One of the major problems related to super intensive aquaculture is the generation of effluents with high organic loads. Feeding in intensive monocultures increment the discharge effluents with a high pollution potential, with a high concentration of nitrogen and phosphorus (Briggs and Funge-Smith, 1994). These problems have raised concerns in many countries due to the likely environmental impacts of this activity (Boyd et al., 2020).

The Integrated Multi-Trophic Aquaculture (IMTA) aims at the integration of species with different trophic levels in the culture system, in order to take advantage of the residues from the production of one species for the culture of other species (Troell et al., 2009). Even organisms that occupy the same trophic level in the system can be produced at IMTA if they make use of different food webs in that system (Boyd et al., 2020). This variety in the combination of organic and inorganic extractive species gives IMTA advantages in terms of biomitigation and economic gain, consequently more sustainability, when compared to monocultures (Biswas et al., 2020).

The biofloc system is another culture technology that has been adopted in shrimp culture. This system gives a new direction for the shrimp monoculture in regard to aquaculture sustainability. Due to a better maintenance of water quality, this system can reduce water use by up to 90% compared to traditional systems (Krummenauer et al., 2014), allowing culture at high stocking densities (Wasielesky et al., 2013), reducing the use of land (Liu et al., 2017) and optimizing the use of artificial food (Santhana Kumar et al., 2018). This is possible through the increase of the C: N ratio in water, with the use of organic carbon sources and constant aeration, stimulating the production of microorganisms that will both work on the metabolization of nitrogenous compounds and serve as natural food for reared animals (Krummenauer et al., 2011; Lara et al., 2017; Khanjani et al., 2023).

Shrimp production in the biofloc system has numerous economic advantages (Rego et al., 2017). Nevertheless, there are still environmental constraints such as the accumulation of organic matter during production in a system with minimal water exchange and high densities. This accumulation produces an excess of suspended solids, which increases the biological oxygen demand due to the high concentration of aerobic microorganisms present in bioflocs (Ray et al., 2010). Therefore, it is recommended that excess total suspended solids (TSS) in the biofloc system should be removed trough clarification processes (Gaona et al., 2017).

Sedimentation (removal of excess biofloc particles by gravity) efficiently removes excess solids suspended in the biofloc system (Gaona et al., 2016) that are normally discarded as effluents. Therefore, the rationale for integrating species in the biofloc system is to take advantage of this excess of microbial protein in the form of suspended solids, to feed other species and thus transform effluent into animal protein, making the system even more sustainable.

Tilapia (Oreochromis niloticus) presents some characteristics that make it an ideal species for culture in an IMTA system based on the biofloc system (Azim and Little, 2008; Monroy-Dosta et al., 2013; Poli et al., 2019). This species has a high tolerance for suspended solids, moderate dissolved oxygen, and high stocking densities (Azim and Little, 2008; Simão et al., 2013; Malpartida Pasco et al., 2018; Khanjani and Sharifinia, 2021). In addition, it is an omnivorous species, presenting filtering structures and a digestive system that allows it to be an organic extractive in potential, feeding on natural productivity of the biofloc system and removing the excess of total suspended solids. It can be used as a bioremediation agent in IMTA systems (Azim et al., 2003; Ekasari et al., 2014; Poli et al., 2019).

Commercial expansion of IMTA has been troublesome. Although the biological and environmental advantages of this practice are generally accepted by producers and society, some economic-related issues have yet to be overcome for its adoption (Ridler et al., 2007). Thus, pilot-scale studies aimed at integrating superintensive production of Pacific white shrimp (Litopenaeus vannamei) and Nile tilapia in the biofloc system are sorely needed for the development of large-scale integrated multitrophic cultures.

There are some studies reporting the integrated culture of shrimp and tilapia, though in low production densities and in clear water systems (Tendencia et al., 2004; Tendencia et al., 2006; Muangkeow et al., 2007; Cruz et al., 2008; Yuan et al., 2010; Muangkeow et al., 2011; Simão et al., 2013). More recently, Poli et al. (2019) studied the integrated culture of shrimp and tilapia in an experimental scale, and this work represents a first step towards a future IMTA using biofloc technology. Nevertheless, until now, there is no work with integrated superintensive production of shrimps and tilapia in a BFT system, on a pilot scale. The objective of this study was to evaluate the effect of different fish stocking densities in the integrated superintensive culture of white shrimp L. vannamei and Nile tilapia O. niloticus reared in biofloc system to promote the maintenance of TSS at the appropriate levels for shrimp culture, through the consumption of the excess of biofloc by the fish.

Throughout the experimental period, the physical and chemical parameters of water quality were at suitable levels both for L. vannamei (Samocha and Prangnell, 2019) and for tilapia growth (Atwood et al., 2001; Azim and Little, 2008). Even with the culture of two species, the dissolved oxygen, a limiting factor for cultured organisms, mainly in a biofloc system, remained above 6 mg L^-1, values above those recommended for reared species (Wasielesky et al., 2006; Tran-Duy et al., 2008).

The alkalinity showed a statistical difference between the treatments during the experimental period, however, the average values registered were within the recommended for L. vannamei (Furtado et al., 2014) and for tilapia (O. niloticus) (Cavalcante et al., 2009). The T65 treatment showed a lower mean alkalinity value than T35, which in turn, presented lower mean nitrite values (0.6 ± 0.6 mg L^-1) than T65 (1.1 ± 1.1 mg L^-1). In T65 was registered a peak of nitrite at 21 days of the study, reaching 4.5 ± 3.31 mg L^-1 and, this possibly happened due to the presence of fish in the system at elevated densities, although with the addition of mature biofloc inoculum. The influence of fish density and the imbalance in the nitrification process are reported by Holanda et al. (2020) in a study integrating shrimp and mullets (Mugil liza). In addition, it is possible that the swimming of a great biomass of fish may have altered the size and shape of bioflocs, which affects the nitrification dynamics in the biofloc systems (Carvalho et al., 2006; Delatolla et al., 2009).

The presence of tilapia integrated in the system did not negatively affect the performance of the shrimp, independently of the fish densities used. The productivity of the shrimp was high, approximately 7.0 kg m^-3, already expected in cultures in biofloc system and above the reported productivity values observed in studies evaluating the biofloc monoculture of L. vannamei in pilot scale (Krummenauer et al., 2011; Gaona et al., 2017). These authors obtained yields close to 4.1 kg m^-3.

Even underfed tilapia showed zootechnical performance similar to that reported for tilapia culture in biofloc system. Zaki et al. (2020) showed an average final weight of 115 g for tilapia at a density of 60 fish m^-³ for 84 days, with an initial weight of 50 grams. Malpartida Pasco et al. (2018), on the other hand, observed productivity of 21 kg m^-3 in tanks of 10m³ growing tilapia at the density of 70 fish m^-3 for 56 days in a biofloc system.

The FCR obtained for shrimp was similar to other studies with monoculture of L. vannamei in biofloc systems (Lara et al., 2017; Reis et al., 2019). In this present study, it is worth noting the low FCR values of tilapia, which were below 1.0 in both T65 (0.7 ± 0.04) and T35 (0.7 ± 0.03) treatments. This indicates that tilapias consumed the natural microbiota present in bioflocs, using it as a substantial part of their diet. A low feed rate was provided to tilapias (1% of biomass), which favored their consumption of bioflocs without impairing their growth despite the low amount of feed provided. A standard feed rate for tilapia in a biofloc system, with an approximate weight of 50 grams, is 3% of wet body weight (Zaki et al., 2020). The values obtained in the present study are not only beneficial from an environmental point of view, but also economically advantageous as it can decrease production costs due to the reduction of food used (a major cost in terms of production), producing two species at the same time. Environmentally, using less feed implies the generation of effluents with less polluting potential, making aquaculture environmentally friendly and socially acceptable (Tacon and Metian, 2008; Naylor et al., 2009). Ekasari et al. (2014) showed the consumption of bioflocs by red tilapia, regardless of the size of the fish. The same study also showed that the absorption capacity is 39 to 117 g of TSS/kg of fish. Poli et al. (2019) also found low FCR values (around 0.2) for Nile tilapia in integrated cultivation with L. vannamei, proving the tilapia’s potential to consume the excess solids produced by the shrimp. These results indicate that tilapias can feed on bioflocs and the integration of the two species in an IMTA system could be implemented to warrant a more sustainable culture system.

Even with the integration of the fish in the system, it was necessary to remove the excess of total suspended solids through sedimentation process. The sedimentation time was different in the two treatments, with T65 being clarified for longer time, probably due to the high total biomass in this treatment. Gaona et al. (2016) performed clarification during 58 ± 12.2h in a monoculture of L. vannamei with a density of 350 shrimp m^-2, with an initial weight of 0.18 ± 0.06g for 17 weeks. These data highlight that tilapias could contribute to the management of TSS, since the stocking density of shrimp used in the present study were higher than the reported previously by the authors. In the present study, the lower density of tilapia in T35 required less clarification time (36 ± 12 h), which corroborates that higher stocking densities of fish could contribute with the excess of organic matter in the water, hence increasing the TSS. In addition, the clarification time in T35 was shorter than that reported by Gaona et al. (2016) for L. vannamei monoculture at a lower density than the present study. In this study, it was observed that in treatment T65, where there was a greater biomass of fish, longer time of clarification were needed, which indicates that the system produced more TSS compared to treatment T35. Similar results were observed by Zaki et al. (2020) in a monoculture of tilapia in biofloc systems, showing that the increase in biofloc volume was directly proportional to the increase in stocking density.

Azim and Little (2008) reported a difficulty in maintaining TSS levels at 500 mg L^-1 even using a clarifier and often the level reached exceeded 1000 mg L^-1 TSS, growing tilapia from 80 to 120 grams and density of 12 kg m^-3. The consumption of bioflocs by fish depends on the species cultivated, the feeding habits of the fish, and the size and density of bioflocs. It is possible that the consumption of bioflocs by fish also depends on the presence and rate of feed added to the tank. Therefore, new studies with different biomass ratios of shrimp and fish should be encouraged.

Most studies of IMTA cultures of L. vannamei and tilapia have worked with low shrimp densities (10 to 120 shrimp m^-2) (Yuan et al., 2010; Muangkeow et al., 2011; Simão et al., 2013) and few are the studies of IMTA cultures in super-intensive densities. Poli et al. (2019) observed shrimp yields of up to 4 kg m^-3 in the integrated culture of shrimp, Nile tilapia and Sarcocornia ambigua on a laboratory scale. Our work provides useful information for implementing biofloc cultures with shrimp and fish in integrated farming systems, diversifying production without loss in performance of the target species, which in the present study are the shrimp. From the productive perspective, the data presented here encourage the development of integrated shrimp and tilapia harvests in a biofloc system, in tanks on land.

The results obtained in the present study lead to the conclusion that the integrated culture of L. vannamei and O. niloticus in super-intensive systems using biofloc is possible, allowing the reduction of fish feeding rates without negatively influencing their growth. When higher densities of tilapia are used (65 fish m³), there is an increase in the total suspended solids concentrations in the water, also increasing the clarification time required to keep these concentrations within the levels suitable for the species. This study, on a pilot scale, proves that the IMTA systems of tilapia with L. vannamei based on bioflocs diversifies production without compromising the productivity of shrimp.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

This experiment was approved by the Ethics and Animal Welfare Committee of the Federal University of Rio Grande - FURG (Case number 23116.005895/2016-42).

Conceptualization: LP and MH. Data curation: LP and MH. Formal analysis: LP, MH and WW. Methodology: All authors. Project administration: ER and LP. Supervision: LP and WW. Writing—original draft: All authors. Writing—review and editing: LP, WW and ER. All authors contributed to the article and approved the submitted version.