for initial stages of growth and maintain the C:N ratio.

The biofloc system is responsible for increasing the growth rate and reducing the feed conversion ratio, which eventually increases profitability and reduces aquaculture costs (22). Megahed (85) found that the per-kilogram production cost of green tiger shrimp (Penaeus semisulcatus) and tilapia was reduced by 33 and 10% (93), respectively, using the BFT depending on diet, species, and price of carbohydrates. The biofloc system is more successful in increasing growth rate and reducing culture period than the clear water system (94). Liu et al. (18) showed that shrimp yield can be increased, and that feed conversion ratio can be lowered by adding maize to stimulate biofloc growth in an integrated culture of shrimp, spotted scat, and water spinach. This process can further reduce the total phosphorous and total nitrogen in the cultured water. Similarly, Ekasari (17) demonstrated that biofloc-based polyculture of shrimps, tilapia, mussel and, seaweed resulted in higher growth rate and reduced waste nutrient and microbial biomass. The biofloc system reduces the cost of organic and inorganic fertilizers and covers the cost of carbon source. The BFT is also responsible for reducing water treatment expenses by 30%, and protein utilization efficiency is double when compared with conventional water treatment technologies (5, 21).

When investing in a new or experimental technology, it is necessary to adapt it to the local environment until the production process can be standardized and survival can be maximized (95). Rego et al. (96), in his study, estimated the financial viability of Litopenaeus vannamei culture in the BFT and found that per hectare BFT operating costs are 10 times higher than those of the conventional aquaculture practice. Among the total variable costs, feed contributed highest in both systems, accounting for 54% in the BFT and 79% in conventional (96). Feed was found to be the most important variable cost for intensive shrimp farming in Asia (97), accounting for between 23 and 46 % of expenditures. Rego et al. (96) found that in the BFT, total production cost per cycle and total production cost per year were US$ 33,294.87 and 124,369, respectively, with stocking density of 113 shrimp per m⁻². In terms of production cost, the BFT system generated an annual operating profit of US$ 51,871.54 per productive hectare, which is 141% greater than that of the conventional approach. In comparison, Hanson (98) estimated an initial expenditure of around US$ 992,000, with total production cost equivalent to US$ 983,950.00 year⁻¹, for a super-intensive BFT system operating with densities of 500 shrimps per m⁻³ in 10 tanks of 500 m³ each, located in Texas, United States. The high overall production cost of the BFT system compared to the conventional system is justified not only by the highest feed expenditure but also by the high labor work and energy values that significantly contribute to this variance. Hargreaves (32) stated that the greater energy consumption in the BFT system is due to the shrimp and heterotrophic bacteria in biofloc having high oxygen demand, necessitating the need for artificial aeration in a minimum ratio of 25 HP ha⁻¹, as opposed to only 3 HP ha⁻¹ in the conventional system.

The concept of BFT goes against the popular understanding that pond water must be clean; thus, convincing farmers to use the approach is a big challenge (21). It is also because of the lack of perfect technology, justifying BFT technology and persuading farmers to implement it is more challenging than with traditional approaches. Several considerations, on the other hand, encourage the use of the technique. To begin with, water has become scarce or expensive to the point that aquaculture development is being hampered. Second, most nations have laws prohibiting the discharge of contaminated effluents into the environment. Finally, significant outbreaks of contagious diseases prompted the implementation of overly restrictive biosecurity measures, such as lowering water exchange rates (21). Monitoring of ponds is a critical part of implementing the technology in aquaculture. The BFT is not yet entirely predictable, making its implementation at the farm level riskier.

Water quality monitoring including determining and stabilizing the total concentration of suspended solids, settling solids, the number of aerators, their type and location in ponds are important for the successful implementation of the technology (5). Various findings have revealed that BFT have the ability to control dangerous chemicals in aquaculture systems, which, if left intact, can reduce productivity (21, 99). The BFT system has been tested for multi-species production systems, like tilapia with vegetables, shrimp with microalgae and seaweed, producing favorable results (100). Aquaculture is a sustainable technique that focuses on environmental, social, and economic concerns as it grows, according to the BFT. The BFT is developed on the principle of recycling and reusing nutrients, particularly nitrogen, into microbial biomass, which may either be utilized by cultured organisms or collected and processed into important feed nutrients (101). The development of such an approach necessitates careful modification and execution, requiring additional research and information from researchers, producers, and customers in order to establish a base for this method, which ultimately is the foundation of sustainable aquaculture.

While the BFT helps in the maintenance of adequate water quality suitable for the survival and general well-being of reared aquatic species without the use of massive water volumes and exchange rates, the accumulation of total suspended solids (TSSs) and organic matter in rearing units may cause a number of environment problems. In the BFT, an integrated multi-trophic aquaculture (IMTA) system can be set up in which the waste of a new organism is used as feed for another (102). The system allows filter feeding organisms like Oreochromis sp. and Mugil liza to incorporate in the culture system, so suspended particles and organic debris that accumulate at the bottom of rearing units are absorbed (102). However, the organisms should not compete for food and space with co-cultured species. The species to be cultured is a crucial consideration when building a biofloc system. Biofloc systems function best with species that can benefit nutritionally from direct floc ingestion. Biofloc systems are also best for species that can withstand high sediment concentrations in water and are tolerant of poor water quality. Physiological changes in shrimp and tilapia allow them to absorb biofloc and digest microbial protein, and to take advantage of biofloc as a food source (82). Earlier studies have revealed an increase in N and P recovery, growth performance, profitability, yield, and immunity in integrated culture of shrimps and Nile tilapia (Oreochromis niloticus) when stocked at various stocking densities (5, 103). Borges et al. (102), in his study on integrated polyculture of BFT in mullet (Mugil liza) and white shrimp (Litopenaeus vannamei), revealed that the organic matter in rearing units were utilized by the mullet when raised in same units or two different units with 41 days of culture. When tropical fish species like tambaqui (Colossoma macropomum) were cultivated in the BFT, no improvement in growth or productivity was noted when compared to conventional culture techniques; however, major water problems like turbidity and amount of nitrite increased in the system (102).

There are many methods that can be adopted for increasing the efficiency of BFT when culturing a particular species or a combination of species. One of them is to use lined ponds like those used in countries like Australia, Indonesia, and Malaysia for commercial shrimp culture (15). The basic strategy is to employ small (0.5 to 1.5 ha) ponds lined with plastic (30- to 40-mil HDPE) and aerate vigorously (28–32 hp/ha) with paddle wheel aerators to keep floc in suspension (15). This system requires high stocking density of PL (125–150/m²) to maximize yield. The culture is carried out usually for 90–120 days with maximum daily feed of 400–600 kg/ha before harvesting. This technique of lining shrimp ponds has produced tremendous yields of 20–25 metric tons/ha per crop (15). Another approach is the use of greenhouse raceways for shrimp, which member universities of the old US Marine Shrimp Farming Consortium developed for intensive lined raceways in standard greenhouses (100 feet long × 25 feet wide), building on the intensification of lined, outdoor shrimp ponds. If supplemental heat is given, these greenhouses can be located inland to avoid expensive coastal land, and in temperate climates (93). The stocking density of these shrimps varies from 300 to 500 PL/m², and yields an output of 3–7 kg/m². Higher yields of around 10 kg/m² are also found when supplied with pure oxygen supplementation (93).

It was in the mid-1990s when first commercial uses of BFT in shrimp aquaculture were reported in Belize. The area of culture ponds were around 1.6 ha producing 11–26 mt of shrimp in each cycle (35). Commercial large-scale and small-scale BFT-based shrimp farms are now spreading across Indonesia, Malaysia, Thailand, South Korea, China, and India (35, 80). It is reported that the commercial size of a biofloc pond varies between 0.1 and 2 ha with proper aerators (paddle- wheel) and aspirators in order to aerate the system well and allow particles to be in suspension for proper mixing (34). The technology often leads to higher productivity without affecting the environment. Production intensity in BFT ponds is far more higher than that in non-BFT ponds, like a tilapia culture in the BFT system that exhibited higher growth and quality. The BFT improves output and productivity by assisting in the supply of high-quality fish juveniles, which is one of the most significant inputs in the production process. The culture system contributed around 45% higher production and individual weight gain than those without BFT (51). In Indonesia alone, the technology is used by almost 20–25% of farmers with an average pond area of 0.5 ha and produces more than 30 metric tons of output per cycle. High-density polyethylene (HDPE) sheets are used for lining ponds with an aeration capacity of 28 hp ha⁻¹ (56). While full HDPE-lined ponds in Malaysia generated an average of 17–23 metric tons of produce every cycle, ponds with lining in dikes had a much lower output of 12 metric tons per cycle (56).

Furthermore, biofloc systems may be created and implemented in conjunction with other forms of food production, resulting in more productive integrated systems that seek to produce more food and feed from the same amount of land with less input. By boosting the reproductive performance of aquaculture animals and increasing the immunity and robustness of larvae, the BFT might help sustain the supply of high-quality seeds (17, 35).