1. Introduction
Shrimp production using biofloc technology (BFT), normally carried out without water renewal, results in the accumulation of nutrients in the system [
1]. This occurs due to the action of microorganisms that transform shrimp excreta and food remains into protein and inorganic nutrients [
2]. Chemoautotrophic and heterotrophic bacteria grow during the culture. The first group of bacteria works in the oxidation of ammonia to nitrite and later to nitrate, which is accumulated in the system, along with phosphorus from the feed [
3]. Heterotrophic bacteria participate in the conversion of ammonia into bacterial biomass and, together with the accumulation of feces and feed remains, increase the concentration of total suspended solids in the system. The production of waste is constant during cultivation, and when it is not properly disposed of or treated, it can generate problems in the water quality in the production system and environmental problems in the release of effluents without treatment [
4].
The total suspended solids are important in the water quality of the system and must be maintained between 100 to 300 mg L
−1 [
5]. In addition to water quality, microbial flocs function as a complementary source of natural food within the crop [
6]. Azim and Little [
7] nutritionally described biofloc containing 38% protein and 3% lipid in dry matter, showing a high nutritional value, which may depend on the carbon source used in the system. Wasielesky et al. [
8] showed that it is possible to reduce the feeding frequency of shrimp
L. vannamei when cultivated in a biofloc system.
Despite the benefits of microbial flocs in the shrimp culture system, its effect on macroalgae growth is unknown. As macroalgae are photosynthetic organisms, the concentration of solids can interfere with the light capture that is essential for their growth. Brito et al. [
9] showed the deposition of solids on macroalgae, which may have a negative effect on its growth. However, the nutritional value of macroalgae can also change when grown in biofloc. Legarda et al. [
10] showed an increase in nitrogen, phosphorus, chlorophyll
a, and carotenoids when macroalgae were cultivated in an integrated system in biofloc.
One way to take advantage of the nitrogen and phosphorus accumulated in the system is integration with other species of different trophic levels in production, as proposed by Chopin [
11] in an integrated multi-trophic aquaculture (IMTA). In the system, residues are reused by different species, increasing the final productivity of the cultivation and sustainability. The IMTA system is composed of a species fed with commercial feed, such as shrimp or fish, and then a species capable of absorbing inorganic compounds dissolved in the water, such as macroalgae, and a species that consumes organic compounds, such as oyster, is inserted into the system, which will feed on suspended particles in the culture [
12].
Considering the precepts in the IMTA and associating them with the biofloc, the presence of macroalgae in integrated cultures with shrimp can promote the absorption of nutrients from the culture, such as ammonia, nitrate and phosphate. Thus, the use of cultivation effluents for the cultivation of macroalgae or integrated cultivations can be an alternative with lower financial and environmental costs, bearing in mind that enrichment culture media, such as von Stosch, have a high cost due to expensive chemical compounds including essential vitamins that make their use unfeasible for large-scale production [
13].
The choice of species for the composition of the systems can be a limiting factor for the success of the production. Macroalgae have rapid growth due to the efficiency on converting solar energy into biomass, due to their simple cellular structures compared to terrestrial plants [
14], generating large biomasses in a short time. Alencar et al. [
15] showed that the use of effluents from a shrimp culture provided a relative growth rate of 8.8% day
−1 of the macroalgae
U. lactuca and an absorption efficiency with an average of 90% for ammonium (NH
4+) and orthophosphate (PO
4−3). Ramos et al. [
16], analyzing integration of another macroalgae,
U. fasciata, with the cultivation of Pacific white shrimp (
L.
vannamei) with sedimentation and filtration systems by oysters, showed that the combination of systems enabled improvements in several aspects of water quality, using macroalgae in the removal of dissolved nutrients in the system.
Another factor to be considered is the commercial importance of the macroalgae cultivated in the system. Seaweed cultivation is economically viable due to the presence of high-value compounds in algae cells, which are extracted and used for the manufacture of cosmetics, pharmaceuticals, and chemical compounds. El-baz et al. [
17] showed that among species of red and green algae,
U. lactuca had a higher lipid concentration and inhibitory actions on viral and bacterial activities. In industry, macroalgae have wide applicability, with them being a good nutritional alternative and having a good acceptability, as shown by Turan and Tekogul [
18].
The use of macroalgae as bioremediators has been widely used and has been shown to be efficient. Copertino et al. [
19], carrying out a study with
U. chlatrata recirculating water from a
L. vannamei culture, showed a maximum relative growth rate of 20% day
−1 and an uptake of 90% of total ammonia nitrogen (TAN) of the system, demonstrating the feasibility of this integration. However, depending on the area and dispersion of the effluent, large macroalgae biomasses are necessary and may be unfeasible [
20]. There has been an attempt to adjust the proportion of macroalgae in the crop so that the absorption of nutrients is still effective. Alencar et al. [
15] found better growth and nutrient absorption results with a density of 3 g L
−1; this high density is convenient in small volumes of water. Del Río et al. [
21], using
U. rigida as a biofilter for fish tanks, found that the results are good at densities between 1.5 and 2.5 g L
−1. However, in shrimp farming in a biofloc system with high organic load and nutrients, little is known about the ideal density for the maintenance of the system and on the performance of macroalgae in cultivation in a biofloc system and alternatives to optimize nutrient absorption. Therefore, the objective of this work is to evaluate the influence of the seaweed
Ulva lactuca in the cultivation of the shrimp
Litopenaeus vannamei in a biofloc system.
4. Discussion
The introduction of species in integrated aquaculture is related to their ability to adapt according to system conditions and their interaction with other organisms [
35]. In offshore systems, with low water turbidity and water renewal by currents, the integration of the macroalgae into the cultures shows better results. Verdian et al. [
4] showed that in these systems the relative growth rate was 14.3 ± 4.3% day
−1, probably due to the easy adaptation of the macroalgae to the system because of the similarity with the natural environment. In land based biofloc systems, the environmental characteristics are different. The system is characterized by a high organic load and nutrient accumulation throughout the production cycle. When cultured under unfavorable environmental conditions, such as high solids and nutrient loading in closed systems, such changes may influence the macroalgae’s performance. Different management protocols should be applied for the best adaptation and performance of macroalgae in these systems.
Our laboratory experiment showed that different concentrations of solids in the biofloc system did not cause biomass loss of macroalgae (see
Table 2). However, the system did not provide the best conditions to seaweed growth compared to offshore cultivation. The relative growth rate of the macroalgae was similar between the treatments, demonstrating the feasibility of using shrimp culture water in biofloc systems as culture medium. The laboratory environmental conditions were controlled, with a direct light source on the macroalgae and temperature regulation. Another way to favor macroalgae growth under these conditions was the use of transparent experimental units that allow greater light incidence, which may have influenced the processes of light absorption by the macroalgae even with the deposition of solids.
In the laboratory experimental conditions, there was also no increase in solids throughout the experiment. Since there were no shrimp present, the feces and feed waste were not being produced in the culture, which was characterized as a static system. In controlled and fixed conditions, the macroalgae were able to adapt to the different culture media and grow. In contrast, the conditions of the greenhouse experiment simulated real culture conditions, resulting in biomass loss (see
Table 4). The integrated culture of shrimp and macroalgae in an intensive system and with a high feed intake generated an increase in solids. Even with intense aeration, the macroalgae culture structure allowed for greater deposition of organic matter on the macroalgae. This effect was smaller in the laboratory experiment, because the macroalgae were loose in the carboy. Such conditions can be stressful to the macroalgae, impacting their growth throughout the culture, which was verified in this experiment with the loss of biomass. A similar result was also observed by Legarda et al. [
10], working in a closed system with integrated culture of the macroalgae
U. fasciata, the shrimp
L. vannamei, and the fish
Mugil liza in a biofloc system, confirming the difficulty of adaptation of the macroalgae in this system.
The availability of nutrients in the biofloc system is advantageous for macroalgae cultures [
9]. However, the presence of solids can be an inhibiting factor for macroalgae growth. In both experiments, solids were deposited on the macroalgae, and lower concentrations of suspended solids were found in the water. In the laboratory experiment, a decrease in turbidity was observed in the treatment with the highest concentration of solids (400 mg L
−1) on the fourth day of culture (
Figure 2), showing that most of the solids were deposited on the macroalgae, even with constant aeration.
In the greenhouse experiment, a similar process was verified, with the aggravating factor that the production of solids was persistent due to the presence of the shrimp in the culture. The settleable solids (ml L
−1) showed a significant difference between the integrated culture and monoculture treatments. The lowest concentrations of settleable solids were observed in the
U. lactuca treatment. As the individuals of this species are sessile organisms, they probably interfered with the dynamic movement of biofloc particles in the water column, causing the deposition of particles on them, unlike in the monoculture where there was no physical barrier for the particles to decant. Brito et al. [
9] also found a decrease in solids levels due to the deposition of flocs on the photosynthetic macroalgae leaves, going from a suspended to a decanted solid. This result also induced the need to use the clarifier to control excess solids in the system. The treatments with the presence of
U. lactuca required less clarification time compared to the shrimp monoculture treatments. However, removing the macroalgae from the system will cause solids to become suspended in the water again. Excess solids in the water can cause rapid oxygen depletion [
36] and growth performance problems to the shrimp [
5]. Some alternatives to control solids presented by Khanjani et al. [
35] would be the use of organic consumers in integrated cultivation. Thus, the solids would be controlled without the use of clarifiers and there would not be a large amount of solids deposited on the macroalgae.
The deposition of solids on the macroalgae can be a stressing factor for the species, which may prevent the absorption of light and the performance of photosynthesis (see
Figure 4). Such factors can trigger reproduction events such as the release of gametes or spores. These events can be initiated due to several environmental factors, such as high temperatures, the concentration of nutrients, and even the short life cycle of the species, thus resulting in the loss of biomass [
19]. During the cultivation period, the presence of “ghost tissues” was also observed in
U. lactuca as a sign of sporulation. The loss of macroalgae biomass was also verified by Legarda et al. [
10] when cultivating
U. fasciata in an integrated system, probably because of the different characteristics of the biofloc system compared to the natural environment where the macroalgae were collected.
The von Stosch nutrient solution used in the laboratory experiment is composed of balanced minerals and nutrients [
13]. Despite the biomass gain, the relative growth rate of
U. lactuca in this experiment in the laboratory condition was lower compared to the studies with a maximum of 16.9% day
−1 for
U. prolifera, when cultivated in the laboratory conditions with the nutrient medium F/2 [
37]. The von Stosch medium used for the cultivation of
U. lactuca and the concentration of the medium used may not be adequate to cause low growth of the algal biomass. In addition, the treatment water was not renewed nor were more nutrients added, which could have limited the growth of the algae. For the production of culture media, specific compounds of high economic value are needed, so alternative culture media and better management practices can facilitate the production of
U. lactuca.
The increase in the protein content of
U. lactuca cultivated in the biofloc system probably occurred because of the nutritional composition of the macroalgae changes according to the physical and chemical factors of the culture environment. For example, higher concentrations of nitrogen available in the culture system, such as in the biofloc system, provide an increase in tissue nitrogen. According to Duke et al. [
38], greater availability of nitrogen in the medium results in its absorption and its transformation into protein, stored in the form of amino acids and pigments [
32]. Treatments with biofloc effluent contained higher concentrations of nitrate and phosphate than treatment with von Stosch culture medium. This is due to the origin of the biofloc effluent, which came from a shrimp culture with 43 days of cultivation, with a gradual accumulation of nutrients. This high availability of nitrogen in the water favored the increase in the protein content of the macroalgae. The results obtained were superior to those observed by Fong et al. [
39], who obtained protein concentrations ranging from 0.6 to 5.4% in algae grown in nutrient solutions in the laboratory. The high protein value of
U. lactuca in the present study shows its importance for human food as a food supplement, for muscle tissue reconstruction, and in vegan foods, similar to the previous study conducted by Bleakley and Hayes [
40].
Even with 20% biofloc inoculum in the greenhouse experiment, the ammonia concentrations in the first days of cultivation exceeded the concentration of 1 mg L
−1 in the tanks without
U. lactuca. Although ammonia concentrations were well controlled in the biofloc system, at times throughout the production cycle it may be necessary to add organic carbon to stimulate the development of heterotrophic bacteria in the system [
26]. When using a low percentage of inoculum diluted in seawater, the bacteria can undergo adaptation in the system. Together with the feed supply and continuous excretion of the animals, these bacteria were not able to convert all the ammonia in the system and its concentration increased, with the use of molasses in shrimp monoculture being necessary to increase the number of heterotrophic bacteria, in this experiment. Such instability of the bacteria also occurred in the integrated treatment, but due to the absorption capacity by
U. lactuca, there was no increase of ammonia concentration in the system. Castelar et al. [
41] observed that the genus Ulva tends to have a preference for ammonia, making its assimilation faster and thus controlling nitrogen in the crop as a consequence.
Nitrate was another nitrogenous compound absorbed by
U. lactuca. Its accumulation is constant throughout the production cycle in the biofloc system, with it reaching high concentrations. The
U. lactuca absorbed the nitrate, as it is the nitrogenous compound with the highest availability in the system, with the best absorption result occurring in the treatment with the lowest density of
U. lactuca (1 g L
−1). High densities, above 1 g L
−1 are likely to increase intraspecific competition, due to
U. lactuca overlap in the structure, and negatively affect nutrient absorption. Alencar et al. [
15], testing different densities with
U. lactuca, also observed that nutrient removal was impaired when the algae density and growth rate increased. Therefore, unlike the gradual accumulation of nitrate that occurred in the monoculture, the treatment with the density of 1 g L
−1 of
U. lactuca resulted in a lower concentration of nitrate at the end of the cultivation. Due to absorption by the macroalgae, this compound is used for the production of biomass and pigments.
In addition to nutrient absorption, the integration of
U. lactuca into shrimp cultivation also interferes with other components in the system. For the biofloc system, calcium carbonate or calcium hydroxide is required to maintain alkalinity [
30]. The integrated cultivation of
L. vannamei and
U. lactuca required a smaller number of corrections with Ca(OH)
2, maintaining a more stable level of alkalinity compared to the monoculture treatment where there was no
U. lactuca. This possibly occurred due to the absorption of carbon dioxide from the medium by
U. lactuca [
36]. Chopin [
11] comments on the decrease in water acidification due to the absorption of gases by macroalgae, acting on the greenhouse effect. The same pattern can occur in cropping systems integrated with
U. lactuca.
The physical and chemical parameters of water quality were kept in the ideal ranges for
L. vannamei cultivation, such as temperature [
42], salinity [
43], dissolved oxygen [
44], pH [
45], alkalinity [
30], and TSS [
5]. The shrimp cultivated in this study had a similar performance to that found in the literature for monoculture systems [
26]. Therefore, the integration of
U. lactuca in the system did not interfere in the performance of the shrimp, with it being environmentally advantageous. In the study of Brito et al. [
9], a higher average final weight of the shrimp was observed when cultivated with the macroalgae, possibly due to nutritional advantages provided by the macroalgae to the shrimp. Although the macroalgae are grown in a separate floating structure from the shrimp, they are both grown in the same tank. Therefore, the reduction in the production of biomass served by Ulva can also be explained by its consumption by shrimp. In future studies, it is recommended to observe and monitor if the shrimp consume algae when they are integrated in the same tank as well as when they are in a separated cultivation structure.
In a conventional farming system, there is a significant loss of nitrogen that is not incorporated by the animals and becomes available in the water as a residue that can be toxic and the main source of environmental pollution [
46]. The use of macroalgae for the absorption of nutrients from the system has been widely used due to greater sustainability and productivity gain [
4]. The use of macroalgae
U. lactuca at a density of 1 g L
−1 in an integrated system with shrimp
L. vannamei in biofloc was also viable due to the incorporation of nitrogen by the algae, resulting in a biomass with higher protein content, in addition to increasing the system productivity and sustainability.