Evaluation of Biofloc-Based Probiotic Isolates on Growth Performance and Physiological Responses in Litopenaeus vannamei

: A comparison of the growth performance of Penaeus vannamei was ascertained by supple-menting the potential probiotics isolated from a bioﬂoc system incorporated through feed. Post-larvae shrimp (0.045 ± 0.005 g) were stocked at a density of 500/m 3 in FRP tanks (500 L) in triplicates for a period of 60 days. A total of 40 bacterial strains were isolated from previous bioﬂoc culture trials and tested for their antimicrobial activity against the pathogen Vibrio parahaemolyticus. Among these, Bacillus megaterium , Exiguobacterium profundum , Pseudomonas balearica , and Pseudomonas stutzeri showed higher antimicrobial activity. The treatment groups included clear water with no probiotics (CW), clear water + isolated probiotic (CW + IP), bioﬂoc alone (BFT), and bioﬂoc + isolated probiotic (BFT + IP), in triplicates. Distillery spent wash was used as a carbon source for bioﬂoc development and maintenance. A probiotic concentration of 1 × 10 9 cfu/g was supplemented throughout the trial. The recorded water quality parameters (pH, alkalinity, calcium, and magnesium) were observed to be signiﬁcant among the experimental groups ( p ≤ 0.05). The highest weight gain (2.43 g), SGR, PER, and lower FCR values were recorded in BFT + IP. The lowest values of total Vibrio were found in BFT. The histology analysis revealed that there was a mild increase in the B and R cell vacuoles in the hepatopancreas of CW and BFT + IP, whereas mild degeneration was found in the intestine of CW and CW + IP. Microbiome analysis of the shrimp gut revealed that Proteobacteria was the most abundant phylum in all experimental groups. P. balearica , K. pneumoniae , P. stutzeri , and E. profundum were present in the gut of C, whereas P. balearica , K. pneumonia , and P. stutzeri were present in the gut of CW + IP and BFT + IP. The results proved that the probiotics isolated from bioﬂoc colonized in shrimp gut could play a promising role in aquaculture.


Introduction
The aquaculture industry, a fast-growing sector, meets the demand of the growing population to balance the decline in capture fisheries. Globally, India is the second largest country in aquaculture production [1], and shrimp farming has gained positive attention due to its export potential compared to other aquatic species. In recent times, Litopenaeus vannamei has attracted farmers with its characteristics of fast growth, resistance to native diseases, availability, and tolerance to a wide salinity range [2]. Despite the prospects, this industry faces hindrances related to the availability of natural resources, feed raw materials, and disease outbreaks.
Deterioration of water quality paves the way for disease outbreak, which affects production and productivity. In addition to other interventions to overcome the issue, biofloc technology, which involves zero or limited water exchange technology, is gaining momentum. Biofloc is an aggregate of biotic and abiotic factors, which in turn acts as a feed to the culture animal [3]. Biofloc, with various advantages, has also been reported with the presence of bacteria that can induce a probiotic effect to the culture animal internally or externally against Vibrio sp. and ectoparasites [4]. Kuhn et al. [5] explained that the healthiest shrimp and their performance in the system were associated with various algae and bacteria. The usage of antibiotics, prebiotics, and probiotics in aquaculture, particularly in shrimp farming, has widely increased for proper growth and maintenance [6]. However, antibiotic use in aquaculture has now been restricted as the pathogenic bacteria have become resistant toward the numerous antibiotics available in the market [7]. The use of antibiotics has been replaced by the use of prebiotics and probiotics due to their inefficiency in treating bacterial diseases [8]. The function of probiotics in aquaculture for disease prevention and nutritional enhancement has been reported by several investigators [9][10][11]. Probiotics act on the principle of rapid multiplication, thereby reducing the levels of pathogenic bacteria. Probiotics supplemented through feed act by rapidly multiplying in the intestine, thereby reducing the infections caused by the pathogens. The supplementation of probiotics in the feed will also help in improving the organ development and intestinal equilibrium of the culture animal [12]. Numerous authors have documented the positive effects of probiotics on growth and survival in various studies. These effects are attributed to their ability to enhance nutrient digestibility and absorption, combat Vibrio pathogens, and stimulate immune components [13][14][15][16][17][18]. In the realm of shrimp aquaculture, several commercial probiotic products are readily available, with a focus on multi-species bacterial preparations, notably lactic acid bacteria (LAB). According to Ninawe and Selvin [19], probiotics are restricted because of their shelf life and sustainability across the entire culture cycle. In contrast, Hauville et al. [20] claimed a higher efficiency of multi-strain probiotics than single-strain probiotics in aquaculture.
Several studies have been reported using Bacillus as a mode of probiotic biofloc to improve shrimp growth and immune response [18][19][20]. As biofloc has proven its various advantages, the isolation of bacteria from biofloc culture water has also been a major challenge. Very few research projects have been carried out on isolating probiotic bacteria from biofloc culture systems [21]. Studies on isolating probiotic bacteria from biofloc systems and microbial characterization of the intestine are less common. Therefore, the proposed study is aimed at proving the advantage of using isolated probiotic strains from a biofloc culture system and its application in L. vannamei culture.

Biofloc Production and Isolation of Probiotic Bacteria
According to Nyan Taw [22], one month was required to maintain the biofloc in 50 ton raceways (DSW) obtained from MS Biosolutions. Coimbatore was used as source of carbon to maintain the C:N ratio at 10:1 [23]. The bacterial isolates were derived from biofloc culture systems and screened for probiotic potential by checking antimicrobial activity against the common pathogen Vibrio parahaemolyticus (ATCC ® 17802™). Isolates from shrimp were procured from the State Referral Lab for Aquatic Animal Health, TNJFU, Chennai. Morphological and biochemical characterization confirmed the bacterial species as a probiotic according to Bergey's Manual of Determinative Bacteriology [24]. The zone of inhibition criterion was utilized for selecting the bacterial strains for further testing.

Molecular Confirmation of Identified Probiotic Bacterial Isolates
The genomic DNA isolation from probiotic bacteria was carried out using the phenolchloroform method. PCR amplification was carried out using the universal forward primer 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and reverse primer 1492R (5 -CGGTTACCTTGT TACGACTT-3 ) [24]. The PCR was performed using the standard protocol. The products of PCR were sequenced and confirmed through BLAST analysis. Once the sequence was confirmed, it was submitted to the NCBI GenBank, and the related numbers were obtained.

Medium Optimization
A Lark Innovative, Chennai brand in situ fermenter was used, to which we added various sources of carbon at the rate of 1%. It was inoculated with probiotic strains in nutrient broth supplemented with DSW under aerobic conditions. The most suitable carbon sources were identified to detect the cell growth and dry weight of probiotic strains. The probiotic strains were further inoculated in 0.5, 1, 2, 4, and 8% DSW to identify the optimal carbon percentage. A bioreactor of 5 L volume was used to incubate the bacteria consortia. The bioreactor in this experiment had a microporous tube at the bottom receiving vigorous aeration.
To sterilize the water, sodium hypochlorite solution was employed after filtering it through microns. For 36 h, these probiotics were incubated at 30 • C. The viability of different bacteria species was attained at 1 × 10 9 CFU/mL under these ideal conditions.
Water (33-35 • C) was sterilized with sodium hypochlorite, filtered through a 5-micron filter, and then neutralized with sodium thiosulfate after 12 h. For 36 h, the probiotics were incubated at 30 • C. The viable heterotrophic bacteria reached 1 × 10 9 CFU/mL under these circumstances.

Experimental Design
Post-larvae Litopenaeus vannamei (0.045 ± 0.005 g) were stocked at 500/m 3 density in triplicate in 500 L FRP tanks for a period of 60 days. The treatment groups included clear water with no probiotics (CW), clear water + isolated probiotic (CW + IP), biofloc alone (BFT), and biofloc + isolated probiotic (BFT + IP), in triplicates. In the biofloc tanks, the carbon-to-nitrogen ratio was maintained at 10:1 [25], using DSW as a carbon source.
The isolated potential probiotics grown in 0.5% DSW as a carbon source were supplemented in the feed. The isolated probiotics were supplemented at the concentration of 1 × 10 9 cfu/g once a week throughout the trial period following the method of Kesselring et al. (2019) [25]. The experimental feeds were incorporated with 35% crude protein content. Feed was prepared, once every five days, to ensure the maximum survival of probiotics in the experimental diets.

Water Quality Parameters
Physicochemical parameters such as temperature, pH, dissolved oxygen, alkalinity, ammonia (NH 4 -N), nitrite (N-NO 2 ), calcium, and magnesium ion concentrations were measured on a weekly basis, according to APHA [26].

Growth Parameters
Different parameters were calculated including weight gain, feed conversion ratio (FCR), specific growth rate (SGR) per day, protein efficiency ratio (PER), and survival percentage using the below formulae.
where W 0 and W t are the initial weight and final weight (in grams) of shrimp, and t is the culture time in days. The total Vibrio count in the water samples of experimental tanks was conducted on a weekly basis by plating on thiosulfate-citrate-bile salts-sucrose (TCBS) agar, Hi Media Laboratories, India, at room temperature for 24 h, and this was expressed as CFU.

Histological Analysis
The histological analysis was performed in the Department of Pathology, Madras Veterinary College, Chennai for the hepatopancreas and intestine of P. vannamei from various treatments [27].

Shrimp Gut Microbiome Analysis
In our research experiment, we analyzed the gut of each experimental animal from each tank and then analyzed the microbiome. Prior to sample collection, the feeding of the experimental animals was stopped before 24 h. To isolate the DNA samples, we used the EXpure Microbial DNA kit. Then, using this DNA, PCR amplification was carried out. A Qubit Fluorometer 3.0 instrument was used to determine the quality and quantity of PCR products. On the basis of the nanopore sequencing and 16s sequences using the NCBI DATABASE, we classified the sequences in terms of percent coverage and identity [28].

Statistical Analysis of Experiment
Results are presented as the mean and standard deviation (SD), and data were analyzed using one-way ANOVA, followed by a Duncan test, with the help of SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). We compared each dataset to the relevant control. The differences were deemed significant at p = 0.05.

Medium Optimization
The growth performance of probiotic strains under various carbon sources is given in Figure 1. DSW was found to be an ideal carbon source when compared with the other commercially available carbon sources. All probiotic isolates showed good performance in terms of animal growth and dry weight of the cell when inoculated with 0.5% distillery spent wash. The OD value at 600 nm was observed, and the wet weight and dry weight of the cell are shown in Figure 2. in Figure 1. DSW was found to be an ideal carbon source when compared with the commercially available carbon sources. All probiotic isolates showed good perform in terms of animal growth and dry weight of the cell when inoculated with 0.5% dist spent wash. The OD value at 600 nm was observed, and the wet weight and dry weig the cell are shown in Figure 2.

Parameters of Water
Various water quality parameters were recorded during the culture trial. The pa eters included pH and alkalinity, and a significant difference was observed in calcium commercially available carbon sources. All probiotic isolates showed good performanc in terms of animal growth and dry weight of the cell when inoculated with 0.5% distiller spent wash. The OD value at 600 nm was observed, and the wet weight and dry weight o the cell are shown in Figure 2.

Parameters of Water
Various water quality parameters were recorded during the culture trial. The param eters included pH and alkalinity, and a significant difference was observed in calcium and

Parameters of Water
Various water quality parameters were recorded during the culture trial. The parameters included pH and alkalinity, and a significant difference was observed in calcium and magnesium among the treatments. There was no significant difference in water temperature among the various treatments, as presented in Table 1.

Growth Parameters
Various growth indices were observed during the culture period. A difference was observed in the experimental animals with respect to final weight, weight gain, PER, and SGR among treatments such as CW, BFT, and BFT + IP, and a significant difference in survival between CW and CW + IP was also recorded, as presented in Table 2.

Total Vibrio Count
To evaluate the various bacterial concentrations, we used the logarithm values of our experimental data. The total count of Vibrio sp. observed in the water sample of various treatments is listed in Figure 3. The population of Vibrio sp. gradually increased with the days of the culture period up to the 49th day and decreased afterward.

Figures 4 and 5 show various histological changes in the shrimp intestine and hepatopancreas system reared under various treatments (10× magnification).
Water 2023, 15, x FOR PEER REVIEW 7

Microbiome Content of Shrimp Gut
After the microbiome study of prawn guts raised under various treatments, CW had a classification of 59%, CW + IP had a classification of 64%, BFT had a classification of 77%, BFT + IP had a classification of 82%, and BFT alone had a classification of 18%. In our research and experiments, we identified Proteobacteria as the major phylum. The cumulative reads for Proteobacteria were as follows: CW-13250, CW + IP-801, BFT-5044, and BFT + IP-4961. The relative abundance of the microbiome is depicted in Figure 6.
Pseudomonas stutzeri, Pseudomonas balearica, and Exiguobacterium profundum were detected at the species level, indicating that the isolated bacterial strains present in the CW also had the capacity to colonize prawn guts. Animals raised in CW + IP and BFT + IP were found to have supplemental Pseudomonas stutzeri, Klebsiella pneumonia, and Pseudomonas balearica in their guts. See the Supplementary Materials for the full results.

Microbiome Content of Shrimp Gut
After the microbiome study of prawn guts raised under various treatments, CW had a classification of 59%, CW + IP had a classification of 64%, BFT had a classification of 77%, BFT + IP had a classification of 82%, and BFT alone had a classification of 18%. In our research and experiments, we identified Proteobacteria as the major phylum. The cumulative reads for Proteobacteria were as follows: CW-13250, CW + IP-801, BFT-5044, and BFT + IP-4961. The relative abundance of the microbiome is depicted in Figure 6.

Discussion
In this study, we evaluated the probiotic bacteria isolated from the biofloc system and their colonization ability in the shrimp gut. Among the 40 bacterial strains, Bacillus megaterium, Pseudomonas balearica, Exiguobacterium profundum, and Pseudomonas stutzeri developed a major zone of inhibition against the tested pathogen Vibrio parahaemolyticus. The presence of these isolated strains in the biofloc culture systems at the genus level was previously reported [29,30]. Martínez Cruz et al. [31] described that microbes act as pro- Pseudomonas stutzeri, Pseudomonas balearica, and Exiguobacterium profundum were detected at the species level, indicating that the isolated bacterial strains present in the CW also had the capacity to colonize prawn guts. Animals raised in CW + IP and BFT + IP were found to have supplemental Pseudomonas stutzeri, Klebsiella pneumonia, and Pseudomonas balearica in their guts. See the Supplementary Materials for the full results.

Discussion
In this study, we evaluated the probiotic bacteria isolated from the biofloc system and their colonization ability in the shrimp gut. Among the 40 bacterial strains, Bacillus megaterium, Pseudomonas balearica, Exiguobacterium profundum, and Pseudomonas stutzeri developed a major zone of inhibition against the tested pathogen Vibrio parahaemolyticus. The presence of these isolated strains in the biofloc culture systems at the genus level was previously reported [29,30]. Martínez Cruz et al. [31] described that microbes act as probiotics such as Pseudomonas and Bacillus sp. in an aquatic environment, helping to maintain the water quality and improve animal growth in the aquaculture environment. In biofloc systems of L. vannamei, Panigrahi et al. [32] identified Exiguobacterium sp. in the biofloc system. Kumar and Suresh [33] determined the extracellular protease activity of these potential probiotics in the biofloc system.
Further, these isolates were inoculated in medium supplemented with different carbon sources such as glycerol, as well as carbohydrates such as glucose, maltose, and sucrose substances. DSW was found to have a better growth performance.
Although the study used simple soluble carbon sources, there may have been major differences in the carbon utilization by the bacteria, and the benefits derived would have varied accordingly. Further dose optimization of the DSW revealed that an increase in the percentage supplementation of DSW showed no improvement in bacterial growth. Panigrahiet al. [34] reported that higher levels of carbon addition have no effect on stimulating the heterotrophic bacterial population. Yuniasari and Ekasari [35] and Krummenauer et al. [36] reported the importance of maintaining optimal levels of basic variables such as pH, DO, temperature, and salinity to maintain the quality of water in all experimental tanks. Adding a carbon source to the system can help to minimize the exchange of water in BFT and BFT +IP. Ebeling et al. [37] stated that a biofloc system including bacteria with a high metabolic rate can reduce pH and alkalinity in BFT + IP and BFT. This study mentioned these factors are reduced because of the inorganic carbon consumption by the various heterotrophic and probiotic nitrifying bacteria. Probiotic addition in the CW + IP treatment had no effect on the pH and alkalinity levels. Ammonia was very low in all treatments. Ebeling et al. [37] described ammonia assimilation in the CW + IP, BFT, and BFT + IP treatments by different bacteria present in the biofloc system, which helped to reduce the ammonia and nitrite concentrations in the water environment. Boyd [38] and Avnimelech [3] described the calcium (Ca) levels in various tanks, identifying that Ca levels were higher in CW because of CaCO 3 precipitation and its concentration in the water culture; sustaining the calcium ions in the water system may be a result of the micro-aggregates of the biofloc acids.
The levels of magnesium were lower in the case of BFT + IP than other treatments, which might be due to the higher levels of uptake by the culture animals compared to other treatments for their growth and survival. However, optimum concentrations of magnesium were maintained throughout the culture trial (10-30 mg/L) [38]. The highest weight gain with survival was found in the BFT + IP treatment, in which the isolated potential probiotic was supplemented in the feed along with the biofloc. Improved growth performance (weight gain, SGR, FCR, and PER) was found in both BFT and BFT + IP, compared to other treatments. This may have been due to the effect of rearing the animals in biofloc systems, as well as the supplementation of probiotics, which would have improved the equilibrium of intestinal activity.
The total Vibrio count was found at reduced levels in CW + IP, BFT, and BFT + IP. The results of the present study are in agreement with the findings of Aguilera-Rivera et al. [39] and Sundaram et al. [40], who recorded a reduction in the population of Vibrio in the rearing system. Zhao et al. [41] also reported that inoculating Bacillus into BFT water resulted in a decrease in the population of total Vibrio. This may have been due to the fact that biofloc displayed bio-control activity against Vibrio, the virulence of which can be regulated by the process of quorum sensing. The study reiterated the fact that biofloc not only maintained the water quality but also helped in reducing the spread of harmful pathogens. Further, supplementation of potential probiotics through the feed helped in the plausible stimulation of heterotrophic bacteria, thereby reducing the levels of pathogens.
As a result of the histological examination, various types of cells were identified in the tissue of the hepatopancreatic tubules. The hepatopancreas sections showed significant changes between the control and the treatment groups. A normal hepatopancreas was observed in the control group. However, the hepatopancreas of the culture animals reared under various treatments showed degeneration and increased B and R cells, especially in the case of BFT + IP. In the experimental animals, the proximal tubule portion showed a greater quantity of ingested material by the animals, indicating a larger proportion of cells with intracellular digestion (B) and absorption (R). However, no such abnormalities were found in the shrimp's intestine in all experimental groups, except CW, where a positive thickening of the epithelial mucosal layer was found. Won et al. [27] also revealed thickening of the muscle layer of shrimp fed with isolated potential probiotics in shrimp culture [25].
According to Pilotto et al. [42], some phyla such as Proteobacteria, Bacteroidetes, Acidobacteria, and Firmicute were identified as important in the intestine of L. vannamei. This was also confirmed in our study. Pilotto et al. [42] stated that the microbiome digestive tract was influenced by the environment in which the animals were reared. Proteobacteria was the abundant taxonomic group at the phylum level, but differed in frequency distribution among the experimental groups, similar to the findings of Sha et al. [43]. This phylum is predominantly found in the marine environment, and it contributes to the nutrient and mineral processing of organic compounds [43]. The relative abundance of Proteobacteria (13,250, 82%) in CW may have been due to the nature of its abundance in biofloc, biofilms, and recirculation systems. Its ability to efficiently degrade complex organic compounds in biofloc systems plays a significant role in maintaining the system's ecological balance and nutrient cycling, which, in turn, sustains its growth and abundance within the microbial community. The lower abundance of this phylum in CW was due to regular water exchange. Firmicutes was present in CW at 12% (1955), CW + IP at 13% (133), BFT at 14.7% (923), and BFT + IP at 11% (643). Acidobacteria was present in CW at 2% (352), CW + IP at 4% (43), BFT at 3% (212), and BFT + IP at 1.98% (115). Bacteriodetes was present in CW at 1.7% (276), CW + IP at 1.4% (15), BFT at 0.85% (54), and BFT + IP at 0.6% (35). Firmicutes, Acidobacteria, and Bacteriodetes were the most abundant phyla with respect to relative abundance, along with Proteobacteria. Vargas-Albores et al. [44] found that biofloc bacterial communities were mainly Planctomycetes, Proteobacteria, Bacteroidetes, and Firmicutes, whose various pathways are essential for nitrogenous and carbonaceous compound processing, similar to our experimental results. Wei et al. [45] reported that Acidobacteria associated with the biofloc system is involved in the ammonia assimilation process. Wobken [46] reviewed that Bacteriodetes is frequently found in colonizing macroscopic organic matter particles. These bacteria use organic and nitrogen compounds for their growth. These two parameters were satisfied in the BFT system, enabling the biofloc-associated bacteria to utilize the organic matter and attach to surfaces. The isolated probiotic bacteria were found in the CW gut, while we also observed species of Pseudomonas in BFT and BFT + IP, such as P. balearica and P. stutzeri, in the diets of shrimp.
Some probiotic bacteria such as Bacillus megaterium and Exiguobacterium profudumin were absent in the intestine walls due to a lower or lack of adherence capacity. The main feed was supplemented with isolated potential probiotics, which may have been due to the competitive inhibition among the various probiotics supplemented in the feed.
Vargas-Albores et al. [44] stated that P. stutzeri acts as a commercial probiotic, revealing its role in the CW, BFT, and BFT + IP guts. The biofloc system is more advantageous, as it is made of rich organic matter and stimulates the population of microbes, thereby acting as a source of nutrients to maintain the culture animals. According to Aguilera et al. [39], this system is activated through colonization by bacteria, phytoplankton, and zooplankton. Tzuc et al. [47] described that the animal intestine populated by microbes gains additional potential benefits toward growth and metabolism. In this research, we aimed to isolate the probiotic bacteria in the biofloc system, which support the development and rearing efficiency of animals in this system. This study proved that we could isolate potential probiotics from the biofloc system and use them to promote the growth and immune response of shrimp.

Conclusions
This study aimed to characterized the probiotics derived from biofloc and determine their efficacy. Distillery spent wash proved to be highly beneficial as it promoted the development of a diverse bacterial community within the biofloc environment. In addition to improved growth performance, the biofloc and isolated probiotic treatments also exhibited reduced levels of ammonia and a decreased abundance of Vibrio. Further, the biofloc treatments demonstrated an increase in B and R cells in the shrimp's hepatopancreas, indicating potential improvements in the shrimp's immune response and nutrient utilization efficiency. The high abundance of Proteobacteria observed in the biofloc system is particularly noteworthy, as these bacteria are known for their ability to consume organic compounds, which contributes to the overall nutrient cycling and bioremediation capacity of the biofloc system. Overall, this study contributes to advancing sustainable aquaculture practices, and the application of biofloc systems and probiotics may hold promising prospects for the future of shrimp farming.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/w15163010/s1, Table S1: Morphological and biochemical characteristics of four different Bacterial sp. Isolated from the biofloc system; Table S2: Determination of antagonism of selected Bacterial sp., against shrimp pathogens (Vibrio parahaemolyticus).