The transporters located on the vacuole membrane are activated to compartmentalize Cd-phytochelatin complexes from the cytosol into vacuoles. The AtCAXs were reported to transfer Cd into vacuoles to mitigate damage to plants under Cd stress. Previous studies reported that ATP-binding cassette transporters located on the vacuole membrane, such as OsHMA3 in rice and AtABCC1 and AtABCC2 in Arabidopsis,promoted Cd vacuolar sequestration during stress, thereby reducing the translocation of Cd from root to shoot. In this research, MdCAX3, MdABCC2, and MdHMA3 transcripts were induced higher in the roots of transgenic plants than in WT under Cd treatment, which might contribute to a much lower Cd concentration in the aboveground part of transgenic plants than in WT.Integrated agri-aquaculture aquaponics systems typically combine a recirculating aquaculture system  with soilless plant production in hydroponics, based on sharing and reusing nutrients and water. Aquaponics has become widespread in aquaculture as a way to increase the efficiency of water and feed use and consequently reduce the discharge of nutrient-rich effluents. Commonly, between 21 and 30% of the dry matter and 40 to 47% of the nitrogen  content in the feed are retained in the biomass of tilapia, and most of the inputted nutrients are discharged into the environment. The discharge of nutrient-rich effluent might result in pollution and eutrophication of water bodies. These problems can be minimized by directing the aquaculture effluents to nourish plants in aquaponics. Most aquaponics systems are run in coupled setups,roll bench in which water and nutrients continuously flow through the aquaculture and hydroponics subsystems. However, a trade-off between the required water quality and required environmental conditions in the respective subsystems has been reported as an issue in coupled aquaponics. 

Decoupled layouts have been proposed to solve this trade-off by separating each subsystem component with a unidirectional flow from the aquaculture to the hydroponics subsystem. This allows for meeting the requirements of all subsystems and achieving high productivity of both fish and plants. In addition to evaluating different layouts, different aquaponics approaches have recently been tested, as well, seeking to improve the sustainable character of food production. FLOCponics is a term proposed by Pinho et al.  as an offshoot of aquaponics in which RAS is replaced by a system based on biofloc technology. BFT aims to manage the water quality in aquaculture systems without the need for costly mechanical and biological filters or for high volumes of water exchange. The growth of specific microbial communities that recycle the nitrogenous waste is directly fomented in the aquaculture tanks,by providing strong aeration and water movement. Besides that, an external carbon source is added to increase the carbonnitrogen ratio of the water and to promote the growth of biofloc microbiota. As a result, extra macro- and micro-nutrients are added to the water via the carbon source. Additionally, lower nutrient loss by minimal solids or sludge removal can be linked to BFT when compared to RAS. The higher accumulation of nutrients in FLOCponics water compared to aquaponics using RAS could directly influence plant nutrition. However, the results presented to date have not reached any consensus on the benefit of using BFT effluents for plant growth in FLOCponics systems.The negative results of plant production in FLOCponics were in general related to nutrient imbalances, high water pH, and the presence of bioflocs in the plant roots. The presence of bioflocs probably affected the breathing process and the absorption of nutrients by the plants. In most of the studies, the FLOCponics systems were run in one loop instead of decoupled layouts. Thus, only sub-optimal conditions for plant growth were achieved. With respect to the effect of FLOCponics on tilapia production, increased zootechnical performance should be expected when comparing it to conventional aquaponics. This is because the BFT microorganisms are a constant and nutrient-rich source of natural food for the fish,resulting in better fish weight gain, survival and feed conversion rate when compared to RAS production. 

The consumption of bio-flocs by tilapia juveniles makes it possible to adapt nutritional strategies. For instance, alternative protein ingredients can be used instead of the conventional high-cost fish meal and soybean meal,or the use of diets with low protein content. Mansour and Esteban  showed that even with a reduction of 30% to 20% of the dietary protein in tilapia reared in BFT, the fish grew more significantly than those cultured in a clear-water system and fed with 30% protein. To date, whether this nutritional benefit of BFT also occurs in FLOCponics has not yet been reported.In these studies low volumes of bioflocs were reported for coupled FLOCponics systems, indicating low availability of natural food. Such a low volume of bioflocs occurred because of the need to limit the quantity of solids in the whole system in order to enable plant production. By individualizing each subsystem in a decoupled layout, proper management of bioflocs in the fish tanks can be carried out. Given the optimal biofloc volume for fish growth in the fish tanks, subsequently optimal nutritional strategies for plant production can be explored. Developing technologies that allow the reduction of the amount of protein in tilapia diet, without undermining the system yields, benefits the aquaculture sector in both economic and environmental terms. This is mainly because the use of low dietary protein may result in:lower feed cost since protein is the most expensive nutrient in fish diets ;lower use of fish meal and, on a large scale, minimizing the over exploitation of natural fish stocks ; and  decreased input of N and, depending on the production system, less discharge of N into the surrounding environment. This last consequence of reducing the amount of protein may also influence plant production in the integrated system. The effect of using less CP in the fish diet on plant growth must still be understood. It is important to note that the amount of dietary protein required by fish depends on the employed system and the production phase. For instance, tilapia in the nursery phase  usually require high dietary CP to ensure optimal growth when they are young and, consequently, to promote rapid growth until harvest. In systems with minimal natural food available, such as in RAS and cages, the recommended CP for tilapia juveniles varies between 30 and 40%,whereas 28% CP has been suggested as enough to achieve high growth performance in BFT. Consequently, decoupled FLOCponics  seems to be an alternative approach to take advantage of the nutritional benefits of BFT in integrated agri-aquaculture systems and thus reduce the amount of protein in the diets of tilapia juveniles. Additionally, testing different CP levels in this new system is necessary to indicate the optimal input of N to meet both plant and fish nutritional needs. The aim of the study was, therefore, to investigate and evaluate the production of lettuce and tilapia juveniles in a decoupled FLOCponics  system using different levels of crude protein  in the fish diets. For this, the zootechnical performance of tilapias in DFP systems receiving diets with 24, 28, 32 and 36% CP were compared to those reared in a traditional decoupled aquaponics system  and in BFT, both fed with a 32% CP diet.

Two cycles of lettuce production were also performed in DFP systems with the different CP levels. Their growth was compared to those in DAPS and traditional hydroponics systems as control treatments. In addition, the physical-chemical parameters of the water were monitored in the aquaculture and hydroponic subsystems.A completely randomized experiment was designed to evaluate the production of tilapia juveniles  and lettuce  under different production techniques or subjected to diets with different crude protein  contents. In total, seven treatments were tested: one treatment was a tilapia culture in BFT without integration with lettuce production ; the second was a lettuce hydroponic treatment ; and the other five were tilapia culture integrated within two-loop decoupled systems. Of these five, one comprised a traditional decoupled aquaponics system  and the other four were DFP systems using different levels of CP. In the two fish control treatments,diets with 32% CP were used, while in the other DFPs the following levels were tested: 24%, 28%, 32%, and 36% CP. There were three replications of each fish treatment and six of each plant treatment. From this, the effluent of one aquaculture subsystem was used to nourish two plant tanks. The experiment lasted 56 days. In this period, two cycles of lettuce production and one of tilapia juveniles were performed.Ingredients usually used in Brazilian commercial feed industries were selected and their nutrient and energy contents were analyzed at the Laboratory of Animal Nutrition  of the Faculty of Agricultural and Veterinary Sciences. After that, four diets for tilapia juveniles were formulated to contain different levels of crude protein according to the tested treatments. The diets were isoenergetic, isophosphoric and the protein ingredients from animal sources were maintained at 25% of the total protein content. In all diets, commercial greenhouse supplies the proportion of the protein ingredients from animal sources was set at 3:2:1 for poultry by-product meal, fish meal, and feather meal, respectively. Soybean meal was the most used protein ingredient among the plant-based sources. The choice of these ingredients and their representation in the diet formulation was based on their availability and quality in Brazil. The diets were formulated to meet some nutritional requirements of tilapia juvenile, i.e., a minimum of 35, 5.5, 17.8, and 4 g kg− 1 of ether extract, methionine, lysine, and phosphorus, respectively, and a maximum of 60 and 80 g kg− 1 of crude fiber and ash, respectively. Diet ingredients were finely ground and sieved in 0.9 mm mesh and 0.5 to 4 mm feed pellets were processed at the Feed Manufacturing Facility of the FCAV-Unesp.The greenhouse hosted individual aquaponics systems run in decoupled mode with unidirectional flow from the aquaculture to the hydroponics subsystems. In each replicate the effluent from the aquaculture subsystem was supplied to two hydroponics subsystems. Thus, in total 18 aquaculture subsystems and 36 hydroponics subsystems were run. The configuration of the aquaculture subsystems differed from each other according to the aquaculture technology employed. 

The aquaculture subsystem of the DAPS treatment was run as a recirculating aquaculture system  and consisted of a circular fish tank,a radial flow settler,a bag filter,and a moving bed bioreactor. When operated as DAPS, the water was recirculated between the aquaculture units using a pump  submerged in the MBBR. The configuration described above was applied in three identical and independent aquaculture subsystems for the DAPS treatment, whereas 15 identical and independent aquaculture subsystems of the biofloc-based treatments were run. These biofloc-based subsystems consisted of a circular fish tank  and a RFS. In contrast to DAPS, in the BFT or DFP treatments the water remained in the fish tank and, in DFPs, was periodically directed to the RFS for the collection of the supernatant for plant nutrition. The plant nutrition management is detailed below, in subsection 2.4.1. The sedimented organic matter  from the RFS-DAPS was removed weekly. In the hydroponics subsystem, 36 individualized production units in a deep-water culture  mode named as plant tanks  were used, totaling 6 PTs for each treatment. The surface of each PT was 0.42 m2,where 8 plants were accommodated in an expanded polystyrene block with an identical area to each tank. It should be noted beforehand that high fish mortality occurred in all replicates of DFP-36 treatment after the middle of the experiment. After two days of exceptionally high temperatures,a combination of high nutrient load, high settleable solids  and high water temperature  caused a sudden drop in the dissolved oxygen  in the water at the end of the fortieth day of the experiment and, subsequently, the death of more than 80% of the fish. Thus, this treatment was discontinued, and its results were not analyzed and presented. Aeration was provided by an air blower  and distributed in each system by micro-perforated diffusers. Circular pieces of diffuser  were placed in the center of each fish tank and a 15 cm length in each MBBR and PT. In each fish tank, a 500 W thermostat heater was used to maintain the water temperature at 27 ◦C.