Effect of Organic or Inorganic Fertilization on Microbial Flocs Production in Integrated Cultivation of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei

Abstract

Different fertilization regimes in biofloc systems influence the predominance of distinct bacterial populations, impacting water quality and organism performance. This study evaluates the growth and nutrient absorption of the macroalgae Ulva lactuca when cultivated in an integrated system with Penaeus vannamei and Oreochromis niloticus in chemoautotrophic and heterotrophic systems. The experiment lasted 45 days and comprised two treatments, each with three replicates: chemoautotrophic—utilizing chemical fertilizers; heterotrophic—employing inoculum from mature biofloc shrimp cultivation, supplemented with organic fertilizers. Each treatment consisted of three systems, each containing a 4 m³ tank for shrimp, 0.7 m³ for tilapia, and 0.35 m³ for macroalgae, with continuous water circulation between tanks and constant aeration. Water quality analyses were carried out during the experiment, as were the performances of the macroalgae and animals. The data were subjected to a statistical analysis. Results revealed an increase in macroalgae biomass and the removal of nitrate (57%) and phosphate (47%) during cultivation, with a higher specific growth rate observed in the chemoautotrophic treatment. Nonetheless, the heterotrophic treatment exhibited higher levels of protein in the macroalgae (18% dry matter) and phosphate removal rates (56%), along with superior maintenance of water quality parameters. Tilapia performance varied across treatments, with a higher final weight and weight gain recorded in the heterotrophic treatment. The recycling of water from an ongoing biofloc cultivation with organic fertilization demonstrated viability for macroalgae cultivation within an integrated system involving shrimp and fish.

1. Introduction

Numerous recent studies have explored marine macroalgae as a source of human food, bioactive compounds, and supplements for marine organisms [1,2]. According to Chopin [3], macroalgae cultivation can be deemed sustainable due to their minimal need for feed, reduced land footprint, and bioremediation capabilities. The genus Ulva exhibits a global distribution, with the morphology and composition adapting to the environmental variables of the production site, rendering it a feasible and intriguing option for cultivation [4]. The cultivation of macroalgae integrated with other aquatic organisms has gained traction in aquaculture, referred to as Integrated Multitrophic Aquaculture (IMTA) [5]. Resende et al. [6] demonstrated the feasibility of incorporating macroalgae for nutrient absorption and biomass production in open cultivation systems with Sea bream Sparus aurata and European sea bass Dicentrarchus labrax. In addition to macroalgae serving as inorganic consumers, the IMTA system also includes organic consumers to consume the solids produced in the system. The tilapia Oreochromis niloticus, known for its ease of handling and feeding habits, has been utilized in integrated systems, showing positive results in solid filtration [7,8]. Due to its robustness, tilapia can also be produced in brackish water without negative effects on its zootechnical performance [9]. Both species could bring benefits when integrated into the cultivation of a main species, as in the farming of the Pacific white shrimp Penaeus vannamei, the most cultivated shrimp in the world, especially because it is an euryhaline [10], easy to manage and adapt [11], and holds significant economic interest [12], which has been employed as a primary species in integrated systems.

In conjunction with integrated systems, the utilization of biofloc technology has intensified production by improving the utilization of accumulated waste in cultivation. Studies by Brito et al. [13], Legarda et al. [14], and Morais et al. [15] have reported positive results from the production of shrimp, tilapia, and macroalgae in an integrated biofloc system. In general, biofloc technology offers enhanced biosecurity with reduced water exchange, facilitates water quality control through microbial activity, and serves as a supplementary food source for cultivated species [16]. Various fertilization approaches promote the growth and dominance of distinct bacterial groups within the system, including heterotrophic bacteria, chemoautotrophic bacteria, or a combination of both in mixed or mature systems [17]. The bacterial groups play a crucial role in nitrogen consumption and oxidation within the system, contributing to the maintenance of water quality for the organisms. Heterotrophic bacteria are favored by daily carbon source fertilization, typically at a ratio of 15 g of carbohydrate per gram of available nitrogen in the system [18]. Ammonia consumption by heterotrophic bacteria leads to bacterial biomass production, increasing the total suspended solids concentration in the water, which should ideally be maintained within the range of 100 to 350 mg L⁻¹, as suggested by Gaona et al. [19] to avoid adverse effects on animal performance.

Another significant bacterial group comprises chemoautotrophs, which, according to Ebeling et al. [18], oxidize nitrogen within the system, converting ammonia to nitrite and eventually to nitrate, a less toxic final product for organisms. Chemical fertilization is utilized for system establishment, requiring approximately 30 to 45 days of chemical fertilization prior to cultivation initiation to maintain low concentrations of ammonia and nitrite [17]. Unlike heterotrophic bacteria, chemoautotrophic bacteria generate fewer solids in the system and consume less oxygen. However, they utilize more inorganic carbon, requiring alkalinity adjustments to maintain levels above 150 mg of CaCO₃ L⁻¹ [20]. Nitrate accumulates as the final nitrogen product in this system’s oxidation process, posing toxicity risks to cultivated organisms at high concentrations [21], and, when discharged untreated, can lead to diseases such as methemoglobinemia in humans [22]. Another viable option is to utilize a biofloc inoculum from an ongoing cultivation, providing enhanced stability in nitrogen control and greater sustainability through water reuse [17]. This approach results in a mixed system containing both heterotrophic and chemoautotrophic bacteria, aimed at regulating water quality by promoting bacterial biomass production and nitrification [23]. Organic fertilization is typically employed at the onset of cultivation to expedite the stabilization of ammonia until nitrifying bacteria become established [24].

The biofloc system is complex and subject to variations based on the fertilization strategy utilized, which can impact water quality, production costs, and animal performance. Brandão et al. [23] reported greater shrimp growth in mixed systems compared to heterotrophic systems. Conversely, tilapia performance was negatively impacted in chemoautotrophic systems due to low organic matter loads [8]. However, limited information exists regarding macroalgae performance within these systems. High concentrations of solids produced by heterotrophic bacteria may accumulate on macroalgae, hindering photosynthesis and affecting their performance [25]. Additionally, elevated nutrient concentrations present in the chemoautotrophic system can induce stress in macroalgae and trigger reproductive events [26]. Choosing the right cultivation system can provide better growing conditions and biomass production for the macroalgae. In addition to growth and nutrient absorption, the specific physical and chemical variables inherent to each cultivation system also influence the nutritional composition of macroalgae [27]. The production of biomass with enhanced nutritional value can offer economic advantages for the system through the generation of valuable by-products. For instance, utilizing the biomass of macroalgae cultivated in the integrated system as a food source for shrimp and fish could prove beneficial for aquaculture [2]. Therefore, the objective of this study was to evaluate the growth performance, nutrient absorption, and bioactive compounds of the macroalga Ulva lactuca when cultivated in an integrated system with Pacific white shrimp Penaeus vannamei and tilapia Oreochromis niloticus using two biofloc fertilization strategies: a chemoautotrophic system and a heterotrophic system.

2. Materials and Methods

2.1. Location and Origin of the Animals

The experiment was conducted in an agricultural greenhouse situated at the Marine Station of Aquaculture (EMA), Institute of Oceanography, Federal University of Rio Grande (IO-FURG), located on Cassino Beach, Rio Grande, Rio Grande do Sul. The greenhouse was devoid of shading, and aeration within the tanks was provided by a blower through continuous air injection via micro-perforated hoses (aerotubes).

2.2. Animal Materials

The shrimp originated from a biofloc cultivation system within a grow-out greenhouse at EMA, with an initial weight of 7.13 ± 0.18 g. Tilapia were sourced from a recirculation system grow-out cultivation, starting with an initial weight of 412.33 ± 72.58 g. The macroalgae were cultivated in a greenhouse in a 1 m³ tank containing water with 35.1 ± 2.74 mg L⁻¹ of nitrate and 2.24 ± 1.2 mg L⁻¹ of phosphate.

2.3. Experimental Design

The experiment, which spanned 45 days, was conducted on six experimental production systems. Each system comprised a 4 m³ tank for shrimp (350 shrimp m⁻²), a 0.7 m³ tank for tilapia (10 fish per m³), and a 0.35 m³ tank for macroalgae cultivation (0.1 g m³ of the useful volume of the entire system). A submerged pump circulated the system, transferring water into the macroalgae tank, which then flowed by gravity into the shrimp tank before returning to the tilapia tank (Figure 1). The macroalgae were contained within the tank using a circular structure with a diameter of 0.60 m positioned near the surface, constructed from polyethylene netting with 5 mm mesh openings.

2.4. Treatments

Two treatments were employed, each with three replicates: chemoautotrophic—a system utilizing chemical inorganic fertilization; heterotrophic—a system supplemented with organic fertilizer. Inoculum preparation for the chemoautotrophic system involved maintaining water with a salinity of 20 ppt in an 8 m³ tank. Over 35 days, daily fertilization with sodium nitrite (Neon Comercial, São Paulo, SP, Brazil) and ammonium chloride (Neon Comercial, São Paulo, SP, Brazil) was conducted to achieve a concentration of 1 mg L⁻¹ for each compound in the water. To establish bacterial populations in the system, six pillow-like structures containing biological media were placed in the main tank and then distributed among the replicates. The tank was continuously aerated and devoid of light, and no heaters were utilized to simulate greenhouse cultivation conditions.

Once the ammonia and nitrite concentrations stabilized and were converted into nitrate, the experiment started. Chemoautotrophic treatment replicates were prepared by blending 40% inoculum with water of salinity 20, up to a useful volume of 5 m³. At the onset of the experiment, the water parameters were as follows: a temperature of 26.0 ± 0.4 °C, dissolved oxygen of 7.2 ± 0.5 mg L⁻¹, pH of 8.18 ± 0.3, alkalinity of 170.0 ± 2. 0 mg CaCO₃ L⁻¹, total ammonia nitrogen of 0.02 ± 0.02 mg L⁻¹, nitrite of 1.5 ± 0.2 mg L⁻¹, nitrate of 64.0 ± 1.7 mg L⁻¹, phosphate of 1.2 ± 0.4 mg L⁻¹, and total suspended solids of 160.0 ± 5.8 mg L⁻¹.

The tanks designated for the heterotrophic treatment were prepared with 40% mature biofloc inoculum and seawater at a salinity of 20 ppt, up to a useful volume of 5 m³. The biofloc inoculum was sourced from a shrimp cultivation system with a useful volume of 237 m³, a density of 184 shrimp m⁻², and an average weight of 7.1 ± 1.2 g, cultivated for 68 days. The initial water quality parameters in the shrimp production tank before the experiment were as follows: a temperature of 25.6 °C, dissolved oxygen of 5.4 mg L⁻¹, pH of 7.47, alkalinity of 215.0 mg CaCO₃ L⁻¹, total ammoniacal nitrogen of 0.20 mg L⁻¹, nitrite of 0.20 mg L⁻¹, nitrate of 147.0 mg L⁻¹, phosphate of 4.0 mg L⁻¹, and total suspended solids of 700.00 mg L⁻¹.

2.5. Chemical and Physical Water Parameters

Water quality analyses were conducted on samples collected from the shrimp tanks, considering water homogenization due to the circulation within the systems. Temperature and dissolved oxygen were measured twice daily using a Pro-20 model (YSI Inc., Yellow Springs, OH, USA), and pH was measured daily with a bench pH meter (Seven2Go S7 Basic, Mettler Toledo, São Paulo, SP, Brazil). Salinity was assessed twice a week using a Pro-20 model (YSI Inc., OH, USA), and, if necessary, fresh water was added to maintain salinity at 20. Alkalinity (mg CaCO₃ L⁻¹) was monitored twice a week following the APHA methodology [28], with calcium hydroxide added to both treatments when alkalinity fell below 150 mg CaCO₃ L⁻¹, as per Furtado et al. [20] recommendation.

Total ammoniacal nitrogen (mg L⁻¹) and nitrite (mg L⁻¹) were initially measured daily and then twice a week after nutrient stabilization, according to UNESCO methodology [29]. Nitrate (mg L⁻¹) and phosphate (mg L⁻¹) were measured twice a week, according to the method proposed by Aminot and Chaussepied [30]. Total suspended solids (mg L⁻¹⁻TSS) and settleable solids (ml L⁻¹⁻SS) were quantified twice a week, using the methodology described by Baumgarten et al. [31] and APHA [28], respectively. For the heterotrophic system, organic carbon (molasses) was added when the total ammoniacal nitrogen exceeded 1 mg L⁻¹ to promote nitrogen uptake through heterotrophic bacteria growth, as proposed by Wasielesky et al. [32]. In the chemoautotrophic system, inorganic carbon (calcium hydroxide) was added when ammonia and nitrite concentrations exceeded 1 mg L⁻¹ and 5 mg L⁻¹, respectively. In this treatment, alkalinity was maintained at 300 mg CaCO₃ L⁻¹ for optimal nitrifying bacteria performance, as recommended by Furtado [20].

2.6. Macroalgae Growth and Biochemical Analysis

Macroalgae biomass was weighed every 15 days. Before the weighing process, the macroalgae were gently shaken inside the holding structure to eliminate any solids adhering to the surface. Subsequently, the circular holding structure was removed from the tank and set aside to air-dry for 10 min to remove excess water before weighing. The initial weight of macroalgae in each replicate was 502.7 ± 0.5 g. After each weighing, the extra macroalgae biomass was removed, ensuring that the initial weight of the macroalgae was maintained. The following formula was used to calculate the macroalgae specific growth rate (SGR) [33]:

SGR (% d⁻¹): 100 × [ln (final weight (g)/initial weight (g))/(final time − initial time)]

(1)

The nutrient absorption efficiency (NRR) of the macroalgae was calculated using the following formula [33]:

NRR (%): 100 × [(nutrient concentration at initial time (mg L⁻¹) − nutrient concentration at final time (mg L⁻¹))/nutrient concentration at initial time (mg L⁻¹)]

(2)

At the conclusion of the experiment, random samples of macroalgae were collected from each replicate. Wet samples were weighed and then subjected to drying in an oven at 60 °C for 24 h after obtaining the dry weight. To determine the concentration of chlorophyll-a, chlorophyll-b, and carotenoids, 500 mg of the dry sample was macerated and then incubated in 5 mL of methanol in the dark for 60 min at 4 °C. After that, the solution was centrifuged (12,000× g, 10 min), and the supernatant was used to quantify the pigments. The wavelengths of 664 and 647 ηm were used to calculate chlorophyll a (Chla = 11.75 × A664 − 2.35 × A647), chlorophyll b (Chlb = 18.61 × A647 − 3.91 × A664), and carotenoids (Car = (1000 × A470 − 2.27 × Chla − 81.4 Chlb)/227), according to the methodology of Lichtenthaler & Wellburn [34].

Protein quantification was conducted using the Bradford method. An extract was obtained from the dried macroalgae, following the protocol of Barbarino & Lourenço [35], with the addition of 1 mL of sodium hydroxide and centrifugation. The extract and TCA (25%) were added in a ratio of 2.5:1 (v/v) to precipitate the protein and kept in an ice-cold bath for 30 min. The solution was then centrifuged and washed with dilutions of TCA (10 and 5%), removing the supernatant, until the protein pellet was formed. To the pellet suspension, 0.5 mL of sodium hydroxide (0.1 N) was added, and 20 µL of the solution was combined with 1 mL of the total protein kit for the final analysis procedure.

2.7. Feed Management and Performance of the Animals

Shrimp were fed twice daily with 1.6 mm feed (Guabi aqua QS 1–2 mm, Guabi Nutrition and Animal Health S.A., Campinas, São Paulo, SP, Brazil), and weekly biometrics were conducted to adjust feed quantities following the method proposed by Jory et al. [36]. The tilapia were fed twice a day with commercial feed containing 40% protein (Guabi Tech, Guabi Nutrition and Animal Health S.A., Campinas, São Paulo, SP, Brazil) at a rate of 1% of the biomass to encourage biofloc consumption. To evaluate shrimp performance, measurements were taken at the beginning, middle, and end of the experiment. Fish biometrics were conducted at the beginning and end of the experiment. The animals’ performance was assessed using the following formulas:

• − Final average weight (g): final biomass of live animals (g)/total number of animals;
• − Weekly weight gain (g week⁻¹): weight gain (g)/number of weeks;
• − Final biomass (g): ∑ final weight of all live animals (g);
• − Feed conversion rate (FCR) = ∑ feed offered (g)/(biomass gain (g));
• − Survival (%) = (final number of animals/initial number of animals) × 100;
• − Yield (kg m⁻³): (final biomass (kg)/tank volume (m³);
• − Weight gain rate (%) = 100 × [(final mean weight − initial mean weight)/initial mean weight].

2.8. Statistical Analysis

The data mean (±standard deviation) values are presented in Table 1, Table 2 and Table 3. Data normality and homoscedasticity were assessed using the Shapiro–Wilk and Levene tests, respectively. Upon meeting these assumptions, a Student’s t-test was employed to compare treatment differences. In cases where the assumptions of the Student’s t-test were not met, the non-parametric Kruskal–Wallis test was utilized. Additionally, a one-way ANOVA followed by a Tukey post-hoc test was conducted to evaluate nitrate and phosphate concentrations over time in each treatment. A significance level of 5% (p ≤ 0.05) was applied to all analyses. The tests were carried out using the PAST 4.03 2020 software [37].

3. Results

3.1. Physical and Chemical Parameters

During the 45-day trial period, there were significant differences (p < 0.05) observed in pH, alkalinity, and calcium hydroxide consumption between the treatments. The chemoautotrophic system exhibited the highest values, along with higher consumption of calcium hydroxide (Table 1).

Regarding nutrient levels, the chemoautotrophic system demonstrated higher concentrations of ammonia and nitrite, reaching maximums of 3.1 and 20.0 mg L⁻¹, respectively. Conversely, the heterotrophic system exhibited higher concentrations of total suspended solids and settleable solids (Table 1). Significant nitrate and phosphate removal (p < 0.05) was observed in both treatments, although the heterotrophic treatment displayed a higher phosphate removal rate compared to the chemoautotrophic system.

Table 1. Water quality parameters (mean ± standard deviation) (maximum–minimum) of chemoautotrophic and heterotrophic biofloc systems during the 45 days of integrated cultivation of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.

	Treatments
Parameters	Chemoautotrophic	Heterotrophic
Temperature (°C)	22.82 ± 0.30 (27.4–15.5)	22.67 ± 0.38 (27.3–14.9)
DO (mg L −1)	7.06 ± 0.04 (9.9–5.3)	6.96 ± 0.06 (9.7–5.2)
pH	8.15 ± 0.02 a (8.9–7.7)	7.89 ± 0.04 b (8.1–7.5)
Salinity (g L−1)	20.20 ± 0.26 (22.1–19.1)	21.83 ± 0.76 (23.3–20.3)
Alkalinity (mg CaCO3 L−1)	280.00 ± 8.19 a (365.0–155.0)	189.58 ± 7.02 b (230.0–150.0)
TAN (mg L−1)	0.95 ± 0.07 b (3.1–0.0)	0.18 ± 0.08 a (1.9–0.0)
N—Nitrite (mg L−1)	7.96 ± 1.16 b (20.0–0.0)	0.86 ± 0.60 a (5.2–0.0)
N—Nitrate (mg L−1)	26.41 ± 2.72 (68.0–10.0)	26.04 ± 4.03 (75.0–15.0)
P—Phosphate (mg L−1)	1.04 ± 0.25 (2.0–0.3)	1.19 ± 0.21 (2.2–0.4)
SS (ml L−1)	0.39 ± 0.21 a (3.0–0.0)	8.44 ± 2.91 b (15.0–3.0)
TSS (mg L−1)	189.22 ± 26.02 a (270.0–70.0)	335.38 ± 47.92 b (452.5–175.0)
Calcium hydroxide (g L−1) #	0.29 ± 0.02 b (0.32–0.28)	0.08 ± 0.03 a (0.10–0.04)
Water exchange (m−3) &	2.0 ± 2.0 a	0.0 ± 0.0 a
Removal rate
Nitrate (%)	56.47 ± 4.93	57.00 ± 7.00
Phosphate (%)	47.75 ± 4.75 b	56.14 ± 1.14 a

DO (dissolved oxygen); TAN (total ammonium nitrogen); SS (settleable solids); TSS (total suspended solids). ^# Use of calcium hydroxide during cultivation. ^& Volume of water used for renovations. Different letters in the same line represent significant differences (p ≤ 0.05) between treatments after Student’s t-test.

Table 1. Water quality parameters (mean ± standard deviation) (maximum–minimum) of chemoautotrophic and heterotrophic biofloc systems during the 45 days of integrated cultivation of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.

	Treatments
Parameters	Chemoautotrophic	Heterotrophic
Temperature (°C)	22.82 ± 0.30 (27.4–15.5)	22.67 ± 0.38 (27.3–14.9)
DO (mg L −1)	7.06 ± 0.04 (9.9–5.3)	6.96 ± 0.06 (9.7–5.2)
pH	8.15 ± 0.02 a (8.9–7.7)	7.89 ± 0.04 b (8.1–7.5)
Salinity (g L−1)	20.20 ± 0.26 (22.1–19.1)	21.83 ± 0.76 (23.3–20.3)
Alkalinity (mg CaCO3 L−1)	280.00 ± 8.19 a (365.0–155.0)	189.58 ± 7.02 b (230.0–150.0)
TAN (mg L−1)	0.95 ± 0.07 b (3.1–0.0)	0.18 ± 0.08 a (1.9–0.0)
N—Nitrite (mg L−1)	7.96 ± 1.16 b (20.0–0.0)	0.86 ± 0.60 a (5.2–0.0)
N—Nitrate (mg L−1)	26.41 ± 2.72 (68.0–10.0)	26.04 ± 4.03 (75.0–15.0)
P—Phosphate (mg L−1)	1.04 ± 0.25 (2.0–0.3)	1.19 ± 0.21 (2.2–0.4)
SS (ml L−1)	0.39 ± 0.21 a (3.0–0.0)	8.44 ± 2.91 b (15.0–3.0)
TSS (mg L−1)	189.22 ± 26.02 a (270.0–70.0)	335.38 ± 47.92 b (452.5–175.0)
Calcium hydroxide (g L−1) #	0.29 ± 0.02 b (0.32–0.28)	0.08 ± 0.03 a (0.10–0.04)
Water exchange (m−3) &	2.0 ± 2.0 a	0.0 ± 0.0 a
Removal rate
Nitrate (%)	56.47 ± 4.93	57.00 ± 7.00
Phosphate (%)	47.75 ± 4.75 b	56.14 ± 1.14 a

DO (dissolved oxygen); TAN (total ammonium nitrogen); SS (settleable solids); TSS (total suspended solids). ^# Use of calcium hydroxide during cultivation. ^& Volume of water used for renovations. Different letters in the same line represent significant differences (p ≤ 0.05) between treatments after Student’s t-test.

Over the weeks of cultivation, there was a reduction in the concentration of nitrate and phosphate (Figure 2 and Figure 3). The highest nitrate concentrations were observed at the beginning of cultivation, with a decrease from day 14 onward in both treatments (Figure 2). Similarly, phosphate concentrations were higher during the initial week, after which they stabilized in the heterotrophic treatment. In contrast, the chemoautotrophic treatment exhibited a decrease in phosphate concentration until the first week, followed by stabilization and a subsequent increase in the final week (Figure 3).

Total suspended solids exhibited significant differences (p < 0.05) between treatments during most of the experimental weeks. High concentrations of solids reaching 452 mg L⁻¹ were observed in the heterotrophic treatment, in contrast to maximum concentrations of 270 mg L⁻¹ in the chemoautotrophic treatment (Figure 4).

3.2. Macroalgae Growth and Biochemical Analysis

There was an increase in macroalgae biomass in both treatments, with higher concentrations of protein in the macroalgae tissue in the heterotrophic treatment (p < 0.05). However, no significant differences (p ≥ 0.05) were found in biomass gain, chlorophyll-a, chlorophyll-b, or carotenoids between the treatments (Table 2).

Table 2. Performance and biochemistry of the macroalgae (mean ± standard deviation) in the chemoautotrophic and heterotrophic treatments at the end of 45 days of an integrated culture of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.

	Treatments
	Chemoautotrophic	Heterotrophic
Initial mean weight (g–FW)	502.76 ± 0.65	502.64 ± 0.44
Biomass gain (g–FW)	964.63 ± 290.57	708.11 ± 141.70
Protein (%)	15.13 ± 0.56 b	18.49 ± 0.56 a
Chlorophyll-a (mg g−1)	2.20 ± 0.02	2.18 ± 0.06
Chlorophyll-b (mg g−1)	3.28 ± 0.02	3.26 ± 0.08
Carotenoids (mg g−1)	0.06 ± 0.00	0.04 ± 0.02

Different letters in the same line represent significant differences (p ≤ 0.05) between treatments after Student’s t-test.

Table 2. Performance and biochemistry of the macroalgae (mean ± standard deviation) in the chemoautotrophic and heterotrophic treatments at the end of 45 days of an integrated culture of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.

	Treatments
	Chemoautotrophic	Heterotrophic
Initial mean weight (g–FW)	502.76 ± 0.65	502.64 ± 0.44
Biomass gain (g–FW)	964.63 ± 290.57	708.11 ± 141.70
Protein (%)	15.13 ± 0.56 b	18.49 ± 0.56 a
Chlorophyll-a (mg g−1)	2.20 ± 0.02	2.18 ± 0.06
Chlorophyll-b (mg g−1)	3.28 ± 0.02	3.26 ± 0.08
Carotenoids (mg g−1)	0.06 ± 0.00	0.04 ± 0.02

Different letters in the same line represent significant differences (p ≤ 0.05) between treatments after Student’s t-test.

The specific growth rate indicates that the growth of macroalgae in the chemoautotrophic treatment remained consistent throughout the entire experiment, with no significant difference (p ≥ 0.05) observed between weighings. In contrast, for the heterotrophic treatment, the highest growth rate was recorded on day 21, followed by a subsequent decrease in growth rate. Notably, a significant difference in growth rate was only detected in the last weighing among the treatments (Figure 5).

3.3. Performance of the Animals

Shrimp performance remained unaffected by the different biofloc strategies, with no discernible differences observed between treatments. However, the performance of the fish exhibited a significant difference (p < 0.05) between treatments, with a higher final weight, weight gain, and weight gain rate recorded in the heterotrophic treatment compared to the chemoautotrophic treatment (Table 3).

Table 3. Animal performance (mean ± standard deviation) in the chemoautotrophic (chemical fertilization prior to stocking) and heterotrophic (use of an inoculum from an ongoing biofloc shrimp cultivation) treatments at the end of 45 days of an integrated cultivation of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.

	Treatments
	Chemoautotrophic	Heterotrophic
Shrimp
Final mean weight (g)	9.60 ± 0.60	10.75 ± 0.49
WWG (g week−1) #	0.41 ± 0.10	0.60 ± 0.08
Final biomass (kg)	10.02 ± 0.43	10.79 ± 0.60
FCR ##	2.16 ± 0.38	2.36 ± 0.24
Survival (%)	84.71 ± 4.50	84.08 ± 5.22
Fish
Final mean weight (g)	447.55 ± 1.05 b	456.25 ± 3.25 a
WWG (g week−1) #	5.93 ± 0.18 b	7.38 ± 0.55 a
Final biomass (kg)	2.89 ± 0.54	2.98 ± 0.25
FCR ##	1.03 ± 0.10	1.18 ± 0.02
Survival (%)	90.48 ± 16.50	95.24 ± 8.25
WGR (%) ###	8.63 ± 0.25 b	10.74 ± 0.79 a

^# WGW (weekly weight gain); ^## FCR (food conversion rate); ^### WGR (weight gain rate). Lowercase letters mean differences between treatments.

Table 3. Animal performance (mean ± standard deviation) in the chemoautotrophic (chemical fertilization prior to stocking) and heterotrophic (use of an inoculum from an ongoing biofloc shrimp cultivation) treatments at the end of 45 days of an integrated cultivation of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.

	Treatments
	Chemoautotrophic	Heterotrophic
Shrimp
Final mean weight (g)	9.60 ± 0.60	10.75 ± 0.49
WWG (g week−1) #	0.41 ± 0.10	0.60 ± 0.08
Final biomass (kg)	10.02 ± 0.43	10.79 ± 0.60
FCR ##	2.16 ± 0.38	2.36 ± 0.24
Survival (%)	84.71 ± 4.50	84.08 ± 5.22
Fish
Final mean weight (g)	447.55 ± 1.05 b	456.25 ± 3.25 a
WWG (g week−1) #	5.93 ± 0.18 b	7.38 ± 0.55 a
Final biomass (kg)	2.89 ± 0.54	2.98 ± 0.25
FCR ##	1.03 ± 0.10	1.18 ± 0.02
Survival (%)	90.48 ± 16.50	95.24 ± 8.25
WGR (%) ###	8.63 ± 0.25 b	10.74 ± 0.79 a

^# WGW (weekly weight gain); ^## FCR (food conversion rate); ^### WGR (weight gain rate). Lowercase letters mean differences between treatments.

4. Discussion

It is widely acknowledged that the utilization of biofloc technology, compared to conventional cultivation methods, leads to reduced water consumption and enhanced control over water quality parameters [16]. The various fertilization strategies employed in this experiment resulted in differences in water quality maintenance, organism performance, water usage, and inputs. The chemoautotrophic system utilizes more inputs, such as sodium hydroxide, compared to the heterotrophic system. This variance is necessary to maintain optimal alkalinity values. In the chemoautotrophic system, the optimal functioning of nitrifying bacteria occurs at values maintained at around 300 mg CaCO₃ L⁻¹, consequently resulting in higher pH values [20]. As a result, there is a more frequent application of sodium hydroxide to correct these values and stimulate the growth of nitrifying bacteria with higher alkalinity. Furthermore, differences in nutrient concentrations were observed between the adopted systems, which play a crucial role in macroalgae development. According to Messyasz et al. [38], most marine Ulva species thrive in environments with high concentrations of ammonia or nitrate. Ammonia, originating from waste feed and animal excretion, is the primary nitrogenous compound formed in the system and can be lethal to cultivated organisms at low levels [39]. In both systems in this study, an initial increase in ammonia concentration was observed due to animal stocking. Wasielesky et al. [32] suggest that the use of organic carbon fertilization could promote the growth of heterotrophic bacteria in the system, which consume produced ammonia and generate bacterial biomass. For the chemoautotrophic system, only inorganic fertilization with calcium hydroxide was employed to encourage the growth of nitrifying bacteria with higher alkalinity [20]. However, the slow establishment of nitrifying bacteria resulted in maximum values of 3.1 mg L⁻¹ of ammonia in the system, which were higher than those found in the heterotrophic system. Nevertheless, according to Lin & Chen [39], the values obtained in our study were not toxic to the organisms.

The produced ammonia is oxidized into nitrite by ammonium-oxidizing bacteria and subsequently into nitrate by nitrite-oxidizing bacteria. However, the observed increase in nitrite levels in the chemoautotrophic treatment suggests that the nitrite-oxidizing bacteria were not fully established in the system to facilitate this transformation. Despite the use of artificial substrate in this experiment, it is likely that the bacterial population was insufficient to oxidize the nitrite produced following the stocking of shrimp. The use of artificial substrate in the system is necessary for bacterial adherence and to increase their numbers [40]. According to Lin & Chen [41], the safe level for nitrite at a salinity of 25 is 15.2 mg L⁻¹, and concentrations exceeding this limit can be lethal to shrimp. Consequently, in our experiment, we carried out partial water exchange, and a reduction in shrimp and fish feeding was necessary to control nitrite levels in the system, resulting in higher water usage than in the heterotrophic system and the dilution of nutrients.

In biofloc systems, elevated concentrations of nitrate and phosphate are common in long-term production due to low water exchange rates and high animal densities, providing an advantageous environment for macroalgae development. Carneiro [42] noted that when macroalgae inhabit eutrophicated environments, they tend to absorb significant nutrient concentrations initially for storage, serving as a precautionary measure in case of sudden nutrient depletion. Additionally, Hanisak et al. [43] suggested that a constant high nitrogen availability in the environment does not necessarily result in increased removal, as macroalgae nitrogen absorption capacity saturates quickly at high concentrations. This phenomenon may have occurred in both treatments in our study, resulting in a substantial reduction in nitrate and phosphate concentrations at the onset of cultivation. Following the second week, nutrient stabilization occurred. It is documented that 57% of nitrogen is lost from the water daily, with an increase over time [44], suggesting that the stabilization of these nutrients in the experiment may be attributed to the continuous absorption carried out by the macroalgae. Studies such as Massocato et al. [45] have demonstrated that 85% of the nitrate from a fish cultivation was absorbed within the first five days of algae cultivation.

Phosphorus is also another compound accumulated in the system and produced daily through waste feed [44]. It is an important element in photosynthesis and the transfer of energy from macroalgae [46], which shows the advantage of integrating macroalgae into closed systems. Phosphorus absorption is connected with nitrogen absorption, with an ideal ratio of 30:1 (nitrogen:phosphorus), so that phosphorus or nitrogen are not limiting [47]. The higher removal rate found in the heterotrophic treatment may be linked to the pH values. According to Rathod et al. [48], higher phosphate absorption occurs at pH levels below neutrality. The maintenance of high alkalinity and pH in the chemoautotrophic treatment may have negatively impacted phosphate absorption.

The utilization of macroalgae as a biofilter has advanced due to their excellent performance in nutrient absorption, ease of management, and high biomass production [49]. Alencar et al. [50] demonstrated that the macroalgae Ulva lactuca absorbed 94% of the ammonia concentration in an integrated cultivation with shrimp. Conversely, the impact of the organic load generated in macroalgae cultivation remains poorly understood. Due to the intensive production of bacterial biomass, the heterotrophic system in this study exhibited higher concentrations of total suspended solids and settleable solids. In contrast, the chemoautotrophic system, with its use of inorganic fertilizers and water exchange, exhibited a lower organic load, with a maximum of 270 mg L⁻¹. Similar outcomes were reported by Ferreira et al. [17] in their study of the two biofloc systems. Despite the absence of a significant difference in macroalgae biomass gain between the treatments, a higher growth rate was observed toward the end of cultivation in the chemoautotrophic treatment, potentially attributable to the lower solids content in the system compared to the heterotrophic system. The accumulation of microbial biomass and waste in the heterotrophic system intensified toward the end of cultivation, likely directly affecting macroalgae growth. Carvalho et al. [51] demonstrated that the presence of macroalgae in the heterotrophic system led to solid deposition due to the formation of a physical barrier, reducing light exposure for the macroalgae and consequently impacting their performance.

Despite the lower concentration of solids in the chemoautotrophic system, they still accumulated on the surface of the macroalgae, representing one of the challenges of biofloc systems. Studies like Resende et al. [6] reported significantly higher growth rates, with a maximum growth rate of 15.33 ± 2.87% day⁻¹ when macroalgae were cultivated freely in tanks with fish farm effluent, characterized by minimal solids concentrations. The results found in our experiment are in agreement with studies by Martins et al. [52], who observed a growth rate of 3.0 ± 0.6% day⁻¹ with the macroalga Ulva ohnoi in a biofloc system. Studies with red algae in biofloc have also been carried out, showing a maximum growth rate of 1.19 ± 0.04% day⁻¹ [53], similar to those observed in our heterotrophic treatment results in the last weeks of cultivation. However, unlike studies such as Carvalho et al. [25] and Legarda et al. [14], which did not observe macroalgae growth in biofloc systems, our use of partial harvests might have reduced macroalgae density in the cultivation structure and minimized shading, resulting in improved biomass production. Biancacci et al. [54] showed that the use of partial harvests in the cultivation of the macroalga Macrocystis pyrifera promoted greater biomass gain, a lower incidence of epiphytes, and a change in the macroalgae biochemical composition.

In addition to serving as a bioremediator, macroalgae possess economic value, as the biomass they produce can be utilized in the pharmaceutical and food industries [55], thereby fostering sustainability and profitability in production. Macroalgae serve as vital sources of nutrients and vitamins and possess antioxidant and immunostimulant properties [56]. The higher protein values observed in macroalgae from the heterotrophic system may be attributed to reduced luminosity in the system due to the gradual accumulation of solids over the cultivation period. Ganesan et al. [57] showed a correlation between high pigment concentrations in low-light and salinity environments in their study on the macroalga Ulva fasciata, indicating potential adaptations to environmental conditions. The observed high values of chlorophyll-a and chlorophyll-b in our study compared to those reported by Silva et al. [58] may be linked to the necessity of increasing pigment concentrations in macroalgae to maximize photosynthesis, likely due to reduced light penetration caused by suspended particles in a biofloc system. Similar trends were noted by Fillit et al. [59], who reported increased pigment concentrations during periods of low light availability.

In the integrated system, all species must have productivity in cultivation and economic potential [60]. Despite the elevated nitrite concentrations in the chemoautotrophic system, shrimp and fish performance was not affected. However, growth outcomes and survival in both treatments were lower than those reported by Ferreira et al. [17] in their study on shrimp cultivation in chemoautotrophic, heterotrophic, and mature systems. This can be attributed to temperature differences between the studies. The minimum temperature recorded in our experiment was 14.9 °C, directly impacting the survival of the organisms. Furthermore, the overall average temperature in our study (22.0 °C) was lower compared to studies conducted with shrimp and fish [61], which also influenced the growth of the animals due to their decreased metabolism. Fish performance in terms of weight gain was superior in the heterotrophic system compared to the chemoautotrophic system, possibly due to the higher availability of suspended organic matter. The reduced feed supply aimed to induce floc consumption in the system, as demonstrated by Holanda et al. [7], with floc serving as a supplementary food source for organisms [62]. Hence, the higher concentration of total suspended solids in the heterotrophic system might have positively influenced fish weight gain. Similar results were reported by Poli et al. [8], who observed lower fish growth in an integrated system with chemoautotrophic floc.

The use of integrated multi-trophic systems aims to balance system productivity with sustainability, ensuring that all organisms adapt to the cultivation conditions. According to Khanjani et al. [63], the utilization of integrated systems has been consistently increasing, highlighting potential species for inclusion in the system, with crustaceans being among the most commonly produced target species. Zimmermann et al. [64] discuss the future of tilapia production, emphasizing multitrophic cultivation and biofloc technology as promising systems for maximizing production, considering greater sustainability, biosecurity, and increased density. However, the integration of macroalgae into biofloc systems has not yet been fully stabilized. The inclusion of macroalgae in biofloc systems has presented challenges due to their low productivity [14,15,25], but their role as bioremediators in nutrient absorption has shown promise, as demonstrated by the data presented in this study. Furthermore, the production of macroalgae biomass with an increase in nitrogen content in tissues, as reported by Legarda et al. [14] and Carvalho et al. [25], adds value to the product and enhances its applicability. The incorporation of macroalgae produced in integrated systems into fish and shrimp feed has yielded significant results, as evidenced by Marinho et al. [65] and Valente et al. [66]. Improved methods for managing the incorporation of macroalgae into biofloc systems are needed to enhance production and sustainability in intensive production systems.

5. Conclusions

The use of macroalgae in an integrated system with organic fertilization proved to be viable for increasing biomass production and nitrate and phosphate absorption, improving the system’s sustainability. The use of a system with a low concentration of solids, as in the chemoautotrophic system, promoted better growth rates for the macroalgae. However, the use of an inoculum from a heterotrophic system intensified the removal of phosphate and nitrate and increased the protein content of the macroalgae. A better maintenance of water quality was found in the heterotrophic system with the use of organic fertilization, without the need for water renewal. Finally, the heterotrophic system contributed to the better performance of the tilapia, with an increase in weight gain and a higher average final weight.