4.1. Experiment I – Milk Powder/Molasses/Calcium carbonate
The significant differences (p < 0.05) in the initial DO may be related to water sources. Freshwater (T1, T3, and T5) contains a higher DO concentration than saltwater due to the number of dissolved salts (salinity). For the final DO, the values of T1 and T2 were statistically higher when compared with the other treatments. This difference may be associated to the higher consumption of DO in treatments T3, T4, T5, and T6, including the experiments with powdered milk and molasses. These products are organic carbon sources used by microorganisms to feed, grow, and multiply. Thus, the rapid development of bacteria using the available carbon sources may have affected DO availability .
Significant differences (p < 0.05) were observed for the growth of Bacillus between the beginning and the end of the experimental period. Within the conditions tested, all treatments showed the same growth trend, regardless of water salinity. The negative treatments (T3 and T4), which did not use the commercial probiotic, were as efficient in the development of Bacillus bacteria as the treatments that contemplate the application only of the probiotic (T1 and T2) and the treatments that rely on the complete application of the protocol (T5 and T6). After 24 h, the growth of Bacillus was statistically similar between treatments. This can be related to the isolated and controlled environment where the experiment was carried out or to the nutritional difference between tested ingredients. The short duration of the experiment may also have affected the appearance of possible statistical differences.
The growth of the group of coccoid bacteria obtained significant differences (p < 0.05) during the experiment, where T1 and T2 treatments showed lower values at time 00 h. This behavior may be related to the lack of available nutrients since only the commercial probiotic was included in those treatments. However, at the end of the experimental period, the growth of coccoid was equal in all treatments, without significant differences.
Filamentous bacteria grew in smaller numbers when compared to Bacillus and coccoid, but in some cases, they may have the pathogenic potential for the cultivated species. Statistical differences (p < 0.05) between the initial and final time for each treatment, showing the lower growth of this group in treatments T1 and T2, may be related to the suppression made by bacteria from commercial probiotics on other microorganisms. Thus, it is noteworthy that its growth was more significant in freshwater treatments. All treatments showed significantly higher growth in the final time when compared to the initial time. Despite this behavior, no statistical differences were observed at the end of the experiment (p < 0.05); that is, the filamentous growth was equal in all treatments, regardless of the products used.
4.2. Experiment II – Rice Bran/Crystal sugar/Sodium bicarbonate
There were no statistical differences for DO at times 00 h and 24 h. However, there were significant differences (p < 0.05) in the final time for oxygen consumption at T6, which differed from the other treatments, recording the lowest mean concentration (0.06 ± 0.01b).
Although we did not identify statistical differences between the other treatments, the lowest final DO concentration was observed in treatment T5 (0.1 ± 0.01a). The levels of DO play an essential role during bacterial processes . Moreover, the fact that the T5 and T6 treatments have the lowest levels may be related to the more significant amount of organic matter developed in the environment, as both had the addition of the complete protocol. The recorded temperature was about 30 ºC, similar to temperature conditions often found in shrimp culture farms.
The growth of the Bacillus group differed statistically from the initial time to the final time, demonstrating that there was growth in all treatments. The count was lower in saltwater treatments T2 and T4 than in the other treatments for the initial time. These differences were not noticed in the final time, where the average growth was statistically equal for all treatments.
Despite the absence of the probiotic in the negative treatment, Bacillus bacteria grew. The protocol contemplates rice bran, which can stimulate the appearance of several groups of microorganisms that make up the primary productivity in the aquatic environment  due to the amount of organic carbon released.
Bacteria of the coccoid morphotype also grew in this experiment presenting similar growth in the initial and final time between all treatments. Thus, in the control treatments T1 and T2, where the dosage of commercial probiotic was high, the rapid domination of the medium by Bacillus from the commercial product may have hindered their growth. Probiotic bacteria of the genus Bacillus spp. produce bacteriocins capable of suppressing and killing a variety of microorganisms . No vibrios were identified during the experiment.
In this experiment, with an average temperature of 30 ºC and an available carbon source, the filamentous also occupied and colonized the water. There was no significant difference in the final time, although T5 and T6 showed significant growth, and the DO reached levels close to zero. The water can be quickly colonized when quickly degraded products, such as sugar, are used, making carbon available to microorganisms.
4.3. Experiment III – Bokashi Bran/Molasses/Sodium bicarbonate
Oxygen consumption was significantly higher in treatment T5 (0.1 ± 0.02) in the final time, followed by T6 (0.22 ± 0.1). Both treatments have the highest total bacterial counts, T5 (7.89X107) and T6 (6.65x107), which may explain the higher overall consumption of DO.
Treatments T3 and T4 had the lowest initial Bacillus count, which may be explained by the fact that these treatments do not include commercial probiotics. Through the quantification of Bacillus, we can observe that probiotics in treatments T1 and T2 were similar to those in treatments where the product was not included, which is the case of negative treatments, T3 and T4. Thus, our findings show that the protocol does not increase the development of Bacillus in treatments with the commercial product.
Bokashi Bran is a compound, acquired through a mixture of bran of vegetable origin (rice bran, wheat,), bran of animal origin (bone meal, meat), energy source (sugar, molasses, etc.), and oilseeds . Commonly used as a fertilizer for growing fruit, it is now used in aquaculture, although it does not yet have a standardized commercial formula. This experiment demonstrates the ability of organic fertilization with Bokashi Bran to favor the appearance of Bacillus bacteria, although its final count is statistically equal between treatments.
Coccoid and filamentous morphotype bacteria showed statistical differences (p < 0.05) at the end of 24 h, obtaining similar growth between all treatments. Oxygen reached rates close to zero near the middle of the experimental period, with no recovery of DO rates, even with a constant mechanical supply of blown air. The abundant presence of microorganisms may have collaborated with the excessive consumption of DO and the increasing amount of organic matter in the medium, which need oxygen to grow and multiply.
4.4. General discussion
The development of microorganisms in water depends on several factors such as available nutrients, temperature, DO, pH, and growing time. Each group of bacteria has its preferences; Bacillus spp. are mesophilic, developing between temperatures of 10–48 ºC. Bacteria with a morphotype of coccoid and filamentous can develop in the most varied temperature ranges. Temperature is considered the most critical environmental factor for microbiological growth .
The bacteria present in commercial probiotics are of the genus Bacillus spp. (B. subtilis and B. licheniformis), classified as gram-positive, sporulation and mandatory or facultative aerobics may occur . During the growth and multiplication of aerobic microorganisms, the introduction of mechanically blown air is traditional as the DO is consumed so quickly that the diffusion of atmospheric oxygen cannot supply the microorganism's respiratory demand. Despite the fact that the experiments had a constant supply of air blown, the microorganisms consumed oxygen, and DO levels dropped to close to 0.00 mg/L between 12 h and 24 h. This increased DO consumption may have been due to the rapid proliferation of different bacteria, which need oxygen to grow and multiply . These results indicate that adding the fertilizer to the protocols may significantly decrease the DO of the shrimp production systems, which may affect their growth or cause their death, depending on the amount added.
The pH can also change during cell growth due to the metabolic reactions of microorganisms that can consume or produce acid or base substances. B. subtilis can produce, for example, acetic acid . The addition of buffer substances is necessary for pH maintenance, such as calcium carbonate and sodium bicarbonate, both added to the tested protocols. The tested protocols showed differences in the composition of their ingredients, thus causing the development of different amounts of bacteria in each experiment. Despite the product differences, the growth behavior of groups of bacteria was similar between experiments I, II, and III. All experiments showed significantly higher Bacillus growth in the final time when compared to the initial time. Coccoid and filamentous bacteria were also quantified, with significantly higher growth at the end of the 24 h period.
There was no identification and quantification of bacteria from the Vibrio group throughout the experiments. The dosage of the commercial probiotics used in this study (1 g/L) was at least 1000x higher than the recommended dosages for nursery phases (1 mg/L) in shrimp cultures, resulting in 50 million of Bacillus per mL. As a hypothesis, the pressure of Bacillus abundance may have excluded the appearance of Vibrios, or the values may have been below the detection limit. This absence can be explained by the total time of the experimental period, which may have been insufficient to allow the appearance of bacteria of this genus. Santos (2020) quantified the appearance of Vibrios on the fifth experimental day, in a culture of L. vannamei in the Aquamimicry System, while the present study lasted only 1 day.
Water temperature of all experiments (I, II, and III) remained within the ideal range of 28–30 ºC for the growth of L. vannamei. This study aimed to maintain temperatures at levels similar to those found on production farms, based on empirical protocols. Different salinities, with fresh and saltwater salinity close to 0 mg/L and 25 mg/L, respectively, also aimed to simulate practices seen on the farms. In some shrimp farms, using fresh water to carry out the multiplication of probiotics is usual; on the other hand, saltwater is often used directly from the culture ponds, without prior disinfection.
When comparing all the experiments, the experiment that presented the most significant total growth of Bacillus was Experiment III (treatment T3), where the base of the protocol was the Bokashi Bran, reaching a value of 1.08x108. Even with the differences between the products used, the development of Bacillus was similar in all control treatments, regardless of water salinity, temperature, and DO.
The closed and controlled environment may have influenced the growth dynamics of bacteria and protozoa.  quantified a more significant presence of Bacillus, coccoid and filamentous bacteria, flagellates, ciliates, amoebas, and rotifers in treatments exposed to light compared to treatments with light restriction. The lack of light exposure in the experiments in this study may have harmed the development of microorganisms. There were no exchanges between indoor and outdoor environments, and no interactions with farm animals. The multiparameter probe and the pipette were disinfected during the experimental period before each measurement and water sampling.
The growth of the bacteria population may also have been limited by the total multiplication time of the protocols. After 24 h, the diversity of microorganisms that developed was not expressive compared to more extended cultures. By carrying out this study for more than 24 h, statistical differences could be evidenced in the quantification of microorganisms between treatments due to the possibility of more significant interactions between microorganisms such as predation and pasture as the natural evolution of the community.
However, the duration of the experiment (24 h) is based on the standard practices of farms from which the empirical protocols were designed. The commercial probiotic multiplication protocols are carried out daily. At the end of 24 h period, the multiplication product is divided among the culture units and included directly in the water or mixed with the feed supplied to the animals.
The development of Bacillus in all treatments can demonstrate the ability of home protocols to collaborate with their growth in water within 24 h. Nonetheless, these products are not able to increase Bacillus quantification when acting along with commercial probiotics. Thus, this confirms that Bacillus bacteria develop regardless of the products added to the water, as there were no statistical differences in the final time between treatments. The structure of microorganisms was similar in all experiments, demonstrating that the development of the microbial loop was similar regardless of salinity and products added to water. The initial abundance of microorganisms, at 00 h, right after the inclusion of the products in the water, may be linked to the possibility that the disinfection technique used was not efficient in eliminating all the microorganisms present in natural water. The optimal salinity ranges used by most bacteria can be quite broad, with natural waters being an important inoculum  that helps form a diverse microbial community capable of assimilating the available nutrients.
Our findings show that the protocols observed in cultivation farms, carried out empirically, do not demonstrate scientific efficacy. The bacterial counts demonstrated that increasing the volume of probiotic bacteria present in the commercial product using various products (powdered milk, molasses, rice bran, bokashi bran, sugar) to provide a food source for the bacteria present in the commercial product was not effective. However, this emphasizes the importance of understanding the action mechanisms of commercial probiotics and investigating the effectiveness of empirical practices so that the producer does not waste financial resources on techniques that do not work.
Validating scientifically and seriously the practices used in different stages of animal production guides the decision-making process of the producer and gives assurance. Ultimately, other empirical protocols are carried out on shrimp farms, but the effectiveness of their applications will be undefined until scientifically tested.