2. Probiotics

2.1. Definition and Application

Microorganisms that confer beneficial effects on their host are known as probiotics, a concept that has gained prominence in recent years [14, 15]. El Saadony et al. [16] noted that probiotics are live microorganisms introduced into the GIT through food or water, which help restore the microbial balance and enhance overall health. The gut microbiota produces metabolites that regulate immune response and metabolism. Accordingly, modulating the intestinal microbiota has emerged as a novel and increasingly significant approach to enhancing health and immunity, attracting considerable scientific attention. For instance, the intestinal microbiota metabolizes short‐chain fatty acids (SCFAs). These SCFAs strengthen the epithelial barrier and can reach distant organs, act on antigen‐presenting cells, and help attenuate inflammation in various diseases [17].

2.2. Sources of Probiotics

Potential probiotic microbes can be isolated from various sources. These include the intestines of healthy fish, aquaculture water, sediment from culture tanks, other aquatic organisms, and various fermented food items [18]. While the effectiveness of commercial probiotics remains ambiguous in the existing scientific literature, research indicates that host‐derived probiotics have significant potential to modulate the microbiota of aquatic organisms ex vivo [19]. These commensal bacteria are integral to the innate immune defenses of healthy hosts [20] and offer a more targeted approach [21]. Non‐marine sources of probiotics include fruits, vegetables, cereals, legumes, and pulses. Lactobacillus plantarum, Bifidobacterium animalis subsp lactis (commonly referred to as Bifidobacterium lactis), and Saccharomyces cerevisiae are effective non‐dairy probiotic microorganisms. These probiotics can improve gut health, help manage inflammatory bowel disease (IBD), and support immune function. The effectiveness, however, depends on the viability and stability of the microorganisms within various food matrices, highlighting the need to maximize their health benefits [22]. In parallel, research has been conducted on probiotic microorganisms isolated from aquatic species, including shrimp and finfish, as well as from terrestrial animals and their cultures. Within aquaculture, the effectiveness of these probiotics in preventing and controlling infectious diseases has been evaluated. The use of such specialized sources can enhance growth performance, improve immune responses, and offer a sustainable alternative to antibiotics in aquaculture [19].

2.3. Selection of Probiotic Strains

The primary objective of probiotic application in marine species is indeed to conserve the beneficial ecological balance between commensal and pathogenic bacteria in their skin mucus and alimentary canals. By modulating the immune responses and improving disease resistance, probiotics can help maintain mucosal homeostasis [23]. This application should also safeguard the surrounding environment, such as water and sediment quality. Historically, the choice of probiotic bacteria has typically been made on an ad hoc, trial‐and‐error basis with a limited scientific rationale. This lack of stringent selection has led to many research failures. While established selection methods are available, they are not universally applicable due to the diverse host species and environmental contexts. Thus, the selection of potential probiotics and their mode of action must be clearly defined [24]. Key determinants of these selection criteria include biosafety considerations, production and processing methods, delivery systems, and the anticipated sites of microbial activity within the host. The selected probiotic strain ought to exert a verifiable beneficial effect on the host animal, such as increased growth or resistance to disease. Additionally, it is crucial that these strains remain effective during production, manufacturing, distribution, and storage [25]. Given that the beneficial effects of many species are species‐specific, it is ideal to isolate candidate bacteria from the same species for which they are intended to be used on. This approach guarantees a better biological fit for specific conditions in the animal’s digestive system [26]. When used as feed additives, probiotic cultures must withstand pelleting conditions, humidity during transportation and storage of feed, and potentially aggressive environments with the risk of exposure to mycotoxins or heavy metals. Probiotics added to feed and premixes should have a high viability period of at least 4 months. Formulations often require encapsulation to ensure an adequate amount of viable probiotic strains reaches the host’s GIT [27]. Safety, efficiency, and technical feasibility are vital to the selection process. Most importantly, selected probiotics should not harbor plasmid‐encoded antibiotic resistance genes. They should also tolerate a broad pH range and bile salt concentrations >2.5% [28]. Addressing these factors can enhance the effectiveness and reliability of probiotics in aquaculture and animal husbandry (Figure 1).

Figure 1: The selection criteria for probiotics.

2.4. Types of Probiotics

Currently, the market offers a variety of commercial probiotic products derived from diverse sources, including bacterial strains, yeast, and microalgae. Among these, gram‐positive bacteria generally demonstrate greater efficacy than gram‐negative ones. The most widely recognized probiotic strains belong to the category of lactic acid bacteria (LAB) group, with notable examples including Bifidobacterium and Streptococcus. Although still a relatively novel concept in aquaculture, the use of probiotics has garnered significant interest for their potential to regulate physiological processes in aquatic species. Several strains, such as Lactobacillus helveticus, Aeromonas media, Bacillus subtilis, and Enterococcus faecium, have been identified as highly effective. Additionally, it is noteworthy that the GIT of fish and shellfish harbors gram‐negative facultative symbiotic anaerobes, including Vibrio, Pseudomonas, Plesiomonas, and Aeromonas, which may also serve as promising probiotic candidates [29].

2.4.1. Bifidobacterium Species

Several species of the Bifidobacterium genus have been identified and approved as potential probiotics. These anaerobic bacteria, which produce antimicrobial proteinaceous substances, are part of the intestinal microbiota of fish and mammals [30]. For example, the probiotic Bifidobacterium animalis subsp. lactis BB‐12 (BB‐12) has been well established and is among the most studied due to its health benefits. This strain has been shown to enhance immune responses, assist in the prevention of diarrhea, and reduce the likelihood of constipation. Due to its widespread and clinical food industry use, understanding its developmental properties is crucial for any product based on this strain [31]. For efficient BB‐12 strain culture and preservation, the choice of culture media is essential. Among these compound reagents used in the preparation of the culture media, nitrogen plays a particularly significant role, serving not only as a growth precursor but also as a substrate for the bacterium’s metabolic pathways. This allows strain BB‐12 to produce diverse metabolites, such as vitamins, SCFAs, and antibiotic compounds, that may contribute to its health benefits [32]. Treatment with Bifidobacterium lactis BL‐99 significantly reduced intestinal inflammation in adult zebrafish (Danio rerio), increased the number of goblet cells, and produced several beneficial metabolites [33]. Immune cells have been reported to be activated by B. longum [34], and when supplemented at 107 CFU/g in diets, B. longum improved Penaeus vannamei’s growth performance [35].

2.4.2. Enterococcus Species

Enterococci, a group of LAB, are considered potential probiotic candidates for aquafeed. They exhibit resistance to stomach acidity and bile salts, possess strong epithelial binding properties, and show immunomodulatory and antagonistic effects against pathogens [36]. Their ability to produce bacteriocins makes them an attractive alternative to the classical antibiotics. For instance, Enterococcus faecium significantly decreased mortality in European eels (Anguilla anguilla) exposed to Edwardsiella tarda [37]. Enterococcus casseliflavus is a promising probiotic for rainbow trout culture due to its impact on immune stimulation and growth performance [38]. According to Akbari et al. [36], a bacteriocinogenic strain of Enterococcus mundtii was isolated from the intestinal contents of Hemiodema spectabilis collected from the Patagonian coast of Argentina. In this study, the marine‐derived strain demonstrated antibacterial activity, showing resistance to most antibiotics tested except cephalothin while exhibiting activity against various pathogens, including Listeria innocua and vancomycin-resistant enterococci. However, the ability of Enterococcus species to develop multiple antibiotic resistances has drawn increasing attention [39].

2.4.3. Lactococcus Species

Lactococcus lactis is a major strain of LAB recognized as safe gram‐positive bacterium. It plays an important role in maintaining intestinal microbiota balance and promoting the immune function of animals [40]. L. lactis is well known for its special uses in improving animal feed, fermenting milk, and producing vaccines [41]. Supplementing L. lactis PH3‐05 at a dose of 106 CFU/g diet significantly stimulated the growth, survival, and digestive status of A. tropicus larvae [42].

2.4.4. Lactobacillus Species

Lactobacillus species are gram‐positive bacteria known to improve immune responses and guard against upper respiratory and gastrointestinal diseases. The protection of fish against pathogen infections is a key benefit of utilizing Lactobacillus [43]. Adhesion is regarded as a beneficial characteristic for a probiotic strain as it can improve the residency of probiotics in the gut and their interactions with the host’s epithelium and immune cells; Lactobacillus species are known to possess this particular trait [44]. Among them, L. fermentum strains have been reported to generate a variety of strong antimicrobial peptides usable as antibiotic substitutes or as food preservatives [45]. An investigation of Lactibacillus plantarum CLY‐05’s effects specifically on sea cucumber (Apostichopus japonicus) demonstrates increased body weight and improved non‐specific immune enzyme activities [46]. As a result of improving intestinal health and integrity, Lactiplantibacillus plantarum E2 significantly increased large yellow croaker (Larimichthys crocea) survival and growth rates. Its supplementation increased intestinal enzyme activities (amylase, trypsin, and lipase) and improved gut microbiota composition by decreasing harmful Sphingomonas while increasing beneficial Lactobacillus and Pseudomonas. In addition, E2 supplementation enhanced disease resistance against Pseudomonas plecoglossicida, suggesting its potential as a probiotic to promote the growth and health of large yellow croaker [47]. According to Feyereisen et al. [48], Levilactobacillus brevis is a heterofermentative, Gram‐positive LAB that thrives at pH 4–6 and 30°C. Additionally, it has been demonstrated that certain strains of L. brevis are able to produce biogenic amines, including tyramine and phenylethylamine [49]. The majority of L. brevis strains exhibit strong antibiotic and proteolytic activity as well as adhesive qualities to GIT cells [50]. Cell‐free supernatants of L. brevis isolates demonstrated strong immunostimulatory effects on Pikeperch and Carp immune cells [51]. Gram‐positive, nonspore‐forming, nonmotile, microaerophilic microbes with a high tolerance to acidic pH conditions make up the species Lactobacillus delbrueckii [52]. L. delbrueckii has an obligately homofermentative metabolism, producing primarily lactic acid, with an ideal growth temperature of 42°C [53]. Furthermore, strains of L. delbrueckii can be isolated from a variety of ecological sources, such as vegetables, dairy products, fermented foods, and human tracts [54]. According to Silvi et al. [55], L. delbrueckii shows a strong ability to colonize the gut of sea bass, altering the gut microbiota, which translates into higher chances of survival. Cyprinus carpio growth, gastrointestinal enzyme activities, and growth‐related gene expression can all be enhanced by supplementing L. delbrueckii in diets at ~1 × 106 CFU/g [56].

2.4.5. Bacillus Species

One of the most commonly used probiotic genera known to produce extracellular enzymes is the Bacillus species. These species are known to produce spores that are impervious to physical and chemical conditions; thus, they are able to withstand the harsh conditions met during feed processing. Juvenile coral trout grouper (Plectropomus leopardus E3S) have been shown to grow more when fed Bacillus cereus [57]. In a study conducted by Yao et al. [58], Bacillus strains were isolated from the intestinal tract of healthy large yellow croaker (Larimichthys crocea), and four strains with protease and lipase activities were identified. Among these, Lysinibacillus sp. strain LYD11 exhibited strong inhibitory activity against two major aquaculture pathogens, Vibrio harveyi and Vibrio alginolyticus. Additionally, strain LYD11 demonstrated effective coaggregation and adhesion exclusion capabilities, suggesting its potential as a promising probiotic candidate for disease prevention in aquaculture, particularly against common bacterial infections [58]. Also, Bacillus licheniformis ATCC 11946 supplementation at a dose of 108 CFU/g diet was reported to enhance the growth, proximate body composition, serum and hepatic immune and antioxidant enzyme activities, intestinal morphology, and assembly of intestinal microbiota by yielding more beneficial bacteria and reducing pathogenic bacteria in Litopenaeus vannamei, which translated to shrimp protective capacity against Vibrio parahaemolyticus [59]. Numerous studies have identified the Gram‐positive bacterium Bacillus subtilis as a traditional probiotic for aquaculture species [60]. Research indicates that B. subtilis enhances host immunity against bacterial infections and viral diseases while also playing a role in immunomodulation [61]. B. subtilis strain HGCC‐1 has been shown to positively influence liver health in golden pompano by regulating the intestinal microbiota, reducing hepatic steatosis, and promoting growth performance [62]. It was found that dietary Bacillus licheniformis significantly improved the physiology and immunity of the sea cucumber (Apostichopus japonicus). Growth performance, digestive and immune enzyme activity, and vibriosis resistance were enhanced as a result of supplementation. By combining Bacillus licheniformis with herbal extracts, the microbial community was optimized by promoting beneficial bacteria and inhibiting pathogens, resulting in reduced cumulative mortality rates and improved overall health [63].

2.4.6. Clostridium Species

Clostridium butyricum is primarily used in the treatment of intestinal inflammation associated with dysbiosis or imbalances in the intestinal flora [16]. The therapeutic benefits of C. butyricum include the repair of intestinal damage, enhancement of nutrient absorption and digestion, and modulation of the immune response [64]. Incorporation of C. butyricum at concentrations ranging from 107 to 1011 CFU/kg improved the growth performance of giant yellow croakers (Larimichthys crocea) [65]. It was identified that Clostridium species were present in Pacific white shrimp (Litopenaeus vannamei), including Clostridium subterminale, Clostridium beijerinckii, Clostridium butyricum GC subgroup A, and Clostridium bifermentans GC subgroup A. Clostridium spp. were detected in 16 strains. In shrimp intestines, these anaerobic bacteria produce enzymes for carbohydrate and protein breakdown, which contribute to the absorption of nutrients [66].

2.4.7. Shewanella Species

Numerous studies have highlighted the beneficial effects of Shewanella species as probiotics in aquaculture. For example, S. putrefaciens (strain Pdp11 or more recently as SpPdp11), isolated from healthy gilthead seabream, has shown probiotic potential for this species [67]. Additionally, Shewanella sp. MR‐7, isolated from the turbot fish gut, significantly enhanced their intestinal health [68]. In Pacific white shrimp, it improved growth performance, bolstered immunity, and increased the population of beneficial gut bacteria. Supplemented animals exhibited greater weight consistency and higher total protein concentrations, alongside elevated alkaline phosphatase activity and enhanced non‐specific immune responses. Furthermore, the administration of S. putrefaciens Pdp11 from the onset of exogenous feeding positively influenced the larval development of Senegalese sole (Solea senegalensis) [69]. Feeding Shewanella haliotis to Litopenaeus vannamei significantly increased respiratory burst activity [70] and Shewanella colwelliana, according to Jiang et al. [71], enhanced abalone’s innate immunity and tolerance to diseases.

2.4.8. Yeast and Algal Species

In recent years, yeasts have gained increasing attention for their potential health benefits as probiotics. Beyond the well‐studied Saccharomyces boulardii, scientists are actively searching for new yeast strains with probiotic qualities, including species such as Debaryomyces hansenii, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia hudriavzevii, and Torulaspora delbrueckii. These are isolated from diverse sources such as traditional fermented foods, the human gut, and natural environments. Research suggests that some of these yeasts could offer support in managing IBD, irritable bowel syndrome, skin conditions, and allergies. Yeast probiotics, such as Debaryomyces hansenii, are utilized in aquaculture to enhance growth, modulate the gut microbiota, and improve the health of farmed fish. They promote somatic growth, improve feed efficiency, and stimulate immune responses without disrupting the intestinal cell organization. D. hansenii has shown the ability to reduce opportunistic Proteobacteria while enhancing beneficial microbial populations, contributing to a healthier intestinal environment. This makes yeast probiotics a valuable addition to sustainable aquaculture feeding strategies [72]. Studies indicate that S. cerevisiae positively affects growth rates, feed efficiency, and water quality in various fish species. It works by stimulating the immune system, altering microbial metabolism, and competing with pathogens for resources [73]. Microalgae, such as Dunaliella salina, Dunaliella tertiolecta, Isochrysis galbana, and Tetraselmis suecica, have been shown to improve growth, health, and survival rates in aquatic animals. These algal probiotics contribute beneficial nutrients and enhance the immune response of cultured species. Their incorporation into aquaculture practices can improve the overall performance and suitability, underscoring their potential as effective probiotics alongside traditional bacterial strains [74].

2.5. Mode of Action of Probiotics

Probiotics play a crucial role in promoting fish health within aquaculture by engaging various biological pathways. They are vital for regulating the gut microbiota and maintaining microbial balance by facilitating the colonization of beneficial bacteria while inhibiting the growth of harmful pathogens through a process known as competitive exclusion. By obstructing pathogens’ access to essential nutrients and binding sites on the gut’s surface, probiotics significantly reduce the incidence of bacterial infections. In addition to combating infections, probiotics produce antimicrobial compounds, including bacteriocins and organic acids, that further suppress the growth of harmful bacteria. They also bolster the host’s immune system, enhancing innate and adaptive immune responses. The primary biological mechanisms by which probiotics exert their effects include increased adhesion to the intestinal mucosa, improved epithelial barrier function, inhibition of other pathogenic microbial adhesion, competitive exclusion of pathogenic microorganisms, production of antimicrobial substances, and modulation of the immune system [75].

The synthesis of inhibitory compounds is another critical aspect of probiotics’ functionality. Among various substances, probiotic bacteria generate bacteriocins, hydrogen peroxide, siderophores, lysozymes (LYZs), and proteases, all of which exhibit bactericidal or bacteriostatic effects on competing microbial populations [76]. Moreover, certain bacteria produce volatile fatty acids and organic acids, such as lactic, acetic, butyric, and propionic acids. These compounds can lower the pH of the gastrointestinal lumen, inhibiting the growth of opportunistic pathogenic microbes [77].

Another critical mechanism of action is the competition for nutrients and available energy. The ability of microbial populations to compete with other organisms in the same environment for essential chemicals and energy sources is vital for their survival [6]. Various microorganisms, including the well‐known probiotic group of LAB, consume nutrients necessary for the growth of certain pathogens. This competitive dynamic can significantly influence the composition of the gut microbiota or the culture water of aquatic organisms. Improving water quality is another important aspect. Utilizing Gram‐positive bacteria, such as Bacillus species, can enhance the quality of aquatic systems. Unlike Gram‐negative bacteria, which tend to convert a larger proportion of organic matter into bacterial biomass or slime, Bacillus species are more efficient at converting organic matter into carbon dioxide [78]. Furthermore, some probiotic bacteria exhibit strong algicidal effects, particularly against various microalgal species. The application of nitrifying cultures in fish habitats also plays a crucial role in mitigating the toxicity of nitrite and ammonia [78]. Likewise, they bolster fish immune systems, aiding the development of natural resistance and improving the survival rates of fish larvae and post‐larvae. Ibrahem [79] highlights the varied stimulatory effects of probiotics on fish immune systems, including enhanced immune cell activity, antibody production, acid phosphatase (ACP) levels, LYZ activity, and the synthesis of antimicrobial peptides. Additionally, quorum sensing is a sophisticated communication mechanism employed by bacterial cells to interact both within their own species and with other species, particularly when their population density surpasses a specific threshold. This intricate process is initiated by the release of signaling molecules known as autoinducers [80]. These autoinducers bind to receptor proteins, triggering the quorum‐sensing transduction pathway. This pathway subsequently activates the expression of genes associated with pathogenicity, spoilage, and biofilm formation. It has been suggested that metabolites produced by probiotics may possess the ability to inhibit quorum sensing [81]. Through coordinated communication, bacteria can effectively regulate collective behaviors that enhance their survival and adaptability in various environments. This innovative approach underscores the potential of probiotics to improve fish health and contribute to sustainable aquaculture practices. Figure 2 illustrates the mode of action of probiotics in aquatic animals.

Figure 2: Mode of action of probiotics in aquatic animals.

2.6. Beneficial Effects of Probiotics

2.6.1. Growth Performance and Immune Response

Incorporating probiotics and prebiotics into aquaculture practices has been shown to increase growth rates and enhance feed conversion ratios (FCR). Probiotics facilitate better nutritional absorption and digestion, enabling farmed fish to utilize feed more efficiently and to accumulate greater biomass. Furthermore, probiotics contribute to the gut’s structural integrity by increasing the villus height and surface area, thereby enhancing nutrient absorption and digestion efficiency and improving growth performance. They also promote microbial balance and reduce intestinal inflammation, benefiting fish health and improving feed consumption [82] (Table 1).

Probiotics | Host animal species | Duration of experiment | Dosage | Region | Effects | References
Bacillus subtilis BS1 and Lactobacillus plantarum | Chinese Perch (Siniperca chuatsi) | 8 weeks | 1.0 × 108 CFU/g | Huazhong, China | Enhance growth performance and gut health by regulating gut microbiota | [83]
Saccharomyces cerevisiae and Lactobacillus bulgaricus | Chelon ramada (Mugil capito) | 60 days | S. cerevisiae, 4 g/kg diet + L. bulgaricus, 2 g/kg diet) | Kafr Elsheikh, Egypt | Improve growth, body composition, antioxidant defenses, and hepatic and intestinal health | [84]
Bacillus subtilis, Bacillus licheniformis, and Trichoderma longibrachiatum | Rohu (Labeo rohita) | 8 weeks | 0.25, 0.50, and 0.75 g/kg | Kasur, Pakistan | Enhance growth, immunity | [85]
Bacillus halophilus | Large yellow croaker (Larimichthys crocea) | 60 days | 108 CFU/mL | Fujian, China | Improve growth, antioxidant immunity ability, and promote the expression of growth‐related metabolites | [86]
Bacillus subtilis, Enterococcus sp., Lactobacillus casei, Lactobacillus rhamnosus, Saccharomyces cerevisiae, and Saccharomyces boulardii | Pacific white shrimp (Penaeus vannamei) | 75 days | 5 × 109 CFU/mL−1 | Tamil Nadu, India | Enhance growth and immunity | [87]
Lactobacillus plantarum and Saccharomyces cerevisiae | Japanese grenadier (Coilia nasus) | 120 days | 1.0 × 108 CFU/g Lactobacillus plantarum: Saccharomyces cerevisiae, 9:1 | Yangzhong, China | Enhance lipid metabolism, suppress fatty acid synthesis, and reduce oxidative stress and inflammation | [88]
Bacillus licheniformis | Sea bass (Lates calcarifer) | 8 weeks | 1 × 106/CFU/g−1 | Santa Maria RS, Brazil | Improve growth, chemical composition of fish, activity of the liver, and digestive enzymes | [89]
PrimaLac | Caspian white fish (Rutilus frisii kutum) | 45 days | 1% | Tehran, Iran | Elevate immune markers for skin mucus | [90]
Clostridium butyricum | Large yellow croaker (Larimichthys crocea) | 30 days | 5 × 109 CFU/g−1 | Qingdao, China | Promote growth | [65]
Clostridium butyricum | Sea Cucumber (Apostichopus japonicus) | 63 days | 1% | Luoyang, China | Enhance growth promotion and nonspecific immunity | [91]
Pseudoalteromonas piscicida | Pacific white shrimp (Litopenaeus vannamei) | 14 days | 10 g/L | Dramaga Bogor, Indonesia | Improved growth performances, immune responses, and disease resistance | [92]
Enterococcus faecium | Shark catfish (Pangasianodon hypophthalus) | 56 days | 2.0 × 1011 CFU | Alexandria, Egypt | Modulates growth, digestive enzymes, immunity, hepatic antioxidant activity, and disease resistance | [93]
Bacillus velezensis V4 and Rhodotorula mucilaginosa | Atlantic salmon (Salmo salar L.) | 56 days | 106 CFU/g−1 | Qingdao, China | Improved growth, immune response, antioxidant capability, and disease resistance | [94]
Clostridium butyricum | Giant river prawn (Macrobrachium rosenbergii) | 60 days | 2 × 109 CFU/g−1 | Khulna, Bangladesh | Promote growth performance, digestive enzyme activity, and immune response | [95]
Bacillus megaterium PTB 1.4 | Catfish (Clarias sp.) | 30 days | 1010 CFU/mL−1 | Bogor, West Java, Indonesia | Enhanced protease and amylase activities | [96]
Debaryomyces hansenii CBS 8339 | Rainbow trout (Oncorhynchus mykiss) | 37 days | 106 CFU/g−1 | Plouzane, France | Promote larval development | [97]
Bacillus licheniformis TC22 | Sea cucumbers (Apostichopus japonicus) | 30 days | 109 CFU/g | Yantai, China | Improve immunity and disease resistance | [98]
Bacillus subtilis and licheniformis | Japanese eel (Anguilla japonica) | 12 weeks | 1 × 108 CFU/g | Busan, Korea | Enhance growth | [99]
Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Escherichia coli, and Candida albicans | African catfish (Clarias gariepinus) | 60 days | 2 mL/kg | Addis Ababa, Ethiopia | Enhance microbial populations | [100]
Bacillus sp., Streptococcus faecalis and Clostridium butyricum | Nile tilapia (Oreochromis niloticus) | 75 days | Basal diet + probiotics, at 10 g/kg | Mymensingh, Bangladesh | Improve gut microbiota and intestinal morphology | [101]

Probiotics also act as immunostimulatory agents, helping mitigate the effects of disease and reduce pathogen entry, thereby providing essential protection for cultured aquatic organisms [29]. Utilizing probiotics as immunostimulants is a highly effective strategy for enhancing the success of aquaculture operations. Research indicates that after being exposed to Pseudomonas and Aeromonas hydrophila, probiotic Staphylococcus edaphicus successfully boosts the immune response of Kelabau fish (Osteochilus melanopleurus), enhancing blood parameters and survival rates [102]. Furthermore, Staphylococcus sp. strain JC20 has demonstrated cellulolytic properties and probiotic potential in red tilapia without adverse effects [103] (Table 1).

2.6.2. Probiotics on Water Quality Improvement

Ensuring high‐quality water is essential for the optimal performance of aquatic species, and probiotics are noted to play a crucial role in this process. To inhibit the growth of harmful bacteria, probiotics release various byproducts like lactic acid, hydrogen peroxide, and bacteriocins [104]. The hydrogen peroxide released by probiotic bacteria triggers chain reactions of superoxide anions, resulting in a powerful oxidizing effect with bactericidal properties [105]. Studies show that by maintaining pH and reducing harmful ammonia and nitrite levels, mixed Bacillus amyloliquefaciens probiotics significantly improve water quality during white shrimp aquaculture [106]. Adding Bacillus subtilis to shrimp farms at a concentration of 1010 CFU/mL has been shown to lower total ammonia levels and improve water quality [106]. A high concentration of probiotics in fish ponds plays a crucial role in balancing phytoplankton production and mitigating the accumulation of dissolved and particulate matter throughout the growing season [19]. Probiotics also release lactic acid, which can completely inhibit pathogens at 0.5% (w/v) and create an unfavorable environment for unwanted microbes in the culturing environment [107]. Also, probiotics can release nitric oxide (NO) that helps regulate biofilm formation [104]. In addition to these benefits, many probiotic bacteria have been found to have significant algicidal effects on microalgae [108]. Nitrifying bacteria play a crucial role in removing harmful ammonia and nitrite from water and improving the water’s overall microbial composition. In aquaculture water, aerobic denitrifiers are considered effective in converting nitrate and/or nitrite into N2 [109].

Studies have shown that treating Penaeid culture with Bacillus pumilus can improve water quality measures and provide protection against marine pathogens, such as Vibrio spp. [110]. By producing spores and metabolites that inhibit pathogenic microbes, probiotics can effectively mitigate water contamination and maintain a suitable water quality for aquaculture activities. In conclusion, probiotic bacteria play a crucial role in enhancing aquaculture water quality by regulating algal growth and inhibiting the growth of pathogenic microbes (Table 1). This can lead to increased productivity and overall success in aquaculture operations [111]. The diagram presented in Figure 3 illustrates the positive impact of probiotics and prebiotics in water.

Figure 3: The positive impact of probiotics and prebiotics in water. This figure demonstrates the beneficial effects of incorporating probiotics and prebiotics into water sources.

2.7. Probiotics and Bioremediation

Beyond serving as feed supplements, certain probiotics also function as bioremediation agents, breaking down toxic compounds in the environment. Bioremediation relies on microorganisms, including bacteria, fungi, yeast, and algae, as well as microbial byproducts, to degrade, reduce, or eradicate harmful environmental substances. In some cases, phytoremediation, which uses plants, is also employed in certain situations [112]. During bioremediation, microorganisms produce enzymes that transform toxic chemicals through biotransformation. This often leads to biodegradation, where pollutants are broken down into simpler structures, ultimately resulting in harmless and non‐toxic byproducts, a process referred to as mineralization. Several bacterial species, including Bacillus, Pseudomonas, Acinetobacter, Cellulomonas, Rhodopseudomonas, Nitrosomonas, and Nitrobacter, are recognized for their effectiveness in remediating organic waste [113]. For example, De Paiva‐Maia et al. [114] studied the effects of a commercial probiotic on bacterial and phytoplankton densities in intensive Litopenaeus vannamei farming using a recirculating method. In 2.6‐hectare ponds stocked at 98 shrimp/m2, probiotics were applied weekly starting 7 days before stocking and continued throughout the 16‐week experimental period. Probiotics were found to greatly influence the levels of heterotrophic bacteria and Pyrrophyta in sediment, ultimately enhancing the overall environmental conditions. In another study, Hassan et al. [115] examined two probiotic bacteria for bioremediation in earthen ponds stocked with Pangasius sutchi, Catla catla, and Labeo rohita for a year. Probiotic application reduced ammonia, nitrite, and phosphate levels in treated ponds, indicating effective remediation. The study showed that Bacillus spp. produced digestive enzymes such as protease, amylase, and lipase, which helped suppress pathogenic Vibrio species without harming shrimp post‐larvae. Figure 4 displays various types of bioremediation techniques.

Figure 4: Illustration of the various methods of bioremediation, which use biological organisms to remove or neutralize contaminants in the environment.

2.8. Recommendation and Factors Affecting Probiotic Effectiveness

The type and quantity of probiotic strains used can significantly influence the effectiveness of probiotic formulations. Several factors contribute to the efficacy of these formulations, including the selection of optimal strains and the appropriate dosage that ensures enough viable cells. Formulations must be stored and handled according to the manufacturer’s instructions to preserve the viability of these live microorganisms as they are sensitive to adverse conditions such as temperature and light [116]. When administering probiotics, it is essential to avoid concurrent use of other medications and to ensure that the dilution water is free of disinfectants, like chlorine. It is recommended that animals receive the probiotic‐infused water within 6–12 h [26]. Furthermore, a 24–48 h waiting period is recommended after antibiotic or other antimicrobial treatment before initiating probiotic therapy. Generally, the most effective probiotic formulations contain a diverse array of ingredients, incorporating a greater number of microbial species. The method of administration is also crucial; common approaches include injection, ingestion, encapsulation, or immersion in water. In aquaculture, effective probiotics should exhibit strong adhesion or colonization capabilities or reflect the microbial communities in the fish mucosa [28, 117, 118].

It is important to recognize that probiotics designed for aquatic animals are distinct from those used in terrestrial systems due to fundamental ecological and physiological differences [56]. While certain bacteria may be harmful to one aquatic species, they can serve as beneficial probiotics for others. In recent decades, the performance of aquatic animals has seen significant improvement using this biological control agent. It is crucial to consider the optimal conditions for probiotics to survive, colonize, proliferate, and effectively provide their benefits to hosts in a specific environment. The notion of a ‘one size fits all’ approach does not apply to probiotics, as specific strains or species are required for target species in particular environments.