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INTRODUCTION
There are two fundamental premises for the nutrition of water organisms: protein levels and their quality. This last is determined by the composition and availability of amino acids, as well as the level of digestive utilization made by the animal (NRC 2011). In addition, it is the main factor that affects growth, water quality and diet costs during shrimp production (Tacon et al. 2013 and Ponce-Palafox et al. 2017).
The application of biofloc technology (BFT) provides flocs composed by bacteria, microalgae, protozoa and other organisms, which together with detritus and dead organic matter can increase the availability of food for shrimp, 24 hours a day (Avnimelech et al. 2009, Kuhn et al. 2010 and Emerenciano et al. 2012). However, there are factors such as the contribution in protein of the concentrated food, biofloc size, associated microorganisms, fertilizer, type of bioreactor and others, which influence on the quality and quantity of generated flocs (Ju et al. 2008, Kuhn et al. 2010 and Ekasari et al. 2014).
Therefore, the objective of this study was to determine the bromatological and amino acid composition of the biofloc of the macrocosm tank, which was generated with water from the Ecuadorian Pacific, for growing L. vannamei in an intensive system.
MATERIALS AND METHODS
In this study, a circular fiberglass tank with a capacity of 3000 L was used for the macrocosm. It had an air diffuser in the center to guarantee oxygenation, continuous movement of water and resuspension of particles.
The Thalassisira sp. microalgae (1.4 x 106 mL / L) was inoculated to form the macrocosm, and 40 shrimp/m2 with an average weight of 12.7 ± 1.2 g were placed. A commercial 35% protein food (Alimentasa) was daily provided. To guarantee bacterial growth, a C: N ratio of 20:1 was maintained through the contribution of sugar cane molasses and nitrogen from feed, as recommended by Avnimelech (2012).
Bromatological composition of biofloc. Dry matter (DM), crude protein (CP), ether extract (EE) and ashes were determined to the biofloc contained in the macrocosm tank, according to AOAC (2016). Three macrocosm tanks were formed and a water sample (10 L) was taken from each tank for filtration (n = 3). Dried and ground samples were stored at -20 °C its analysis.
Amino acid profile. Samples were analyzed by triplicate to determine the amino acid profile by high performance liquid chromatography (HPLC) with sodium ion exchange column and post-column derivatization with ninhydrin in a visible-ultraviolet detector, according to the 994.12 regulation (AOAC 2016). For the quantification of the amino acids, samples were hydrolyzed with 6N hydrochloric acid, for 22 h at 110 oC, according to the method described by Moore and Stein (1963). For tryptophan, samples were hydrolyzed with 4N lithium hydroxide according to methodology described by Lucas and Sotelo (1980).
Chemical score and essential amino acid index (EAAI). The chemical score was determined as the relation of the value of each essential amino acid (EAA) of the experimental food (biofloc) with the corresponding amino acid value of a pattern protein as described by Peñaflorida (1989). The recommended essential amino acid requirements for shrimp (L. vannamei and P. monodon) were used as the standard protein (Xie et al. 2012, Zhou et al. 2013 and Lin et al. 2015).
The essential amino acid index (EAAI) evaluates the degree of EAA of the entire sample in relation to the composition or requirements of the EAA of the animal (Ju et al. 2008). The formula proposed by Peñaflorida (1989) was used to calculate the EAAI:
RESULTS AND DISCUSSION
Bromatological composition of biofloc. The biofloc developed in the macrocosm showed high protein levels (table 1). These values were comparable to those found by Wasielesky et al. (2006) and Li et al. (2018) although lower than 38.41% of CP, reported by Luo et al. (2014) and 43% of CP found by Maicá et al. (2011). Meanwhile, lipid contribution was low when compared with 2.6% found by Tacon et al. (2002) and 7.5% reported by Ekasari et al. (2014).
Table 1 Proximal composition of the biofloc of the macrocosm tank with white shrimp culture (g / 100g DM, n = 3)
| Indicator | Mean | DS | VC (%) |
|---|---|---|---|
| Dry matter | 97.46 | 0.74 | 0.76 |
| Crude protein | 36.53 | 1.75 | 4.79 |
| Ether extract | 0.97 | 1.14 | 14.18 |
| Ashes | 33.39 | 2.60 | 7.79 |
According to Azim and Little (2008) and Hargreaves (2006), dry weight of protein content can vary from 25 to 50%, although the most common is between 30 and 45%. Fat content varies from 0.5 to 10% and the most common range is between 1.0 to 5%. These authors also agreed that biofloc is a good source of vitamins, minerals and has a probiotic effect.
Reports on CP levels (about 33%) in microbial flocs by Emerenciano et al. (2011) and Suita et al. (2015) were lower than those found in this study, which could be associated with the composition of the microorganism communities present in the biofloc, that are more productive in the waters of the Pacific Ocean (Maridueña 2004). On the other hand, the lipid contents were slightly higher compared to those reported by Wasielesky et al. (2006) and Emerenciano et al. (2011), with 0.47 and 0.49%, respectively, and similar to those of Suita et al. (2015), of 1.6%. These authors attribute these lipid levels to the presence of ciliates and microalgae, specifically diatoms that are a source of essential fatty acids and lipids.
In addition, Schneider et al. (2005) pointed out that the intake of bioflocs can increase food utilization efficiency by recovering a fraction of the excreted nutrient and retaining nitrogen from aggregate foods between 7 and 13%. In this sense, Xu et al. (2012) confirmed the contribution of bioflocs in the protein nutrition of L. vannamei in culture tanks with BFT. These authors concluded that the formation and development of bioflocs can have an important function as in situ supplementary food source and in the improvement of food utilization, protein digestion and retention by the shrimp, contributing even more to improve growth.
The ash content of this study (table 1) reached high values (33.39%) and are similar to other studies, where ash content varied from 7 to 32% of DM (Ju et al. 2008, Ekasari et al. 2010 and Xu et al. 2012). In this regard, Tacon et al. (2002) stated that the high ash content in bioflocs is probably related to the presence of acid soluble oxides and mixed silicates. In addition, these authors stated that they are a good source of essential minerals and trace elements.
Maia et al. (2016) reported that, in commercial ponds with TBF, the use of molasses influenced on water quality and on the natural food source (biofloc), with good nutritional composition for shrimp. At the same time, it should be mentioned that sugar cane molasses used for fertilizing favored the increase of ashes since it is rich in this indicator (Valdivié et al. 2012).
Li et al. (2018) reported that the choice of carbon source is of paramount importance when using biofloc as a food source to reach optimal nutritional indicators in shrimp farming. In this sense, Kuhn et al. (2010) stated that molasses is the most used carbon source to produce biofloc. This is because it is a source of simple carbohydrates, easily assimilated by microorganisms that develop in the biofloc and can promote the growth of culture species (Martínez-Córdova et al. 2016). In addition, it is a source of easy acquisition and low costs (Moss et al. 2010).
Amino acid profile. The amino acid composition of biofloc that occurred in the macrocosms is presented in table 2. It can be seen that the 12 limiting amino acids were present and at levels according to the literature (Ekasari et al. 2010 and Kuhn et al. 2016). Similarly, the concentration of amino acids found corresponds to the shrimp requirements (Xie et al. 2012 and Lin et al. 2015). This suggests that this natural food circulating in the culture medium may be a good supplementary source of EAA for white shrimp (Sabry-Neto et al. 2015).
Table 2 Amino acid composition of biofloc in the macrocosm tank for growing white shrimp (%, n=3) with waters from the Ecuadorian Pacific
| Amino acids | Mean | SD |
|---|---|---|
| Essential amino acids | ||
| Lysine | 1.31 | 0.10 |
| Methionine | 0.69 | 0.15 |
| Arginine | 1.89 | 0.21 |
| Leucine | 2.17 | 0.57 |
| Valine | 1.68 | 0.39 |
| Threonine | 1.6 | 0.02 |
| Histidine | 0.86 | 0.22 |
| Isoleucine | 1.22 | 0.38 |
| Phenylalanine | 1.45 | 0.18 |
| Tryptophan | 0.28 | 0.12 |
| Non essential amino acids | ||
| Glutamic acid | 3.08 | 0.18 |
| Aspartic acid | 2.61 | 0.52 |
| Alanine | 2.30 | 0.37 |
| Serine | 1.45 | 0.29 |
| Proline | 1.58 | 0.10 |
| Glycine | 2.01 | 0.24 |
| Tyrosine | 1.20 | 0.07 |
| Cystine | 0.52 | 0.17 |
Out of the EAA, the highest levels were found for leucine, arginine, valine, threonine and phenylalanine, in contrast to tryptophan, which was the one with the lowest level. These results generally coincide with other studies that analyzed the EAA present in biofloc (Ju et al. 2008).
It is important to state that one of the main reasons that reduce the growth of water organisms in studies where fish meal is partially or totally replaced by alternative protein sources is attributed to an inadequate balance of EAA that lead to a decreased of protein synthesis, essential for animal growth (Toledo et al. 2015). Therefore, the biofloc that is produced in the culture medium should not affect the productive performance of shrimp when the protein levels in the balanced foods are reduced, given the contribution of essential amino acids that it presents.
Ju et al. (2008) reported that the flocs have a considerable amount of non-amino acid nitrogen as ammonium salts, but also possibly urea, nucleotides and amino sugar, which may arise from the metabolic activity of microorganisms and shrimp. These same authors compared the EAA profile of three biofloc samples, two dominated by microalgae (chlorophytes and diatoms) and the other by bacteria versus the EAA profile of two balanced shrimp feeds, and reported that they were similar. This indicates that protein quality of the organisms present in the biofloc was high.
Chemical score and essential amino acid index (EAAI). Table 3 shows the chemical score and the EAAI of amino acids present in the biofloc. The chemical score of tryptophan was the lowest (0.56), resulting in the first limiting amino acid, which means that biofloc can only meet approximately half of the shrimp requirement for this amino acid. Similarly, the isoleucine (0.70) and lysine (0.80) scores were found to be second and third limiting amino acids in the macrocosm biofloc sample.
Table 3 Chemical score and essential amino acid index (EAAI) of the biofloc generated in the macrocosm tank with water from the Ecuadorian Pacific.
| EAA | Biofloc | Shrimp requirements | Chemical score 1 |
|---|---|---|---|
| Lysine | 1.31 | 1.64 | 0.80 |
| Methionine | 0.69 | 0.66 | 1.00 |
| Arginine | 1.89 | 1.96 | 0.96 |
| Leucine | 2.17 | 2.37 | 0.91 |
| Valine | 1.68 | 1.40 | 1.00 |
| Threonine | 1.6 | 1.18 | 1.00 |
| Histidine | 0.86 | 0.80 | 1.00 |
| Isoleucine | 1.22 | 1.73 | 0.70 |
| Phenylalanine | 1.45 | 1.40 | 1.00 |
| Tryptophan | 0.28 | 0.50 | 0.56 |
| EAAI | - | - | 0.89 |
1EAA of the sample / EAA of shrimp requirements. An amount of 1.00 was taken as maximum
Some factors influence on the quality of protein sources, such as amino acid profile (EAA score) and digestibility (Toledo et al. 2015). The chemical score of EAA evaluates the level of each EAA individually and the value 1.00 indicates that the level of the AEA, particularly within the dietary protein, is identical to the requirement of that essential amino acid of the animal. The lowest value, indicates the first limiting amino acid. On the other hand, the essential amino acid index (EAAI) evaluates the degree of EAA of the entire sample in relation to the composition or requirements of EAA of the animal (Montoya-Martínez et al. 2016).
The EAAI found in this study was 0.89, which classifies as useful protein material according to Castell and Tiews (1980). This EAAI was equal to the biofloc preferably of bacteria reported by Ju et al. (2008) and higher than that shown by soy meal (0.87) in Penaeus monodon, according to Peñaflorida (1989). However, they were inferior to bioflocs dominated by microalgae (0.91 and 0.92) (Ju et al. 2008). These values suggest that the experimental biofloc can provide a high quality protein source for shrimp growth and may supplement a balanced food.
Ju et al. (2008) stated that an EAAI close or equal to 1.0 indicates that the diet contains an amino acid profile similar to that found throughout the body of the shrimp and it is assumed to be equivalent to its diet requirements. They also refer that, in general, ingredients or feedstuffs with an EAAI of more than 0.90 are considered to be foods of good protein quality or protein materials, with an EAAI of 0.80 are useful, and below 0.70 are inadequate, according to the classification described by Oser (1959).
However, the ability of a food to meet the amino acid needs of an animal depends on the intake rate, bioavailability (digestibility) and its composition (Toledo et al. 2015). In this regard, Forster and Dominy (2005) report that foods with a low EAA score will require their addition, either by other sources of proteins or synthetic amino acid supplements, to obtain an animal yield comparable to that of a diet of high quality. Biofloc, however, arises naturally from the by-products of shrimp farming (metabolic residues, feces and nutrients lixiviated from feed, etc.), providing a renewable source of nutrients for shrimps at no additional cost to fish farmers.
CONCLUSIONS
Biofloc generated with water from the Ecuadorian Pacific presented an adequate protein quality, in terms of its amino acid composition, chemical score and index of essential amino acids. Hence, it can be used as a supplement to the balanced feed for shrimp culture with biofloc technology.
