Evaluation of Salipiger thiooxidans and Exiguobacterium aestuarii from the Saemangeum Reservoir as Potential Probiotics for Pacific White Shrimp (Litopenaeus vannamei)

In this study, two bacterial species, Salipiger thiooxidans and Exiguobacterium aestuarii, were extracted and screened from the Saemangeum Reservoir. This study examined these species’ suitability as a probiotic by confirming the effects of S. thiooxidans and E. aestuarii added to rearing water for L. vannamei. Three experimental groups were evaluated for 6 weeks: (1) a control group reared in natural (i.e., untreated) water (CON), (2) an experimental group in which S. thiooxidans was added to natural water (SMG-A), and (3) natural water inoculated with E. aestuarii (SMG-B). The SMG-B group inoculated with E. aestuarii showed significantly higher final body weight, weight gain, specific growth rates, and feed efficiency than the control group. The SMG-B group inoculated with E. aestuarii exhibited significantly higher levels of serum lysozyme, and ACP and ALP activity than the control and SMG-A groups. The SMG-A and SMG-B groups inoculated with probiotics showed significantly lower total ammonia nitrogen and nitrite than the control group. Our findings suggest that S. thiooxidans and E. aestuarii extracted from the Saemangeum Reservoir can improve the water quality of aquaculture water, and, in particular, E. aestuarii is a potential probiotic for L. vannamei.


Introduction
Aquaculture is one of the most promising sources of animal protein [1]. However, in recent years, due to indiscriminate intensive aquaculture, water quality deterioration and diseases (viruses and bacteria) have become a serious threat to the aquaculture industry [2]. Although antibiotics and other therapeutic compounds have traditionally been used to treat diseases, these methods are no longer recommended, as they have been linked to negative effects such as antibiotic resistance, antibiotic residues, and environmental pollution [3]. These limitations thus highlight the urgent need to develop alternatives to antibiotics.
An alternative to antibiotic treatment is to apply probiotics [4]. Probiotics are defined as "living microorganisms that, when ingested in adequate amounts, confer a health benefit on the host" [5]. In the past few decades, probiotics have been actively studied for a variety of purposes, including pharmaceutical products, functional foods, wastewater treatment, livestock, and aquaculture [6][7][8][9][10]. Probiotics are being actively studied as an alternative to antibiotics due to their advantages of not generating residues or drug resistance in the body of animals [11]. Probiotics are promising and environmentally friendly alternatives to antibiotics in preventing aquaculture diseases [12]. In the field of aquaculture, probiotics are

Ethics Statement
This study followed the guidelines of the Institutional Animal Care and Use Committee regulations issued by the Kunsan National University, Gunsan, Republic of Korea. Every effort was taken to minimize shrimp suffering.

Extraction of Microorganisms from Saemangeum Reservoir
Bacteria samples were collected from the Saemangeum Reservoir (35 • 52 07.3 N, 126 • 30 29.8 E) located in Jeonbuk Province, Republic of Korea. Both bacterial strains were inoculated as a spot (diameter 2 mm) on the surface of a LB agar plate spread with cell suspension of a given indicator strain. Cells were incubated 48 h at 25 • C, and bacterial species were identified using 16S rDNA sequencing. The microorganism genomes were sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Hampton, NY, USA) according to the manufacturer's instructions after PCR amplification using the 27F-CM (5 -AGA GTT TGA TCM TGG CTC AG-3 ) and 1492R (5 -TAC GGY TAC  CTT GTT A-C GAC TT-3 ) primer pairs [37,38]. A BLAST similarity search of the 16S rRNA was conducted of the National Centre for Biotechnology Information (NCBI) database using BLASTN. Analysis of the phylogenetic tree construction was generated using the neighbor-joining statistical method. The sequences were submitted to GenBank, where they have been assigned the S. thiooxidans strain SMG1 (GenBank Accession No. ON566159) and the E. aestuarii strain SMG2 (GenBank Accession No. ON566158) (Figure 1). inoculated as a spot (diameter 2 mm) on the surface of a LB agar plate spread with cell suspension of a given indicator strain. Cells were incubated 48 h at 25 °C, and bacterial species were identified using 16S rDNA sequencing. The microorganism genomes were sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Hampton, NY, USA) according to the manufacturer's instructions after PCR amplification using the 27F-CM (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGY TAC  CTT GTT A-C GAC TT-3′) primer pairs [37,38]. A BLAST similarity search of the 16S rRNA was conducted of the National Centre for Biotechnology Information (NCBI) database using BLASTN. Analysis of the phylogenetic tree construction was generated using the neighborjoining statistical method. The sequences were submitted to GenBank, where they have been assigned the S. thiooxidans strain SMG1 (GenBank Accession No. ON566159) and the E. aestuarii strain SMG2 (GenBank Accession No. ON566158) (Figure 1).

Shrimp and Experimental Condition
The experiment was carried out in an indoor shrimp farming facility at Kunsan National University (Gunsan, Korea). L. vannamei juveniles were purchased from a shrimp farm (Taean, Korea). The shrimps were allowed to acclimatize to the experimental conditions and facility for 15 d, during which they were fed with shrimp feed containing 38.5% crude protein and 5.4% crude lipid. After the acclimatization period, similarly sized shrimp (mean ± standard deviation, 0.40 g ± 0.01) were randomly stocked in 50 L acrylic tanks (9 tanks with 40 shrimps per tank). Three experimental groups were evaluated in this experiment: (1) a control group reared in natural (i.e., untreated) water (CON), (2) an experimental group in which Salipiger thiooxidans was added to natural water (SMG-A), and (3) natural water inoculated with Exiguobacterium aestuarii (SMG-B). The two experimental groups were raised in water inoculated weekly with probiotics (10 6 CFU/mL) containing S. thiooxidans (SMG-A) or E. aestuarii (SMG-B). Both bacterial strains were cultured using LB medium (Luria-Bertani, LB Broth, Miller, BD DifcoTM, Sparks, MD, USA), and both species were cultured at 25-26 • C, pH 7 ± 0.5, and 15 psu (salinity). The shrimp were fed four times a day (at 8:00, 12:00, 16:00, and 20:00), and the feed ratios were adjusted to 8-16% of the body-weight of the shrimps for 6 weeks. All the experiments were conducted using a static system and the rearing water was not exchanged (zero-exchange water). The average water temperature during the rearing experiments was maintained at 29.5-30.0 • C. Appropriate dissolved oxygen levels were maintained by installing air stones in all experimental water tanks, and a 12:12 (light:dark) photoperiod was maintained using a fluorescent lamp.

Growth Performance and Sample Collection
After undergoing weight measurements, the shrimps were fasted for 20 h, and growth was measured in terms of total shrimp weight. After the final weighing, five shrimps from each tank were randomly selected and anesthetized in ice water for innate immunity analysis. The hemolymph of the shrimp was collected using a syringe treated with Alsever's solution (Sigma-Aldrich, St. Louis, MO, USA), and the samples were allowed to clot at room temperature for 30 min. The serum was then separated by centrifugation (4 • C, 5000× g, 10 min). Microorganisms (100 g) were freeze-dried and used for component analysis.

Proximate Composition Analysis
Proximate composition analysis was performed using the standard AOAC methods [39]. The samples were first freeze-dried for 48 h before the proximate analyses. Moisture level was determined by drying the samples in an oven at 105 • C, and ash content was quantified by combustion at 550 • C. Crude protein content was quantified via the Kjeldahl method, and crude lipid levels were determined via Soxhlet extraction by using a Tecator 1046 Soxhlet system (Tecator AB, Munkedal, Sweden) [40].

Amino Acid Analysis
The two bacterial strains (probiotics) and carcasses of shrimp were freeze-dried for amino acid analysis [37]. Each sample (0.02 g) was hydrolyzed with 15 mL of 6 M HCl at 110 • C for 24 h. The samples were hydrolyzed in distilled water in a 50 mL flask, then evaporated and recovered in sodium citrate buffer (pH 2.2). After filtration (0.2 µm), the samples were analyzed using ninhydrin at 570 nm and 440 nm in an S433 amino acid analyzer (Sykam, Gilching, Germany). For methionine and cystine hydrolysis, performic acid was used instead of 6 M HCl.

Innate Immunity Analysis
The parameters of acid phosphatase (ACP), alkaline phosphatase (ALP), and lysozyme activity in hemolymph were determined by a GENESYS™ 10S UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc., Vantaa, Finland). The activity of TRACP and ALP was analyzed using the TRACP & ALP Assay Kit (Takara Korea, Seoul, Korea). Lysozyme Microorganisms 2022, 10, 1113 5 of 12 activity was analyzed using the Lysozyme Activity Assay Kit (BioVision Inc., Milpitas, CA, USA). The parameters of all measured indexes were adjusted according to the instructions.

Water Quality Analysis
The water of all experimental tanks was collected every three days and used for water quality analysis. The rearing water of tanks was passed through a 0.2 µm syringe filter, and the total ammonia nitrogen (TAN, NH 4 + -N and NH 3 -N), nitrite (NO 2 − ), nitrate (NO 3 − ), and phosphate (PO 4 3− ) concentrations of water samples were quantified by Water-quality Assay Kit (Humas Co Ltd., Daejeon, Korea). All samples were measured according to the manufacturer's instructions. Dissolved oxygen (DO) and pH were measured using a YSI MultiLab 4010-3 water quality analyzer (Yellow Springs, OH, USA).

Statistical Analysis
All data were analyzed using one-way analysis of variance (ANOVA), followed by the Tukey HSD test in SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, USA). Statistical significance was determined at p < 0.05. All the data are reported as mean ± SD. Percentage data were arcsine transformed prior to analysis. Table 1 summarizes the growth, feed efficiency (FE), and survival rate of the CON, SMG-A, and SMG-B groups after the 6-week culture period. The SMG-B group inoculated with E. aestuarii showed significantly higher final body weight (FBW) and weight gain (WG) than the control and SMG-A groups (p < 0.05). The SMG-B group showed significantly higher FE than the control group (p < 0.05). There was no significant difference in the survival rate among all the groups. Table 1. Growth performance of the Pacific white shrimp in the control, SMG-A, and SMG-B groups throughout the 6-week culture period 1 .

SMG-A SMG-B
Initial body weight (g) 0. 40 Tables 2 and 3 show the results of the whole-body proximate and amino acid composition of shrimp after the 6-week culture period. The crude protein level of the shrimp in the SMG-B group was significantly higher than that of the control and SMG-A groups. There was no significant difference in the levels of the crude lipid and crude ash among the all groups. There was no significant difference in the proximate compositions between the two microorganisms S. thiooxidans and E. aestuarii in the experiment. The levels of arginine, histidine, glutamic acid, and glycine in the whole body of the shrimp in the SMG-A or SMG-B groups were significantly higher than those in the control group. Leucine and phenylalanine levels were significantly higher in the shrimp whole body of the SMG-B group than in the control group. In addition, the SMG-B group showed the highest lysine and glycine levels among all the groups. However, the whole body of the shrimp in the control group showed a significantly higher proline level than that in any other group.   Table 4 shows the results of the assessment of the innate immunity in the hemolymph of Pacific white shrimp after the 6-week culture period. The shrimp in the SMG-B group, inoculated with E. aestuarii, showed significantly higher serum lysozyme levels than the control and SMG-A groups. Additionally, serum ACP and ALP activity was significantly higher in the SMG-B group than in the control and SMG-A groups. Table 4. Non-specific immune parameters of the Pacific white shrimp in the control, SMG-A, and SMG-B groups after 6 weeks of culture.

SMG-A SMG-B
Lysozyme 1 0.128 ± 0.006 b 0.141 ± 0.017 b 0.206 ± 0.011 a ACP 2 11.95 ± 0.23 b 12.38 ± 0.48 b 20.35 ± 0.30 a ALP 3 2.08 ± 0.08 b 2.19 ± 0.13 b 4.32 ± 0.58 a Values are means of triplicate groups and presented as mean ± S.D. Values with different superscripts in the same column are significantly different (p < 0.05). 1 Lysozyme activity (U mL −1 ). 2 Acid phosphatase (U mL −1 ). 3 Alkaline phosphatase (U mL −1 ). Figure 2 shows the change in the water quality of the rearing water in the Pacific white shrimp experimental tank during the 6-week culture period. There was no significant difference in phosphate, DO, and pH levels in rearing water in all groups. The SMG-A and SMG-B groups showed significantly lower TAN and nitrite levels than the control group. The nitrate level in the control group was significantly lower than in the SMG-A and SMG-B groups. The phosphate levels in all the groups similarly increased during the culture period.

Water Quality
Values are means of triplicate groups and presented as mean ± S.D. Values with different superscripts in the same column are significantly different (p < 0.05). 1 Lysozyme activity (U mL −1 ). 2 Acid phosphatase (U mL −1 ). 3 Alkaline phosphatase (U mL −1 ). Figure 2 shows the change in the water quality of the rearing water in the Pacific white shrimp experimental tank during the 6-week culture period. There was no significant difference in phosphate, DO, and pH levels in rearing water in all groups. The SMG-A and SMG-B groups showed significantly lower TAN and nitrite levels than the control group. The nitrate level in the control group was significantly lower than in the SMG-A and SMG-B groups. The phosphate levels in all the groups similarly increased during the culture period.

Discussion
Research utilizing probiotics in water, such as the biofloc system, mostly relies on local water resources (unspecified microbial communities such as heterotrophs, microalgae, protozoa, and yeast, presumably present in the influent). Since the microbial species constituting the biofloc technology cannot be clearly identified, it is practically difficult to utilize probiotics in shrimp farms in other regions. Microorganisms in natural seawater may have a positive effect on shrimp or may coexist with pathogenic microorganisms, such as Vibrio species, that also proliferate using carbon sources [42]. If the probiotic bacteria are relatively dominant, the growth of pathogenic microorganisms may be inhibited; however, the opposite case may cause mass death of shrimp, so caution is required. Therefore, many shrimp farms that cannot find a water resource with an effective microbial community in a nearby watershed purchase expensive probiotic products and use them for aquaculture. However, there are no strict regulations for probiotic products for aquaculture in some regions, and there are concerns about labeling reliability because additional species other than those specified by manufacturers in commercial probiotic products are being detected [33,43]. Commercial probiotics often detect more than one microbial species, in addition to multiple additional microorganisms other than those specified in the product, making it difficult to characterize the mechanism for the probiotic effect, or to attribute the effect to a single species [33]. Therefore, for sustainable aquaculture, accurate information disclosure and research on microbial strains used in probiotics are required, and it is necessary to discover and develop probiotics optimized for each aquaculture species.
In numerous studies, probiotic bacteria have been reported to provide nutrients and increase digestive enzyme activity, thereby improving host growth and feed efficiency [44]. Probiotics, such as Bacillus spp. and Lactobacillus spp., have been reported to improve shrimp growth and reduce feed costs by producing amylases, proteases, lipases, and cellulases in the intestine to improve the digestion and absorption of proteins by the shrimp [17,45,46]. The study presented here showed that the administration of E. aestuarii to the rearing water can significantly improve the growth and FE of L. vannamei. Exiguobacterium sp. has been shown to produce protease over a wide temperature range and has been reported to produce several effective enzymes, including proteolytic enzymes [47]. In this study, the levels of crude protein, essential amino acids (lysine, leucine, phenylalanine, histidine, and arginine) and non-essential amino acids (glutamic acid and glycine) of the shrimp reared with E. aestuarii were significantly higher than the control levels. Essential amino acids are nutrients that must be supplied as factors that affect the growth of shrimp [48][49][50]. Probiotics provide essential amino acids to the host by breaking down complex nutrients in the intestine for growth, so intake of probiotics improves intestinal microbial balance to increase nutrition absorption, increase feed efficiency, and improve the growth rate of shrimp [51]. It is considered that E. aestuarii administrated in the water was ingested by L. vannamei and produced proteolytic enzymes in the body of the shrimp to improve the absorption of proteins and essential amino acids by the shrimp. Therefore, the significant increases in the growth performance of the shrimp in the SMG-B group were likely due to the nutritional improvement caused by the administration of E. aestuarii. In this study, various amino acid levels were increased according to E. aestuarii administration, except for the proline level, which was significantly higher in the control group (not inoculated with any probiotic) than in the other groups. After adding proline to the feed for 8 weeks, no significant change in the growth of the shrimp was detected [52]. According to a previous study, the blood proline level of infants fed formula containing the probiotic Bifidobacterium lactis was significantly lower than that of infants fed formula without probiotics [53]. Therefore, it is presumed that probiotics can affect the reduction of proline in the body, but further research is needed on the relationship between probiotics and proline in L. vannamei. This is the first study in which E. aestuarii alone was inoculated into rearing water and tested on L. vannamei, and E. aestuarii was found to improve the growth performance, FE, and nutritional composition of L. vannamei.
The results of this study showed that the serum lysozyme level, and ACP and ALP activity, of E. aestuarii were significantly increased upon inoculation of the rearing water with either probiotic. Lysozyme catalyzes the hydrolysis of the β-(1,4)-glycosidic linkage of n-acetyl-glucosamine and n-acetyl-muramic acid, and thereby protects the host from bacterial infections [54]. Lysozyme activity is one of the most important immune factors in shrimp, and lysozyme is an antibacterial enzyme that acts on the cell wall of pathogens [55]. Similar to our study results, when L. vannamei was inoculated with B. subtilis and bred for 2 weeks, the lysozyme gene expression in L. vannamei was significantly increased [56]. In addition, when Pediococcus pentasaceus was added to the feed of L. vannamei for 8 weeks, the lysozyme activity of L. vannamei was significantly increased [57]. Phosphatase enzymes can hydrolyze organophosphate esters, and ACP and ALP are composed of several phos-phomonoesterases that are very important for the crustacean immune system [58,59]. ACP is a lysosomal enzyme involved in killing and digesting invading organisms during the immune response, and ALP is a multifunctional enzyme that hydrolyzes various types of phosphomonoester substrates and acts as a transphosphorylase at alkaline pH [60,61]. Both ACP and ALP have been reported to correlate with the immune capacity of aquatic animals in immune defense mechanisms [59,62]. Similar to the results of this study, the rearing water of Macrobrachium nipponense was inoculated with B. velezensis CPA1-1. After 20 d, ACP and AKP activities were found to have significantly increased, and resistance to Vibrio infection was improved [63]. In addition, as a result of feeding L. vannamei with Enterobacter hominis and Lactobacillus sp. for 4 weeks, the activity of immune-related enzymes including ACP and AKP tended to increase [9]. These findings showed that probiotics enhance the immunity of shrimp by inducing the host's immune enzyme levels [64]. Therefore, it is considered that E. aestuarii can increase the activity of immune-related enzymes and improve the non-specific immunity of L. vannamei.
In this study, the SMG-A and SMG-B groups, which were inoculated with the probiotics, had significantly lower TAN and nitrite levels than the control group. As the feed is supplied to the shrimp, the concentration of toxic ions, such as ammonia and nitrite, which increases due to the accumulation of feed residues and excrement in the rearing water, may increase, causing shrimp disease, and high mortality may occur [65]. Therefore, water quality management is an important factor in the success of shrimp aquaculture. Multiple studies have demonstrated that probiotics have great potential for reducing toxic ion concentrations in rearing water [65][66][67]. Similar to the results of this study, B. subtilis CM3.1 was inoculated into the rearing water of L. vannamei, and the growth parameters of the shrimp were observed for 120 h. TAN and nitrite levels were found to be significantly reduced [68]. In a similar study, the rearing water of the Pacific white shrimp was inoculated with B. amyloliquefaciens, and the growth parameters of the shrimp were observed for 169 d. Consequently, it was found that this bacterium can significantly improve water quality [69]. In a previous study, it was reported that Salipiger sp. can remove organic compounds, such as nitrite, from wastewater, and E. aestuarii has recently been shown to effectively eliminate such compounds from aquaculture wastewater through extracellular enzymatic activity [36,70]. In this study, it was confirmed that the water quality in the rearing water was improved due to the denitrification action by the two microorganisms. However, research on water quality improvement by these two microorganisms is insufficient, so it is considered that supplementary studies related to TSS and alkalinity are needed in the future.

Conclusions
Both bacterial strains were shown to improve the water quality in the aquaculture of L. vannamei. In particular, E. aestuarii can improve the growth, FE, nutritional content, and innate immunity of L. vannamei, so it can be recommended as a probiotic for L. vannamei. The synergistic effect of mixing the two bacterial strains, and mixing them with other proven probiotics, needs to be studied. Our findings suggest that S. thiooxidans and E. aestuarii from the Saemangeum Reservoir can improve the quality of aquaculture water, and, in particular, E. aestuarii is a potential probiotic for L. vannamei.