Evaluation of the Probiotic Potential of Bacillus spp. Strains Isolated from River Bottom Sediments

Abstract

The study aimed to evaluate bottom sediments as a promising source of probiotic bacteria for aquaculture applications. Bacillus strains were selected as the most suitable bacterial species for application in the food industry. Initially, seven Bacillus spp. strains were isolated from the intestinal contents of healthy sterlet specimens; however, none of them demonstrated high potential probiotic properties. Subsequently, bottom sediments were considered as a source of probiotic strains. In the bottom sediments, bacilli exist in a vegetative form and constitute an integral part of the microbial community. A total of 120 Bacillus spp. strains were isolated and comprehensively analyzed. Proteolytic and amylolytic activities were detected at moderate levels in almost all isolated strains. Most isolates exhibited low or negligible antioxidant, DNA-protective, and antimicrobial activities; however, a small group of strains showed high values of these properties. Principal component and cluster analyses indicated the co-existence of three life strategies of bacilli in bottom sediments. These findings highlight the high probiotic potential of bacilli from bottom sediments and support their suitability as novel probiotics for enhancing the health and productivity of aquatic organisms in aquaculture.

1. Introduction

The development of aquaculture is currently a critical task for ensuring food security, especially given the disruption of marine and river ecosystems due to pollution and climate change [1,2,3]. To enhance the efficiency of aquaculture, alternative strategies that reduce the negative impacts of artificial fish farming and the use of artificial feeds are essential. One of these factors is the application of probiotics.

Utilizing probiotics in aquaculture can increase biomass growth and reduce feed conversion rates [4,5,6]. Probiotics enhance fish immunity, increase disease resistance, improve food digestion, maintain intestinal integrity, and reduce stress levels [4,5,6,7,8,9].

Although the exact mechanisms of action of specific probiotic strains are not always fully understood, known mechanisms include competitive adhesion, the production of digestive enzymes, vitamins, antioxidants, antimicrobial compounds, and biologically active compounds that modulate the host’s physiology [4].

The application of probiotics in aquaculture presents certain challenges, such as the need to consider the low body temperature of fish, which may reduce the physiological activity of many strains. Additionally, fish are characterized by a rapid food passage rate through the intestines, meaning some strains may not have sufficient time to act [10,11].

Bacteria isolated from bottom sediment deposits are increasingly employed in aquaculture. They are primarily used as water additives or within biofilters to improve water quality, particularly in closed-loop aquaculture systems. Such systems lack a fundamental component of natural ecosystems—native sediments with their established microbiota—and these additives are therefore intended to mitigate this deficiency [12]. Additionally, these bacteria can be applied in pond aquaculture systems [13]. In aquaculture, sediment-derived bacteria are mainly utilized to enhance water quality by accelerating the decomposition of organic waste and reducing concentrations of harmful nitrogenous compounds, such as ammonia and nitrites [14].

Among the bacteria used for these purposes, species of Bacillus are the most common, owing to their capacity to lower ammonia and nitrite levels, degrade organic waste, and improve water clarity. These effects contribute to the achievement of a healthier environment for fish farming. Furthermore, the introduction of Bacillus species helps balance microbial communities and suppress pathogens, thereby indirectly enhancing the health of fish and crustaceans [12,15,16].

Previous studies [17] indicated that bacteria with probiotic potential may be present within the microbiota of bottom sediment. These include bacterial genera already recognized for their probiotic activity, such as Bacillus, as well as emerging genera that could serve as novel specialized probiotics for aquaculture, including Priestia, Lysinibacillus, Kocuria, and Heyndrickxia [17].

It has been demonstrated that Bacillus species isolated from bottom sediments can inhibit fish pathogens such as Aeromonas and Pseudomonas [18]. Other Bacillus species exhibit both antagonistic and enzymatic activities, highlighting their high potential as probiotic strains [15,19]. Furthermore, it has been observed that the addition of bacteria as sediment improvers can enhance the productivity of aquatic organisms, even when the water purification effect is limited [20].

This study aimed to compare the probiotic potential of Bacillus spp. strains isolated from fish intestines and bottom sediments, and to assess the suitability of bottom sediments as a promising source of probiotic bacilli for aquaculture. A comprehensive phenotypic characterization of the isolated Bacillus spp. strains was performed, including evaluation of their proteolytic and amylolytic activities, antioxidant capacity, and DNA-protective effects. Additionally, the antimicrobial activity of these strains was assessed. To integratively characterize Bacillus spp. strains from bottom sediments, principal component analysis (PCA) was applied, allowing identification of key parameters that define the functional potential of the isolates.

2. Materials and Methods

2.1. Isolation of Potential Probiotics from the Intestinal Contents of Fish

Bacillus spp. strains were isolated from the intestinal contents of healthy adult sterlets (Acipenser ruthenus Linnaeus, 1758). The fish were raised in recirculating aquaculture system (RAS) conditions at the Experimental Research Center of the Southern Scientific Center of the Russian Academy of Sciences in Kagalnik, Rostov region, Russia. Intestinal contents from five individuals were used in the study. The intestines were delivered to the laboratory within two hours in a cooled state. The intestinal contents were extracted using sterile instruments and placed into sterile plastic tubes and weighed. Then, equal volumes from each individual were taken and pooled to obtain a composite sample.

The composite sample was diluted with 10 mL of sterile physiological solution and heated to 95 °C for 5 min. Subsequently, a series of consecutive decimal dilutions were prepared, and the suspension was plated on solid Luria–Bertani (LB) medium. The plates were incubated at 37 °C for 24 h. The resulting colonies were isolated using a streak method on solid LB medium and evaluated for potential probiotic properties.

2.2. Isolation of Potential Probiotics from the Natural Fish Habitat

Sediment samples were collected from the Don River at the Experimental Research Center of the Southern Scientific Center of the Russian Academy of Sciences in Kagalnik, Rostov region, Russia, at a depth of 0–5 cm.

To determine the abundance of Bacillus spp. cells in vegetative and sporulated forms, a method described in [21] was employed. Immediately after receiving the sediment samples, a portion of sediments was pasteurized (95 °C, 5 min). Subsequently, a series of consecutive decimal dilutions were prepared, and samples were inoculated onto solid Luria–Bertani medium to determine the number of Bacillus spp. cells in sporulated form. The sediment samples were then maintained at +4 °C for 2 days to promote sporulation of Bacillus spp. After pasteurization, the total bacterial count, including those in spores and those transitioning to spores upon cooling, was determined. The difference between these counts represented the colony-forming units (CFU) of vegetative cells.

2.3. Isolation of Potential Probiotic Strains with Specified Enzymatic Activity from Bottom Sediment Samples

Proteolytic activity was assessed on solid Luria–Bertani (LB) agar supplemented with 200 mL of skim milk on 1 L of media. Following incubation, strains showing colonies with clear zones in the medium were selected.

Amylolytic activity was tested on solid Luria–Bertani (LB) agar supplemented with 10 g/L starch. After incubation, strains displaying colonies surrounded by clear zones were selected.

Lipolytic and lecithinase activities were tested on solid Luria–Bertani (LB) agar supplemented with a 1:1 suspension of raw chicken egg yolk. The egg yolk suspension was prepared by mixing 90 g of yolks with 210 mL of saline at 50 °C. After incubation, strains with colonies surrounded by an iridescent halo were selected. Lecithinase activity was indicated by the formation of a white opaque zone around the colony.

In each case, we measured only the hydrolysis zone, without accounting for the diameter of the colony itself.

Bacterial colonies exhibiting the targeted activities were streaked onto solid Luria–Bertani (LB) agar for purity assessment.

2.4. Isolation of Potential Probiotic Strains with Antimicrobial Activity Against Fish Pathogens

For strains isolated from intestinal contents, plates from Section 2.1 were employed. For strains from sediment samples, plates used for enzymatic activity testing (Section 2.3) were employed. Plates with developed colonies were carefully inverted using sterile spatulas and forceps such that colonies faced downward and the agar surface was on top. Pathogens from the laboratory collection, Pseudomonas aeruginosa and Aeromonas veronii, were inoculated onto this agar surface. The plates were incubated at 37 °C for 24 h.

After 24 h, zones of inhibited growth were observed on the pathogen-inoculated agar above colonies exhibiting antimicrobial activity. Agar from above such colonies was then excised with a loop, bacterial biomass collected, and transferred to Luria–Bertani agar. Bacterial colonies exhibiting the targeted activities were streaked onto solid Luria–Bertani (LB) agar for purity assessment.

2.5. Cross-Assessment of Enzymatic Activity of Strains

All isolated strains exhibiting any of the potential probiotic activities described above (proteolytic, amylolytic, lipolytic, antimicrobial) were further tested for amylase, protease, and lipase activities. All strains isolated from fish intestines were also examined. Using the media described in Section 2.3, each strain was inoculated and incubated at 37 °C for 48 h. Any resulting clearance zones were recorded. In each case, we measured only the hydrolysis zone, without accounting for the diameter of the colony itself. Strains from the laboratory collection with previously confirmed probiotic activity, Bacillus amyloliquefaciens B1895a and Bacillus subtilis KATMIRA 1933, were used as controls.

2.6. Evaluation of Antimicrobial Activity of Strains

Strains showing antimicrobial activity were examined using the method of consecutive two-fold dilutions. Overnight cultures of all fish pathogen test strains (Pseudomonas aeruginosa, Aeromonas veronii) were prepared in their respective liquid nutrient media and incubated at 37 °C with shaking.

Overnight cultures of bacilli were also prepared. For this, 5 mL of liquid LB medium was added to a test tube, inoculated with a loopful of the strain biomass, and placed at an angle to increase the surface area of the medium. Biofilm formed on the surface in all samples. After 24 h, the test tubes were centrifuged (5 min, 3900 rpm) and the supernatant was filtered through a 0.22 µm cellulose filter (MilliporeSigma, Burlington, MA, USA) to separate it from remaining bacilli cells.

In a sterile 96-well microplate (Greiner Bio-One GmbH, Kremsmünster, Upper Austria, Austria), 200 uL of sterile supernatant was pipetted in each well. Then, 100 µL was taken from it and transferred to the next well. To this well, 100 µL of nutrient medium was added and thoroughly mixed to obtain a 1:2 dilution. Subsequently, 100 µL of the two-fold diluted sample was taken and transferred to the next well; this allowed us to obtain a 1:4 dilution with consecutive two-fold dilutions. Wells containing nutrient medium without supernatant served as controls.

The biomass of pathogen strains from the overnight culture was centrifuged (5 min, 3600 rpm) and resuspended in fresh nutrient medium to an OD600 of 0.1. Then, 100 µL of the resulting suspension was added to each well. Each plate was sealed with a sterile plate seal and incubated at 37 °C for 24 h.

After 24 h, growth of the pathogen strains in the presence of each supernatant concentration was evaluated. The degree of cell growth was assessed by measuring the optical density at 600 nm using a multimodal plate reader FLUOstar Omega (version 5.70 R2 BMG LABTECH, GmbH, Ortenberg, Germany).

2.7. Determination of Antioxidant and Genoprotective Activity

All strains isolated in the previous stages were examined.

The suspensions of the tested strains were prepared as described in Section 2.6.

Lux biosensors were used to determine the antioxidant and antimutagenic activity. These biosensors are living bacterial cells containing a hybrid plasmid with two main elements embedded: a regulatory region (promoter and operator) and reporter genes. LuxCDABE genes, isolated from the genomes of luminescent bacteria and encoding luciferase and reductase, were used as reporters.

E. coli strain MG1655 pKatG (Lux biosensor of oxidative stress, detecting the presence of hydrogen peroxide and organic peroxides in the environment) was used to determine antioxidant activity, while E. coli strain MG1655 pRecA (Lux biosensor of factors causing DNA damage and SOS response) was utilized to determine genoprotective activity, using an approach described in our earlier work [22].

Strains of biosensors were cultivated in a complete Luria–Bertani (LB) medium supplemented with ampicillin at 100 µg/mL. An overnight culture was diluted in fresh LB with ampicillin to a density of 0.1 (for E. coli MG1655 pKatG) and 0.01 (for E. coli MG1655 pRecA) MacFarland units using a DEN-1B densitometer (Biosan), followed by incubation in a thermostat at 37 °C for 2 h.

For the negative control, a 160 µL aliquot of the biosensor culture was transferred to sterile microplate wells and supplemented with 20 µL of liquid LB medium and 20 µL of distilled water. For the positive control, 20 µL of liquid LB medium and 20 µL of inducers were added to the aliquot: 10⁻² M hydrogen peroxide (equivalent to 10⁻³ M in the well) for E. coli MG1655 pKatG, and 2.25∙10⁻⁴ M dioxidin (corresponding to 2.25∙10⁻⁵ M in the well) for E. coli MG1655 pRecA.

As a control sample for pro-oxidant and promutagenic activities, 20 uL, 20 µL of the supernatant of the tested strains and 20 µL of distilled water were added. For antioxidant and genoprotective activity determination, 20 µL of the supernatant and 20 µL of the corresponding inducer were added.

Microplates were placed in a FLUOstar Omega (version 5.70 R2 BMG LABTECH, GmbH, Ortenberg, Germany) multimodal reader and incubated at 37 °C. The bioluminescence intensity was measured every 10 min for 2 h.

The induction coefficient (I_C) of the SOS response (I^S) was calculated using the following formula:

$$I={\displaystyle \frac{L_e}{L_c}}-1$$

(1)

where L_e—luminescence intensity of the sample with the inducer (in arbitrary units);

L_c—luminescence intensity of the control sample (in arbitrary units).

Statistical significance of the SOS induction effect was determined by the significant increase in L_e over L_c, assessed using the t-criterion. The standard deviation of induction was calculated as the statistical error of the ratio using the following formula:

$$S_{\tilde{z}}=\left|\overline{I}\right|\sqrt{{\left({\displaystyle \frac{s_{\overline{L_e}}}{\overline{L_e}}}\right)}^2+{\left({\displaystyle \frac{s_{\overline{L_c}}}{\overline{L_c}}}\right)}^2}$$

(2)

where indices e and c refer to the experiment and control, respectively.

Statistically significant differences from the control were identified based on the values of confidence intervals calculated using the following formula:

$$\left(\overline{I}-t_{\mathrm{kp}}\times {\displaystyle \frac{s}{\sqrt{n}}}; \overline{I}+t_{\mathrm{kp}}\times {\displaystyle \frac{s}{\sqrt{n}}}\right)$$

(3)

where Ī is the mean value of the induction coefficient; t_kp is determined from Student’s distribution table.

Protective activity was calculated using the following formula:

$$P=\left(1-{\displaystyle \frac{I_e}{I_c}}\right)\times 100\%$$

(4)

where I_e—induction coefficient of the sample with the inducer (in arbitrary units); I_c—induction coefficient of the control sample (in arbitrary units).

2.8. Statistical Processing

All measurements were performed in triplicate. Violin plots were created in R 4.4.2 using the ggplot2 package with the geom_violin function. Principal component analysis (PCA) and cluster analysis were performed using the FactoMineR and pcaMethods packages.

3. Results

3.1. Bacteria from Fish Intestines and Their Probiotic Potential

During the isolation of potential probiotic Bacillus spp. strains from natural sources and intestinal contents, colony morphology on solid media was carefully evaluated. Bacillus species display considerable variability in colony characteristics [23,24,25,26]. Accordingly, colonies grown on the same nutrient medium differed in size, edge shape, elevation, color, density, surface texture, presence, abundance, thickness, height of wrinkles and spatial distribution. To avoid redundancy, morphologically identical strains originating from the same sample were excluded from further selection.

Sterlet intestinal contents contained 2.3 ± 0.3 × 10³ CFU/g of Bacillus spp. belonging to 7 morphotypes. All of these were isolated and tested (Strains BT1-7). The sediment showed higher Bacillus spp. concentrations, with most bacteria in the vegetative state (Table 1).

Table 1. Bacillus spp. Abundance in Bottom Sediments in Vegetative and Sporulated Forms.

	Total Abundance	Vegetative Form	Sporulated Form
Abundance, CFU/g	6.4 ± 0.2 × 107	5.7 ± 0.2 × 107	7.1 ± 0.4 × 106
Percentage	100%	89%	11%

All seven Bacillus spp. strains isolated from fish intestines were evaluated for potential probiotic properties, including proteolytic, amylolytic, and lipolytic activities, as well as antioxidant and antimutagenic capacities. However, none of the seven isolated strains displayed significant probiotic potential. Consequently, the search for probiotic Bacillus spp. strains was extended to isolates from sediments within the natural habitats of sterlet (Acipenser ruthenus).

3.2. Bacteria from Sediments and Their Probiotic Potential

To isolate only Bacillus strains with at least one potentially interesting probiotic property from all the bacilli present, a suspension from the bottom sediment was directly inoculated onto plates containing test media to determine enzymatic activity. This method allowed selection of strains capable of secreting at least one group of exoenzymes. A total of 120 strains were selected: 61 strains secreting exoproteases, 52 strains secreting exoamylases, and 5 strains secreting exolipases.

After selecting these strains, agar test plates with the remaining colonies were inverted and inoculated with a suspension of fish pathogens. The pathogens grew as a continuous lawn, with clear zones appearing over Bacillus spp. colonies exhibiting antimicrobial activity. This process resulted in the selection of 10 additional strains with antimicrobial activity against at least one of the test pathogens.

In total, 120 strains were isolated from the sediments (strains MT1-116, MT141, MT142, MT155, MT186). After purification, each strain was tested for all potential probiotic properties: exoenzyme secretion, antimicrobial activity, antioxidant activity, and antimutagenic activity (Figure 1). These strains were compared to two reference laboratory strains, B. subtilis KATMIRA 1933 and B. amyloliquefaciens B1895, which have demonstrated probiotic efficacy in in vivo studies. The reference strains exhibit both enzymatic and antioxidant activities and have been reported to enhance productivity in broiler chickens and laying hens [27,28].

Figure 1. Evaluation of the extent of potential probiotic properties in Bacillus spp. strains isolated from bottom sediments. Grey dots represent values for individual strains, and black dots indicate the mean value averaged over all strains.

The objective of our study was to isolate strains exhibiting probiotic activities comparable to or exceeding those of the reference strains, while being naturally adapted to the fish intestinal environment and aquatic conditions.

Data for each strain can be found in the Supplementary Materials. Comparative data for two strains with confirmed probiotic activity, B. subtilis KATMIRA 1933 and B. amyloliquefaciens B1895, are also provided.

Proteolytic and amylolytic activities among the strains isolated from sediment exhibited considerable variability, with their distribution approaching a normal pattern within the population. Of the 120 strains analyzed, some showed no notable exoenzyme activity (the assessment method specifically measures exoenzyme activity), whereas others demonstrated strong enzymatic activity, characterized by a clearance zone exceeding 9 mm around the colony. The majority of strains exhibited moderate activity, with clearance zones ranging between 3 and 6 mm.

Lipolytic activity was generally low among the Bacillus spp. strains isolated from bottom sediments. Only 5 out of 120 strains exhibited detectable lipolytic activity, with the width of the iridescent halo surrounding the colonies not exceeding 2 mm.

LUX-biosensor assays enable the detection of both antioxidant and pro-oxidant activities. Antioxidant activity is reflected by a decrease in catalase expression in the LUX-biosensor E. coli MG1655 pKatG in the presence of hydrogen peroxide upon addition of metabolites produced by the test strain. Conversely, pro-oxidant activity is indicated by increased catalase expression in the presence of metabolites produced by the test strain alone.

Most strains produced metabolites with varying degrees of antioxidant activity. However, a small subset (7 strains) exhibited antioxidant activity exceeding 50–55%, as clearly indicated by the upper peak in the violin plot (Figure 1). The majority of strains either lacked pro-oxidant activity or displayed only weak pro-oxidant activity (up to 10%). Nonetheless, some individual Bacillus spp. isolates (20 strains) showed more pronounced pro-oxidant activity, reaching 40–50%, as reflected by the narrow spike in the plot (Figure 1).

DNA-protective and pro-mutagenic activities were evaluated using the same approach as for antioxidant and pro-oxidant activities, assessing the expression level of the Rec operon. Most Bacillus spp. strains isolated from bottom sediments exhibited limited DNA-protective activity, not exceeding 40%, while only a few strains (5) demonstrated high DNA-protective activity, reaching 60–70%. Similarly, most strains showed either no or weak pro-mutagenic activity (up to 10%). However, a small subgroup (21 strains) displayed elevated pro-mutagenic activity, reaching up to 30%.

Most of the isolated strains did not exhibit antimicrobial activity against Gram-negative fish pathogens. Among the 120 strains tested, only 10 demonstrated varying degrees of antimicrobial activity against Pseudomonas aeruginosa and Aeromonas veronii. These strains and their respective inhibition levels are summarized in Table 2.

Table 2. Antimicrobial activity of cell-free supernatants from potential probiotic strains against fish pathogens.

Strain	Dilution		Pseudomonas Aeruginosa	Aeromonas Veronii
Control	-	OD600	0.356 ± 0.036	0.733 ± 0.072
		%	100%	100%
MT36	1/2	OD600	0.043 ± 0.40	0
		%	12% *	0% *
	1/4	OD600	0.185 ± 0.020	0.346 ± 0.40
		%	52%	47% *
MT49	1/2	OD600	0.157 ± 0.027	0.418 ± 0.062
		%	44% *	57% *
	1/4	OD600	0.349 ± 0.024	0.696 ± 0.027
		%	98%	95%
MT55	1/2	OD600	0	0
		%	0% *	0% *
	1/4	OD600	0	0.413 ± 0.029
		%	0% *	56% *
MT67	1/2	OD600	0	0
		%	0% *	0% *
	1/4	OD600	0.210 ± 0.024	0.323 ± 0.024
		%	59% *	44% *
MT72	1/2	OD600	0.097 ± 0.067	0.059 ± 0.017
		%	27% *	8% *
	1/4	OD600	0.140 ± 0.042	0.265 ± 0.036
		%	39% *	36% *
MT78	1/2	OD600	0.045 ± 0.020	0.110 ± 0.078
		%	12% *	15% *
	1/4	OD600	0.190 ± 0.019	0.346 ± 0.027
		%	52% *	47% *
MT86	1/2	OD600	0.157 ± 0.040	0
		%	44% *	0% *
	1/4	OD600	0.191 ± 0.053	0.116 ± 0.024
		%	54% *	16% *
MT97	1/2	OD600	0.278 ± 0.034	0.367 ± 0.041
		%	78% *	50% *
	1/4	OD600	0.358 ± 0.037	0.420 ± 0.034
		%	100%	57% *
MT155	1/2	OD600	0	0
		%	0% *	0% *
	1/4	OD600	0	0.263 ± 0.088
		%	0% *	36% *
MT186	1/2	OD600	0.163 ± 0.076	0
		%	46% *	0% *
	1/4	OD600	0.199 ± 0.36	0.117 ± 0.028
		%	56% *	16% *

* p < 0.05.

Thus, all isolated strains were characterized. However, to describe the Bacillus community as a whole, principal component analysis (PCA) was performed on the obtained data.

3.3. Principal Component Analysis of Bacillus spp. Properties

For a comprehensive analysis of the enzymatic, antioxidant and pro-oxidant, antimutagenic and promutagenic, and antimicrobial properties of the strains isolated from bottom sediments, principal component analysis (PCA) was employed (Figure 2). The first principal component (PC1) accounted for 25.5% of the total variance, the second principal component (PC2) explained 17.6%, and the third principal component (PC3) accounted for 16.7%. Thus, the three principal components collectively explained approximately 60% of the total variability. The identified strains clustered into three distinct groups according to PCA.

Figure 2. Probiotic properties of the strains from bottom sediments via principal component analysis (PCA). (a) Loading plot. Dots represent individual strains. (b) Scree plot. (c) Score plot. (d) Optimal number of clusters plot.

4. Discussion

4.1. Potential Probiotics Isolated from the Fish Intestine

When identifying potential probiotics, it is crucial to define the specific function the probiotic is intended to perform within the host organism and the challenges it aims to mitigate. Based on prior research, the following probiotic functions and properties can be distinguished:

1. Enhancement of feed digestibility during dietary transitions in livestock and poultry [5], during weaning and shifts in diet composition in fish fry [29,30,31,32], as well as during phases of intensive growth in meat-producing animals and poultry [33,34]. In these contexts, selection should prioritize probiotics exhibiting enzymatic activities, particularly the production of proteases, lipases, amylases, and related enzymes.

2. Prevention of bacterial and fungal infections, notably gastrointestinal diseases. Here, probiotics with antibacterial and antifungal activities are sought. Mechanisms include direct inhibition through antimicrobial agents of both specific (e.g., antibiotics, bacteriocins) and nonspecific (e.g., organic acids, reactive oxygen species) nature, as well as interference with quorum sensing and competitive exclusion of pathogenic bacteria [4,5,6,7,8,9].

3. Protection against adverse environmental factors, including exposure to feed contaminated with mycotoxins, bacterial toxins, herbicide residues, and similar compounds. Probiotics with antioxidant and antimutagenic properties [32], alongside the capacity to degrade toxins enzymatically [33,34], are particularly valuable in this context.

4. Modulation of host biochemical processes. The role of gut microbiota in influencing the host’s immune and nervous systems is well established. Although direct bacterial signaling pathways affecting the host remain incompletely understood, several molecular mechanisms have been elucidated. Therefore, it is imperative to screen probiotic candidates for the production of relevant signaling molecules using ELISA techniques or for the presence of genes implicated in the biosynthesis of such molecules [4,5,6,7,8,9].

In this study, the search for potential probiotics for aquaculture focused primarily on the first three criteria, given the limited current understanding of the mechanisms by which gut microbiota influence fish biochemistry.

A critical factor in selecting probiotic strains is their accessibility to end users. This consideration is particularly important when probiotics are intended for large-scale use in agriculture rather than individual medical applications. Such probiotic strains must exhibit a combination of practical traits beyond their immediate functional benefits. These include robust and rapid growth, the ability to proliferate on inexpensive nutrient media, low drying and processing costs, independence from stringent storage conditions, and long shelf life. Collectively, these traits contribute to reducing the overall production costs of probiotic formulations, a major factor in sectors such as aquaculture. Consequently, Bacillus spp. strains exhibiting these characteristics are of particular industrial interest [35,36].

The objective of this study was to identify potential probiotic strains suitable for aquaculture. Initially, we intended to isolate strains from the gut of healthy sterlet individuals. Gut-derived strains are inherently adapted to the gastrointestinal environment, having survived exposure to gastric acidity, digestive enzymes and metabolism adapted to low oxygen levels [37]. The intestinal microbiota is regarded as one of the most promising sources of probiotic candidates [38]. However, the abundance of Bacillus spp. in the intestinal contents of sterlets was low—2.3 ± 0.3 × 10³ CFU/g—and their morphological diversity was limited.

To evaluate the probiotic potential of the isolated strains, their activities were compared with those of B. subtilis KATMIRA 1933 and B. amyloliquefaciens B1895, which served as reference strains. These Bacillus strains have demonstrated probiotic efficacy in previous studies with poultry, enhancing broiler weight gain, increasing egg production in laying hens, and protecting mitochondrial DNA [27,28,29,30,31,32,33,34,35,36,37,38,39].

Unfortunately, none of the strains isolated from the fish gut exhibited probiotic properties comparable to or exceeding those of the reference strains. Measurements of lytic, antioxidant, and DNA-protective activities were uniformly low across all isolates, leading to their exclusion from subsequent investigations.

Consequently, alternative sources of Bacillus were sought for probiotic screening, leading to the examination of microbiota from river bottom sediments.

4.2. Potential Probiotic Bacteria Isolated from Bottom Sediments

It is well established that Bacillus spp. strains isolated from bottom sediments are used as so-called “probiotics” for aquatic environments. Bacilli efficiently absorb organic matter from water and are capable of assimilating excess nitrogen by converting it into a bound form within their own biomass. For this reason, they are commonly utilized in high-density fishpond systems and recirculating aquaculture setups, either as water additives or as part of the biofilter microbial community [12,15,16]. Certain studies have demonstrated that Bacillus-based supplements achieve a less pronounced reduction in detrimental water quality parameters compared to chemical treatments, but concurrently promoting the growth and productivity of aquatic organisms [20]. In this context, Bacillus likely functioned not only as a water purification agent but also as probiotics for crustaceans.

The authors propose that, due to the characteristics of aquatic environments, a unified fish–water–sediment microbiota system exists in aquaculture species, particularly among freshwater and marine coastal and benthic dwellers. Fish harbor microbiomes in their intestines, on their skin, and gills. These microorganisms are released into the surrounding water and subsequently settle in the sediment, thereby becoming integral components of the sediment microbiota. Over time, these bacteria may be resuspended into the water column, especially through the activities of benthic organisms that disturb the sediment. Subsequently, bacteria can recolonize the fish skin, gills, and intestines. This dynamic process supports the concept of a continuous exchange of bacteria between the environment and the associated biota.

Metagenomic data further support the existence of such a unified system. It has been shown that the composition of the microbiota of bottom sediments, water, and the microbiota of aquatic organisms inhabiting the water body largely overlaps [39]. Similarity is lower in fish residing in the water column and predatory species; however, in some cases, the intestinal microbiota and bottom sediments share more than 70% similarity in benthic fish species [40]. Additionally, filter-feeding shrimps from both freshwater and saltwater environments show a high degree of similarity between their intestinal microbiota and that of bottom sediments [41].

Zhang X et al. demonstrated that the fish microbiota is one of the primary sources of bacteria for the microbiota of bottom sediment [42]. Conversely, Zhang B. et al. reported that the microbiota of water and bottom sediments plays a significant role in shaping the microbiota of fish inhabiting these environments. For certain species of fish and crustaceans, the microbiota of bottom sediment serves as the main source for the establishment of intestinal microbiota [40,41].

Therefore, if under natural conditions the microbiota of bottom sediments constitutes a source of intestinal microbiota for fish, the utilization of bottom sediments as a resource for isolating novel probiotics for aquaculture is not only justified but also desirable.

A total of 120 Bacillus spp. strains were isolated from bottom sediments of the Don River, Russia. Only 11% of the population was in the spore form, with the remainder existing in the vegetative state. This suggests that bacilli in bottom sediments are metabolically active and represent an important part of the benthic microbial community.

All 120 isolated strains were tested for potential probiotic properties. It was found that, based on the pattern of property distribution within the Bacillus population, the properties of bacterial isolates can be divided in two groups.

In the first group of properties (lytic activities), the property was present to some extent in all members of the population, and its value distribution was close to a normal distribution. This is illustrated by proteolytic and amylolytic activities, where most of the isolated Bacillus spp. strains exhibited average levels of these properties. Since bacilli are typical r-strategists, focused on rapid consumption of large amounts of organic substrates and fast growth until resource depletion, the presence of extracellular enzyme is essential for their survival [43].

In the second group of properties (antioxidant and DNA-protective activities antimicrobial activity), most of the population either lacked the trait entirely or exhibited weak activity, with only a small subset of strains (6–17%) displaying high activity for the trait. This pattern is characteristic of both advantageous probiotic properties, such as antioxidant and DNA-protective activities and antimicrobial activity, and undesirable features, such as pro-oxidant and pro-mutagenic activities. These traits do not appear to be essential for microbial survival but may offer adaptive advantages under specific environmental conditions.

4.3. Principal Component Analysis of Properties of Bacillus spp. Strains Isolated from Bottom Sediments

To characterize the entire collection of Bacillus spp. strains isolated and to evaluate the relationships among their various potential probiotic properties, principal component analysis (PCA) and cluster analysis were performed.

Principal component analysis provides an effective overview of large datasets and has been widely used in the visualization of metagenomic, meta-transcriptomic, meta-proteomic, and metabolomic data [44,45,46,47].

Among its advantages, PCA enables the identification of correlations between individual traits. The loading plot displays the contributions of variables to the first two principal components. As shown in Figure 2a, antimicrobial activities against Pseudomonas aeruginosa and Aeromonas veronii are strongly correlated. Given that both bacteria are Gram-negative, it is likely that antimicrobial strains produce compounds effective against a broad range of Gram-negative bacteria.

Proteolytic and amylolytic activities exhibited a moderate positive correlation Interestingly, total lipolytic and lecithinase activities were positively correlated with each other but negatively correlated with proteolytic and amylolytic activities. The isolated Bacillus spp. strains generally displayed low lipolytic activity, observed in only a small subset of strains, as was the case for lecithinase activity. Lecithinase belongs to the lipase group (also known as phospholipase C), which explains the correlation between lecithinase activity and total lipolytic activity. Additionally, lecithinase is recognized as a virulence factor [48]. This suggests two alternative life strategies of Bacillus: either active uptake of nutrients or adaptation to pathogenicity.

Antioxidant and pro-oxidant activities were not correlated. At first glance, this may seem unusual; however, some strains simultaneously produced both antioxidant and pro-oxidant metabolites, which was reflected in the arrangement of variable vectors in the loading plot.

Cluster analysis showed that Bacillus spp. strains isolated from bottom sediments can be classified into three distinct clusters. Cluster #2 (Figure 2c) comprised antimicrobial bacilli and strains exhibiting pronounced antioxidant properties. Cluster #3 (#3 in Figure 2c) comprised bacilli with high levels of extracellular enzyme. The last cluster (#1 in Figure 2c) consisted of strains lacking prominent traits, although many displayed lipolytic and lecithinase activities.

These findings suggest the existence of three potential ecological strategies among Bacillus spp. from bottom sediments: (1) growth and development driven by active exoenzyme production, enabling rapid utilization of complex organic compounds entering the sediment; (2) competitive advantage through the expression of additional traits, including antimicrobial activities targeting competitors or antioxidant activities conferring protection against reactive oxygen species; and (3) advantage via conditional pathogenicity and the capacity to utilize other organisms as habitats. Our study focused on Bacillus belonging to the first two groups.

4.4. Efficacy of Strains in In Vivo Experiments

For further studies, eleven strains exhibiting either enzymatic activity, high antioxidant and antimutagenic activity, or antimicrobial properties were selected. These strains correspond to the prominent peaks in the violin plot (Figure 1), surpassing test strains B. subtilis KATMIRA 1933 and B. amyloliquefaciens B1895 in the manifestation of potential probiotic properties.

Some of the selected strains underwent safety testing and sequencing and were used for the formulation of probiotic preparations. These preparations were evaluated in experiments on sterlet (Acipenser ruthenus Linnaeus, 1758) and carp (Cyprinus carpio Linnaeus, 1758) [49,50]. Although comprehensive results are not presented here and can be found in the cited references, it is notable that B. velezensis strains MT14, MT42, MT141, and MT142—characterized by pronounced proteolytic and amylolytic activities—significantly increased biomass growth in carp juveniles. B. velezensis strains MT14 and MT42, as well as B. subtilis MT48 and MT74, promoted biomass increase in juvenile sterlet, accompanied by upregulated expression of the interleukin-1 beta (IL-1β) gene, which is associated with anti-inflammatory effects. Additionally, B. subtilis strains MT48 and MT74 reduced the expression of genes encoding insulin-like growth factor 1 (IGF-1), 70 kDa heat shock proteins (HSP70), and glutathione S-transferases (GST), suggesting a potential role of their antioxidant activity in stress alleviation.

These findings indicate that Bacillus spp. strains isolated from bottom sediments possess significant probiotic potential and may exert stimulatory effects in in vivo tests with live fish.

5. Conclusions

This study evaluated the potential of bottom sediments as a promising source of probiotic Bacillus strains, offering advantages over isolation from fish intestines. We conducted a comprehensive phenotypic analysis of the isolated Bacillus spp. strains, including assessments of their proteolytic and amylolytic activities, as well as antioxidant and DNA-protective properties. Additionally, the antimicrobial activity of these microorganisms was investigated. Principal component analysis was employed for integrative data description and dimensionality reduction, allowing the identification of key parameters characterizing the functional potential of Bacillus strains from bottom sediments. Based on the results, it can be concluded that bottom sediments represent a valuable and efficient reservoir for isolating probiotic Bacillus with a broad spectrum of beneficial biological properties.