**2. Probiotics establish a balanced aquaculture environment management**

### **2.1 Probiotics in aquaculture**

The biggest challenge faced by the aquaculture industry is the malignant changes in the farming environment, whether it is changed by man-made or natural conditions. Aquaculture farming environmental degradation often leads to the accumulation of toxic substances or the dominance of pathogenic microorganisms. Since the industrial operator has only paid attention to the improvement of technology and production yield for a long time, and neglected how to maintain the ecological balance of the culture pond and the surrounding environment, when a disease breaks out, the first strategy is to apply animal drugs, especially antibiotics; undeniably, drug treatment for a disease is the most direct and immediate practice [5], but its damage to the environment is difficult to estimate. In the aquaculture industry, the negative impact of the application of drugs on the environment has received attention increasingly. In addition, drug residues cause food safety concerns and may lead pathogenic microorganisms to become drug resistant [5], which has an irreversible effect on nutrients in the culture environment [6].

So, the main purpose of adding probiotics or microbial products in the aquaculture pond is to decompose organic substances and at the same time to inhibit the growth of pathogenic microorganisms, reduce the risk of disease occurrence [7], convert toxic substances, reduce the accumulation of organic matter, stabilize the water color, and balance ecosystem in water. In the early days, the definition of probiotics was limited to microorganisms that were beneficial to the health of the organism. With the continuous revision, the current broad definition of probiotics not only increases the health of the organism but also improves the survival rate and the quality of the water, improving the immune mechanism of animals and even stabilizing the balance of the bacteria in the body [8], which are all functions of probiotics, and these benefits are very important for the aquaculture process, especially when the fry of aquatic animal are incubated, using probiotics as microbial additives to improve the final aquaculture production yield has important benefits [9].

Microbial additives usually do not have only a single strain. A single strain will have some disadvantages in the treatment of the aquaculture environment. A single strain usually only has the ability to handle certain substances, and the organic components in the aquaculture water are extremely complicated. Although the single strain treatment will change the figures or water quality numbers in the research, the improvement of the overall water quality is not great, so the addition of compound strains has become one of the important concepts of microbial additives. It is one of the important goals of microbial additives to treat different wastes in aquaculture environment with different strains to achieve the goal of purifying and stabilizing the whole culture system. The classification of probiotics in culture is distinguished by the currently widely used probiotics, which can be distinguished by their efficacy and timing of use: as follows: (i) probiotics for fry incubation (as show in **Table 1**); (ii) probiotics for cultured adult or subadult organisms (as show in **Table 2**); (iii) antipathogenic probiotics screened in the environment (as show in **Table 3**); and (iv) probiotics that increase the immune mechanism for aquatic animals.

The immune mechanisms of many aquatic animals are not as complete as those of higher mammals, and most aquatic products rely heavily on nonspecific immune mechanisms due to the lack of an active mechanism for antibody immunity. In the case of shrimps, in general, nonspecific immune mechanisms mainly include phagocytosis in the blood cells, nodule formation, encapsulation, and the interaction of other substances in the plasma, which include the cytotoxicity of cytotoxins, complement activated by the action of lectin, and the role of proPO system and

**65**

**Table 1.**

*Probiotics for aquatic animal fry incubation.*

antibacterial protein/peptides [10, 11]. The biggest disadvantages of these effects are in two parts: one is the lack of memory, and the biggest disadvantage of the mechanism of nonspecific immunity is this. Due to the lack of memorable specificity, the phenomenon of repeated infection becomes a big burden in the aquaculture breeding industry. The second disadvantage is that some immune functions usually do not start real time and they need some special activating substances, such as the

*Application of Novel Technology in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.90142* *Emerging Technologies, Environment and Research for Sustainable Aquaculture*

irreversible effect on nutrients in the culture environment [6].

**2.1 Probiotics in aquaculture**

**2. Probiotics establish a balanced aquaculture environment management**

The biggest challenge faced by the aquaculture industry is the malignant changes in the farming environment, whether it is changed by man-made or natural conditions. Aquaculture farming environmental degradation often leads to the accumulation of toxic substances or the dominance of pathogenic microorganisms. Since the industrial operator has only paid attention to the improvement of technology and production yield for a long time, and neglected how to maintain the ecological balance of the culture pond and the surrounding environment, when a disease breaks out, the first strategy is to apply animal drugs, especially antibiotics; undeniably, drug treatment for a disease is the most direct and immediate practice [5], but its damage to the environment is difficult to estimate. In the aquaculture industry, the negative impact of the application of drugs on the environment has received attention increasingly. In addition, drug residues cause food safety concerns and may lead pathogenic microorganisms to become drug resistant [5], which has an

So, the main purpose of adding probiotics or microbial products in the aquaculture pond is to decompose organic substances and at the same time to inhibit the growth of pathogenic microorganisms, reduce the risk of disease occurrence [7], convert toxic substances, reduce the accumulation of organic matter, stabilize the water color, and balance ecosystem in water. In the early days, the definition of probiotics was limited to microorganisms that were beneficial to the health of the organism. With the continuous revision, the current broad definition of probiotics not only increases the health of the organism but also improves the survival rate and the quality of the water, improving the immune mechanism of animals and even stabilizing the balance of the bacteria in the body [8], which are all functions of probiotics, and these benefits are very important for the aquaculture process, especially when the fry of aquatic animal are incubated, using probiotics as microbial additives to improve the final aquaculture production yield has important benefits [9]. Microbial additives usually do not have only a single strain. A single strain will have some disadvantages in the treatment of the aquaculture environment. A single strain usually only has the ability to handle certain substances, and the organic components in the aquaculture water are extremely complicated. Although the single strain treatment will change the figures or water quality numbers in the research, the improvement of the overall water quality is not great, so the addition of compound strains has become one of the important concepts of microbial additives. It is one of the important goals of microbial additives to treat different wastes in aquaculture environment with different strains to achieve the goal of purifying and stabilizing the whole culture system. The classification of probiotics in culture is distinguished by the currently widely used probiotics, which can be distinguished by their efficacy and timing of use: as follows: (i) probiotics for fry incubation (as show in **Table 1**); (ii) probiotics for cultured adult or subadult organisms (as show in **Table 2**); (iii) antipathogenic probiotics screened in the environment (as show in **Table 3**); and

(iv) probiotics that increase the immune mechanism for aquatic animals.

The immune mechanisms of many aquatic animals are not as complete as those of higher mammals, and most aquatic products rely heavily on nonspecific immune mechanisms due to the lack of an active mechanism for antibody immunity. In the case of shrimps, in general, nonspecific immune mechanisms mainly include phagocytosis in the blood cells, nodule formation, encapsulation, and the interaction of other substances in the plasma, which include the cytotoxicity of cytotoxins, complement activated by the action of lectin, and the role of proPO system and

**64**


#### **Table 1.**

*Probiotics for aquatic animal fry incubation.*

antibacterial protein/peptides [10, 11]. The biggest disadvantages of these effects are in two parts: one is the lack of memory, and the biggest disadvantage of the mechanism of nonspecific immunity is this. Due to the lack of memorable specificity, the phenomenon of repeated infection becomes a big burden in the aquaculture breeding industry. The second disadvantage is that some immune functions usually do not start real time and they need some special activating substances, such as the


**Table 2.** *Probiotics for cultured adult or sub-adult aquatic organisms.*


#### **Table 3.**

*Anti-pathogenic probiotics screened in the environment.*

participation of immune-stimulator to initiate the original immune state, so the reaction time is much delayed. This is also one of the reasons for the sudden death of the aquaculture animals.

Recently, many studies have found that the addition of probiotics can improve the immunity of cultured organisms. Such a phenomenon is very helpful for aquaculture organisms. Especially for the cultured organisms of shrimps and crabs, because they mainly rely on nonspecific immune mechanisms, how to

**67**

*Application of Novel Technology in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.90142*

been found [12, 13].

the use of artificial feed.

activate their nonspecific immune system and improve their efficacy has become an important issue in aquaculture management. According to the report, using the source of immune activation, substances such as β-glucan can significantly stimulate the nonspecific immune mechanism of many water-generating substances, and these related polysaccharides are separated from the secondary metabolites of microorganisms. Such results have led to many ideas for making related microorganisms into biological additives, and many studies have confirmed that active yeasts and polysaccharides produced by beneficial bacilli do have positive effects on improving the survival rate and yield of cultured organisms. Feed was added with probiotics or directly added with polysaccharides, and positive benefits in improving the disease resistance and survival rate of cultured organisms have

**2.2 Effects of probiotics on aquaculture environmental indicators**

At present, probiotics are being used in the treatment of industrial wastewater, domestic sewage, animal waste, soil improvement, etc., and their functions in the aquaculture environment are as follows: (i) Inhibiting the occurrence of diseases: beneficial microorganisms can inhibit the growth of pathogenic bacteria by secreting inhibitory substances, competing nutrients, and competing for attachment sites. (ii) Maintaining good water quality: adding beneficial microorganisms can promote the decomposition of organic matter or the conversion of toxic substances, reduce the accumulation of organic matter in the culture pond, and convert toxic substances into nutrient compounds with low toxicity or even reusability, such as ammonia. It is converted to nitrous acid by nitrification and then converted to nitric acid, which can be used for the growth and utilization of plant plankton, which has the effect of stabilizing water quality and balancing the ecosystem. (iii) Improving health and growth: probiotics can improve the bacterial phase in the digestive tract of cultured organisms, promote the digestion and absorption of cultured organisms, help the health and growth of cultured organisms, and increase the survival rate to increase production yield. (iv) Increasing natural bait: the use of photosynthetic bacteria to culture animal plankton can increase the survival rate of brine shrimp, and the added bacteria can be used as a food for protozoa, and the bacteria themselves are a good source of bait to reduce

Probiotics are used in aquaculture environments in the following ways: (i) combined with artificial feed, (ii) put in culture pond water, (iii) immersion, (iv) via live baits, and (v) via medium release. The usage timing can be divided into preculture and postculture. The former is used as the whole pool rearranges with the hope of utilizing the benefit bacteria phase, while the latter is used as the maintenance of the stable culture environment. The additives in feed can be divided into mixed and mixed for fermentation. It is added after fermentation to increase the digestion and absorption rate of the organisms, thereby reducing the excretion and pollution of the water. The addition of biological wastewater treatment system can be regarded as the microbial planting in the water system, which can ensure the stability of the water quality of the system and increase the effect of treatment. The easiest way to use probiotics to treat water quality is to put the microbial products directly into the culture pond. However, this kind of externally introduced probiotics must be made into a dominant species to function, so it must be continued every other day. To achieve the desired results, the probiotics mass and quantity of the products used should be especially confirmed. In addition, the effect of probiotics is quite susceptible to environmental factors such as salinity, temperature, and pH value. Therefore, it is best to activate the probiotics first and

#### *Application of Novel Technology in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.90142*

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

participation of immune-stimulator to initiate the original immune state, so the reaction time is much delayed. This is also one of the reasons for the sudden death

the immunity of cultured organisms. Such a phenomenon is very helpful for aquaculture organisms. Especially for the cultured organisms of shrimps and crabs, because they mainly rely on nonspecific immune mechanisms, how to

Recently, many studies have found that the addition of probiotics can improve

**66**

**Table 3.**

**Table 2.**

of the aquaculture animals.

*Anti-pathogenic probiotics screened in the environment.*

*Probiotics for cultured adult or sub-adult aquatic organisms.*

activate their nonspecific immune system and improve their efficacy has become an important issue in aquaculture management. According to the report, using the source of immune activation, substances such as β-glucan can significantly stimulate the nonspecific immune mechanism of many water-generating substances, and these related polysaccharides are separated from the secondary metabolites of microorganisms. Such results have led to many ideas for making related microorganisms into biological additives, and many studies have confirmed that active yeasts and polysaccharides produced by beneficial bacilli do have positive effects on improving the survival rate and yield of cultured organisms. Feed was added with probiotics or directly added with polysaccharides, and positive benefits in improving the disease resistance and survival rate of cultured organisms have been found [12, 13].

#### **2.2 Effects of probiotics on aquaculture environmental indicators**

At present, probiotics are being used in the treatment of industrial wastewater, domestic sewage, animal waste, soil improvement, etc., and their functions in the aquaculture environment are as follows: (i) Inhibiting the occurrence of diseases: beneficial microorganisms can inhibit the growth of pathogenic bacteria by secreting inhibitory substances, competing nutrients, and competing for attachment sites. (ii) Maintaining good water quality: adding beneficial microorganisms can promote the decomposition of organic matter or the conversion of toxic substances, reduce the accumulation of organic matter in the culture pond, and convert toxic substances into nutrient compounds with low toxicity or even reusability, such as ammonia. It is converted to nitrous acid by nitrification and then converted to nitric acid, which can be used for the growth and utilization of plant plankton, which has the effect of stabilizing water quality and balancing the ecosystem. (iii) Improving health and growth: probiotics can improve the bacterial phase in the digestive tract of cultured organisms, promote the digestion and absorption of cultured organisms, help the health and growth of cultured organisms, and increase the survival rate to increase production yield. (iv) Increasing natural bait: the use of photosynthetic bacteria to culture animal plankton can increase the survival rate of brine shrimp, and the added bacteria can be used as a food for protozoa, and the bacteria themselves are a good source of bait to reduce the use of artificial feed.

Probiotics are used in aquaculture environments in the following ways: (i) combined with artificial feed, (ii) put in culture pond water, (iii) immersion, (iv) via live baits, and (v) via medium release. The usage timing can be divided into preculture and postculture. The former is used as the whole pool rearranges with the hope of utilizing the benefit bacteria phase, while the latter is used as the maintenance of the stable culture environment. The additives in feed can be divided into mixed and mixed for fermentation. It is added after fermentation to increase the digestion and absorption rate of the organisms, thereby reducing the excretion and pollution of the water. The addition of biological wastewater treatment system can be regarded as the microbial planting in the water system, which can ensure the stability of the water quality of the system and increase the effect of treatment. The easiest way to use probiotics to treat water quality is to put the microbial products directly into the culture pond. However, this kind of externally introduced probiotics must be made into a dominant species to function, so it must be continued every other day. To achieve the desired results, the probiotics mass and quantity of the products used should be especially confirmed. In addition, the effect of probiotics is quite susceptible to environmental factors such as salinity, temperature, and pH value. Therefore, it is best to activate the probiotics first and

#### *Emerging Technologies, Environment and Research for Sustainable Aquaculture*

#### **Figure 1.**

*Effectiveness of photosynthetic bacteria applied in white shrimp culture. Change in the total amount of* Vibrio *in the culture pond within 14 days of continuous use of photosynthetic bacteria in the white shrimp culture process. The results showed that in the experimental group with more than 1 ppm of photosynthetic bacteria added continuously, the total amount of* Vibrio *was significantly reduced, which proved that the use of photosynthetic bacteria can effectively reduce the number of* Vibrio *in water.*


#### **Table 4.**

*Probits stabilizes the water quality, but also reduces the accumulation of residual.*

adjust the temperature of the mixture to be similar to the temperature of the pool water, and because the probiotics themselves are live bacteria, they should not be mixed with antibacterial agents, sulfonamides, antibiotics, and disinfectant water or in high temperature process.

An example of our use in white shrimp (*Litopenaeus vannamei*) culture farms, we use a variety of special strains such as Photosynthetic bacteria *Rhodobacter capsulatus* and *Micrococcus luteus*. Photosynthetic bacteria can utilize the hydrogen sulfide at the bottom of the pool as a nutrient to remove harmful substances and absorb heavy metals and inhibit the growth of Vibrio (**Figure 1**), while *M. luteus* can rapidly decompose proteins and increase the speed of nitrogen circulation in water to effectively reduce the occurrence and damage of sediments at the bottom of the pool. The interactive use of different strains not only effectively stabilizes the water quality but also reduces the accumulation of residual bait and organic matter in the shrimp culture process (**Table 4**) and greatly inhibits the proliferation of pathogenic bacteria such as Vibrio in the water. Such technology has been widely used in aquaculture industry in Taiwan and in Surabaya, Indonesia, and Thailand. Photosynthetic bacteria have become one of the most beneficial microbial strains in today's aquaculture industry (**Figure 2**).

**69**

*Application of Novel Technology in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.90142*

**immune regulation**

**Figure 2.**

**3. Strategies for cultured aquatic animals' immunization and** 

*Photosynthetic bacteria culture and mass production. Purification of photosynthetic bacteria on agar plate* 

Animal congenital immune cells or other nonimmune cells, such as epithelial cells that secrete cytokines, mononuclear cells, macrophages, dendritic cells, and natural killer cells, have many nonspecific pattern recognition receptors, targeting identification and binding of antigen-related molecular patterns on invading pathogens. Complement receptor type 3 (CR3 receptor) on these cells is a receptor for the recognition of β-glucan [14], and when β-glucan binds to CR3 receptor, it triggers a series of signaling cascades to activate transcription factors, which translates cellular transcription into cytokines that trigger inflammatory responses, and incrementally regulates the expression of the major histocompatibility complex (MHC) of the antigen. In turn, other immune cells are activated to achieve immunomodulatory functions [15]. The polysaccharide extracted from the yeast (*Saccharomyces cerevisiae*) was orally administered for 4 weeks in dogs. The expression of total IgA, IgM, and IgG in serum and lacrimal gland was detected, and the expression of IgA in serum was found to be significantly decreased and IgM increased significantly, while IgG did not change significantly; the amount of IgA in the lacrimal gland was also significantly reduced. In the second part, the Bordetella vaccine was injected subcutaneously, and the specific IgA, IgM, and IgG showed the same trend as in the first part. Oral administration of β-glucan to Miguel dog changed the expression of IgA and IgM in serum, and there was no difference in the expression of IgG, indicating that the intestinal mucosal immune response was stimulated through this way. Glucan enters the gut-associated lymphoid tissue from M cells and binds to dentin 1 and TLR-2 on macrophages and dendritic cells, secreting IL-12 and TNF-α and other cytokines, which alter the cytokine microenvironment to stimulate the homologous transformation of B cells, affecting the secretion of immunoglobulins of different isoforms [16]. Dendritic cells are one of the important antigen-presenting cells in the immune system. Lu et al. used a polysaccharide extracted from *Antrodia camphorate* to carry out cell experiments and cocultured it with immature dendritic cells and T cells isolated from healthy human blood to investigate the maturation of dendritic cells and T-cell proliferation. The result showed polysaccharides promote maturation of dendritic cells to stimulate T-cell proliferation and IFN-γ expression [17]. Coculture of polysaccharides with macrophages can promote macrophage secretion of immune-related factors and

**3.1 Aquatic animals' immunity and immune regulation**

*(left). Commercial mass production of photosynthetic bacteria (right).*

#### **Figure 2.**

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

adjust the temperature of the mixture to be similar to the temperature of the pool water, and because the probiotics themselves are live bacteria, they should not be mixed with antibacterial agents, sulfonamides, antibiotics, and disinfectant water

*Effectiveness of photosynthetic bacteria applied in white shrimp culture. Change in the total amount of* Vibrio *in the culture pond within 14 days of continuous use of photosynthetic bacteria in the white shrimp culture process. The results showed that in the experimental group with more than 1 ppm of photosynthetic bacteria added continuously, the total amount of* Vibrio *was significantly reduced, which proved that the use of* 

*Probits stabilizes the water quality, but also reduces the accumulation of residual.*

*photosynthetic bacteria can effectively reduce the number of* Vibrio *in water.*

An example of our use in white shrimp (*Litopenaeus vannamei*) culture farms, we use a variety of special strains such as Photosynthetic bacteria *Rhodobacter capsulatus* and *Micrococcus luteus*. Photosynthetic bacteria can utilize the hydrogen sulfide at the bottom of the pool as a nutrient to remove harmful substances and absorb heavy metals and inhibit the growth of Vibrio (**Figure 1**), while *M. luteus* can rapidly decompose proteins and increase the speed of nitrogen circulation in water to effectively reduce the occurrence and damage of sediments at the bottom of the pool. The interactive use of different strains not only effectively stabilizes the water quality but also reduces the accumulation of residual bait and organic matter in the shrimp culture process (**Table 4**) and greatly inhibits the proliferation of pathogenic bacteria such as Vibrio in the water. Such technology has been widely used in aquaculture industry in Taiwan and in Surabaya, Indonesia, and Thailand. Photosynthetic bacteria have become one of the most beneficial microbial strains in

**68**

**Table 4.**

**Figure 1.**

or in high temperature process.

today's aquaculture industry (**Figure 2**).

*Photosynthetic bacteria culture and mass production. Purification of photosynthetic bacteria on agar plate (left). Commercial mass production of photosynthetic bacteria (right).*
