2.2. Experimental Design

A 62-day factorial experiment was conducted, in which two factors, each with two levels, were evaluated in quadruplicate: the addition of the microalgae Scenedesmus obliquus to the tank water and the supplementation of fish feed, totaling four treatments, namely: (1) No addition of microalgae and no addition of fish feed; (2) no addition of microalgae and addition of fish feed; (3) addition of microalgae and no addition of fish feed; and (4) addition of microalgae and addition of fish feed. No alteration was made in the feeding of shrimp in any of the treatments.

The microalgae addition was performed twice a week with the aim of maintaining a stable concentration throughout the experiment. The final S. obliquus concentration of 5 mg L⁻¹ (dry biomass) at the experimental units was chosen so as to not affect the production of solids.

The shrimp (2.16 ± 0.01 g) were stocked under a density of 400 shrimp m⁻³ (320 shrimp tank⁻¹) and the fish (1.53 ± 0.12 g) were stocked under a density of 522 fish m⁻³ (47 fish tank⁻¹).

In the treatments in which the fish received feed, they were fed once a day at the rate of 1% of their biomass, using a commercial feed (Guabitech Inicial 1 mm, 45% crude protein). Shrimp in all treatments were fed four times a day according to a feeding table [29] with a 40% crude protein commercial feed (Guabitech Inicial J40) until they reached a mean weight of 3 g, after which the feed was substituted with a 35% crude protein one (Guabi Poti Guaçu 1.6 mm).

The experimental units (Figure 1) were composed of 1000 L tanks (800 L of useful volume) and 100 L tanks (90 L of useful volume) for the shrimp and fish, respectively, located in an agricultural greenhouse and covered with shade cloth. A submerged pump (Sarlo-Better 650 L h⁻¹) placed in the shrimp tanks pumped the water to the fish tanks, which returned to the former by gravity, maintaining a 24 h day⁻¹ recirculation. The shrimp units were equipped with artificial substrates made of high-density polyester (Needlona^®), comprising 80% of the tank surface area. A circular microperforated hose in the shrimp unit and four air-stones in the fish unit connected to a central blower maintained dissolved oxygen concentrations above 5 mg L⁻¹ and the bioflocs in suspension. Water temperature was kept at 29.2 ± 1.2 °C through an 800 W heater in the shrimp units.

Schematic drawing of the experimental units used in a Pacific white shrimp (Litopenaeus vannamei) and Nile tilapia (Oreochromis niloticus) integrated culture using biofloc technology for 62 days, in which the addition or no addition of both microalgae and fish feed were evaluated in a factorial design.

Before the experiment began, the shrimp tanks were filled with approximately 40% of water from a matrix biofloc tank. Then, shrimp were stocked and acclimatized to a salinity of 15 g L⁻¹ with freshwater according to the procedure of Van Wyk [30].

Similarly, the fish tanks were filled with 40% of water from the biofloc matrix tank. The remaining was filled with freshwater before the animals were stocked.

The physico-chemical characteristics of the biofloc matrix tank water were: total ammonia-N 0.37 mg L⁻¹; nitrite (N-NO₂⁻) 0.34 mg L⁻¹; nitrate (N-NO₃⁻) 11.15 mg L⁻¹; TSS 465 mg L⁻¹; pH 8.00; alkalinity 160 mg L⁻¹ and salinity 35.2 g L⁻¹. Freshwater used in the acclimation was provided by the water works company of Florianópolis, SC, Brazil. Salinity was controlled through the addition of freshwater to compensate water lost through evaporation, while alkalinity was kept above 120 mg L⁻¹ by the addition of calcium hydroxide.