Novel Technologies for Salmon Culture

#### **Chapter 1**

## New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture

*Paul Gümpel, Urs Dornbierer, Arnulf Hörtnagl and Torsten Bogatzky*

#### **Abstract**

This paper presents the current state of development and selected technological challenges in the application of ecologically and economically sustainable nets for aquaculture based on ongoing development projects. These aim at the development of a new material system of high-strength stainless steel wires as net material with environmentally compatible antifouling properties for nearshore and offshore aquacultures. Current plastic netting materials will be replaced with high-strength stainless steel to provide a more environmentally friendly system that can withstand more severe mechanical stresses (waves, storms, tides and predators). A new antifouling strategy is expected to solve current challenges, such as ecological damage (e.g., due to pollution from copper-containing antifouling substances or microplastics), high maintenance costs (e.g., cleaning and repairs), and shorter service life. Approaches for the next development steps are presented based on previous experience as well as calculation models based on this experience.

**Keywords:** sustainability, stainless steel, high strength, redesign, predator net

#### **1. Introduction**

Of the fish consumed worldwide, the total amount since 2016 has been more than 150 million tons per year, almost half is now produced in aquacultures. While the amount of freely caught fish is stagnating or slightly declining, the amount of fish farmed in aquacultures is continuously increasing. Currently, about 150 species of fish are farmed in aquacultures, and the proportion of seawater farms is constantly increasing [1].

A crucial challenge of today's aquaculture is to protect the farmed fish but also to protect the environment and the surrounding ecosystems. The goal of sustainable aquaculture operations should therefore be to achieve as limited an impact on the environment as possible. At the same time, in the sense of economic operation, care must be taken to ensure that the fish farms are subject to as limited a maintenance and repair effort as possible. In addition, the conditions under which the fish are kept play a decisive role, so that the fish do not suffer from diseases and a sufficient exchange of water is ensured.

The massive expansion of fish farms poses major challenges for operators. Sustainable criteria for aquacultures are indispensable and it is important to avoid negative impacts on the environment in order to meet consumer confidence as well as current and future national and international requirements. The materials used for the construction of the nets play a major role. Generally, a net enclosure consists of a buoyant support system and a net that encloses the animals. There are only minor differences in the basic principles between the classic "nearshore" and the "offshore" applications that have been implemented for a few years. However, the individual installations can vary greatly in size, shape, and materials used [2, 3].

Plastics are predominantly used as the netting material. To counteract the growth of undesirable organisms on the surface of man-made structures, also better known as biofouling, these plastic nets are usually protected from fouling with anti-fouling agents. These industrially available products are currently often made of copper and zinc. It should be noted that both metals are listed by the EU under the Dangerous Substances Directive (67/548/EEC). Especially the application of copper in the field of anti-fouling has a significant impact on the fish in the fish farm as well as on the organisms in the direct environment of the fish farms [4, 5]. Thus, significantly elevated concentrations of copper in sediments can be detected in the vicinity of salmon aquaculture operations that use conventional anti-fouling strategies with copper. Individual scientific studies also indicate that the high concentrations of copper may also lead to sub-lethal effects for the fish kept in the aquacultures [4–6]. The application of copper-containing solutions to the net materials used is mainly by copper-containing dip coatings, which impregnates the nets. This results in a continuous release of copper into the environment, causing the effects listed above to occur. For example, in the OSPAR Commission reporting area, this resulted in an annual emission of at least 454 metric tons (MT) of zinc, copper, and chemical compounds based on them as early as 2009 [7]. Due to the large increase in aquaculture since 2009, a much higher number can be expected today on a global scale. These considerable amounts of valuable raw materials are consumed in this application and cannot be directly recovered by conventional recycling. The search for suitable and practical alternatives to this end has been ongoing in the field of research for several years [8]. Nevertheless, the use of antifouling strategies based on copper and zinc remains the state of the art.

Moreover, the polymer materials used are hardly recyclable and in turn also contribute to plastic pollution of the oceans. Plastics, and in particular the fine fibers of the nets or the ropes and braids, are also very susceptible to the biofouling already mentioned above (substance deposits on the material surfaces). They therefore require complex and costly maintenance and cleaning. In addition, the susceptibility to fouling places a burden on the fish living in the aquacultures. This in turn leads to costly treatment, which also stresses the fish and slows their growth. Omitting the substances that protect against anti-fouling leads to a considerable reduction in the cross-section of the nets within a short time due to the colonization of algae, mussels or barnacles. This fouling can adhere very well to conventional plastic nets and can only be removed with great technical effort, if at all. In addition, the fouling on the nets leads to a significant increase in the mass of the net systems. This results in a significantly higher mechanical load and, in conjunction with the other loads that occur (wave action, tides, currents), can also lead to corresponding damage to the nets [9].

Damaged nets, but also nets that can be easily bitten through by predators, do not provide sufficient protection against classical predators of fish in aquaculture. The often-used setup with two net systems (predator net to keep predators away and fish net inside the aquaculture) is also only limited suitable to keep away the highly specialized and intelligent predators. Exemplary for many other cases are seals as a challenge for salmon farms in Scotland. An adult harbor seal is an extremely efficient hunter, eating between 3 and 7 kg of fish per day, depending on *New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

species and size. In addition, continuous attacks by predators in the aquacultures lead to a significantly increased stress level of the fish, which not only inhibits their growth, but can also lead to their death. The sometimes widespread method of selectively hunting and killing corresponding predators in the environment of aquacultures is restricted by increasingly restrictive regulations at national and international level, so that there is a need here for innovative and sustainable methods to protect fish in aquacultures [10, 11].

All aspects listed here suggest that for an ecologically and economically sustainable operation of aquacultures, both "nearshore" and "offshore", it is necessary to resort to further technological approaches and concepts. The application of highstrength metallic mesh systems represents a promising alternative technology in this respect. The state of the art, the development work carried out to date and the prospects are reported on below.

#### **2. Material selection and mesh production**

The selection and configuration of the net materials as well as their long-term behavior in seawater determine decisive parameters for aquaculture operations. As already described above, the flow of fresh water through the cages, the hygiene of the facility, the protection against external predators and, last but not least, the safety against escape of the farmed fish into the open sea are all significantly influenced by this. For all the above criteria, stainless steel is an ideal material for several reasons. The high specific strength of steel compared to polymers also makes it possible to produce larger net systems and increases the water flow. The technical challenge of producing a mesh from steel wires has now been solved very well on an industrial scale. It seems to be important that only the pure stainless steel surface is used without any coating or that no toxic substances can be used to prevent fouling of the stainless steel surface. The great advantage of stainless steel is the good and easy removal of biological fouling. To build a complete plant with stainless steel cages is in principle technologically feasible, but still represents a major challenge. In particular, the design of the plant, the connection of the nets and the associated force transmission, as well as the handling of the nets must be adapted to the properties of the stainless steel.

In order to find a fundamentally suitable material for aquaculture nets in nearshore and offshore fish farming, several development steps were carried out. The first step was to compare the properties of different stainless steels to meet both wire and net manufacturing and equipment operation requirements. The properties of different types of stainless steels were investigated and evaluated. The sometimes contradictory requirements for high strength with sufficient residual ductility for the manufacture of nets and, in particular, the corresponding corrosion resistance for use in seawater can best be met by the so-called corrosion-resistant duplex stainless steels (**Figure 1**).

Duplex stainless steels also offer the advantage of high resistance to stress corrosion cracking in seawater. In the oil and gas industry, these steels have been used in seawater for many years with very good experience.

Replace the entirety of this text with the main body of your chapter. The body is where the author explains experiments, presents and interprets data of one's research. Authors are free to decide how the main body will be structured. However, you are required to have at least one heading. Please ensure that either British or American English is used consistently in your chapter.

By using duplex stainless steels in the cold-worked condition, all requirements regarding mechanical strength and corrosion resistance can be met. Laboratory

#### **Figure 1.**

*Schematic overview of the relation between corrosion resistance and mechanical strength for the different types of stainless steels [12].*

tests have shown that both materials A (alloy 2304 (UNS S 2304/ 1.4362)) and B (alloy 2205 (UNS S2205/1.4462)) have corrosion resistance in artificial seawater at the usual temperatures. However, the resistance of the molybdenum-containing material B is higher, resulting in greater safety against additional stress factors such as higher temperature, concentration of chloride and/or biologically influenced effects on the corrosion process. A highly specialized manufacturing process was used to produce and further process meshes made of these high-strength materials with a tensile strength of up to 2000 MPa. Therefore, the mesh system manufactured from stainless steels has much higher mechanical strength compared to the conventional plastic meshes. The use of stainless steels with the much higher strength compared to polymers results in thinner mesh bars or larger mesh sizes. The area ratio of web material to flow opening becomes more favorable and water exchange, which is so important for the quality of the plant, becomes much better compared to polymer nets. Regardless of the degree of biological growth, this improves the living conditions of the fish. This positive condition is maintained by a continuous cleaning process, which leads to a higher sustainability.

#### **3. Practical testing**

A selection of different net systems (material and antifouling strategy) represented the second step of the development carried out. For this purpose, samples were outsourced at eight locations worldwide (**Figure 2**) over a period of at least 6 months in order to investigate the individual fouling behavior in comparison to existing net systems in practical use. For this purpose, samples were outsourced in the area of fish farms or shellfish farms in order to simulate local conditions in the farm area as well (see **Figure 3**). Contamination was documented and evaluated at defined intervals using photography and light microscopy. In addition, the cleanability of different mesh systems was tested and evaluated using standardized cleaning procedures.

After several test cycles, the samples were evaluated for their corrosion and antifouling behavior. In addition to these immersion tests in natural fish farm environments, laboratory tests as well as microbiological and corrosion tests were performed to investigate and evaluate the different net systems and AF strategies.

*New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

#### **Figure 2.**

*Sites for natural outsourcing in aquaculture and in artificial seawater for simulation experiments in the laboratory.*

#### **Figure 3.** *Test arrangement of the outsourced samples.*

The results of the laboratory tests as well as the natural deposition in seawater farms clearly showed that the development and also the growth of biofilms (**Figure 4**) is most safely hindered by the application of toxic substances, e.g. of copper as a common antifouling strategy for a limited time. In polymer nets, this is achieved by infiltration of copper. Metal nets, for example, may be made of copper alloys, or the surface of the steel may be coated with a layer of copper or copper alloys. Surface coatings of nonstick materials such as PTFE (polytetrafluoroethylene) or nanostructured materials (sharkskin effect) can reduce biofouling compared to the surface of pure steel, but fouling is only delayed but not prevented (**Figure 5**).

Another essential part of the investigations carried out was the comparison of the possibilities to clean the overgrown structures after a defined aging period. Different cleaning systems were used for these tests. The results can be described as follows: Steel/metal surfaces can be cleaned thoroughly and almost residue-free with a water jet/hydrojetting (see **Figure 6**). Cleaning with this common method is also possible in principle for polymer meshes. However, a closer examination of the surfaces showed that the cleaning result is significantly worse. In this context, biological studies have shown that considerable residues of biofilm material always remain between the individual plastic threads. Such residues of biological material accelerate re-biofouling. Thus, in these cases, a comparatively rapid re-growth of the nets is to be expected. This effect sometimes leads to a reduction in the service life of conventional plastic nets. A comparable acceleration of re-biofouling could not be determined for the steel nets used.

**Figure 4.** *Proportion of the covered area after long-term exposition in seawater (4 months).*

#### **Figure 5.**

*Method for optical evaluation of the change in the area fraction of the biofilm on the outsourced network structures [12].*

On the basis of these preliminary investigations and the material selection made, the first stainless steel nets for aquaculture could be used as predator nets. These coarse-meshed nets keep predators such as seals away from the fish nets inside. The first field trial took place in South America on the Pacific coast. **Figure 7** shows the supplied net rolls that were assembled into cages in the field. Based on the results of the previous laboratory and exposure tests, the nets were made from Material A.

Surprisingly, after only a few months of real-world application in the Pacific Ocean, corrosion attack occurred mainly at the nodes of the mesh (**Figure 8**). Some isolated corrosion attacks were caused by small, invisible defects in the surface. In general, the surface quality of the cold-drawn wires in this highly reinforced condition is a problem. These manufacturing problems were solved by the steel supplier.

The reason for the systematic failures in the node areas could be determined by fault analysis and simulation of corrosion experiments. During field operation, there is high friction at the nodes in the meshes. This friction in the nodes under

*New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

**Figure 6.** *Proven cleaning method to remove biofouling from surfaces.*

**Figure 7.** *Installations in South America - Total 112′000 sqm.*

#### **Figure 8.**

*Typical tribocorrosion in the friction area of a stainless steel net after some months in the Pacific Ocean [12].*

high mechanical stress and seawater environment has led to tribocorrosion. In this particular case, the material in the friction area was activated and could not passivate again under the given conditions. Simulation tests showed the activation. The OCP breaks down during frictional loading and increases when friction stops

(**Figure 9**). A dependence on the time between activation is also evident. The tests showed very clearly that material B has a much higher resistance than material A under these test conditions (**Figure 10**). Temporary activation can occur with material B, but repassivation occurs quickly and reliably. The stainless steel with the higher alloy content and thus a higher pitting resistance value definitely has a higher tolerance to any type of corrosion [12].

The susceptibility of individual materials to the tribocorrosion that occurs led to a further and more detailed investigation of possible locations for aquacultures. Using specially developed measuring buoys, it was possible to compare the corrosivity at different locations. This clearly showed that, in addition to influencing factors such as water temperature or salt concentration, the potential shift caused by microorganisms, also better known as ennoblement, plays a decisive role in the corrosion mechanisms that occur. Since, as already explained above, a correspondingly high input of microorganisms is always to be expected in the direct environment of aquacultures, the ennoblements must always be taken into account when selecting materials. As already listed above, this is possible with the listed alloy B, which could be confirmed by laboratory tests as well as by application in aquacultures [12].

In order to better understand the advantages but also the application limits of steel nets compared to plastic nets, a consideration of specific technical properties is necessary. However, it is important that properties such as density are not considered in isolation. The density of a conventional plastic such as PA 6 (polyamide 6) is 1.1 g/cm3 , whereas the density of a stainless steel is approx. 7.8 g/cm3 . This serious difference in density is put into perspective when the achievable tensile strength of these materials is taken into account. For reconditioned PA 6 ropes, a considerable tensile strength of 110–175 N/mm2 can be achieved. A work-hardened duplex

**Figure 9.** *Open circuit potential during friction simulation – peaks show repassivation when friction stops [12].*

*New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

**Figure 10.** *Current-potential-curves after different times of friction simulation (see phases in Figure 9) [12].*

stainless steel 1.4462 achieves tensile strengths of 1200–1350 N/mm2 , in the case of the highly specialized applications listed here up to 2000 N/mm2 . If these figures are put in relation to each other, the strength achieved per mass used is comparable for both steel and plastic.

Another significant difference lies in the modulus of elasticity of the materials. For the PA 6 material mentioned above as an example, this is approx. 3 GPa. For the steel 1.4462, the value is approx. 200 GPa. This difference is of particular importance, as it clearly shows that a simple substitution of plastic meshes by high-strength steel meshes can be problematic when using the same joining technology. **Figure 11** shows an example of the deformation occurring in a salmon aquaculture. It can be clearly seen that in the application, a significant deformation compared to the original prefabricated geometry occurs due to the dead weight of the mesh panels.

Subsequently, the occurring sea loads, which are composed of the external influencing factors (e.g. water current, wind and tides), result in a time dependency of the occurring forces and thus also of the occurring deformation. This can be seen from the differences in the occurring deflection recorded by measurement on three consecutive days in **Figure 12**.

While conventional plastic nets achieve high deformation even at low forces due to the comparatively low modulus of elasticity of the materials used, steel nets behave comparatively rigidly in most cases. As a result, the load distribution of plastic nets is generally more benign and stress peaks are evenly reduced, even when the mesh panels are rigidly connected to each other. If a correspondingly rigid connection is made in the case of steel nets, there is a risk of wire breaks occurring in the area of load application or at connection points between the nets. This can be the case both as a result of a one-time overload and as a result of a high cyclic load. Taking into account the corrosive environmental conditions in

#### **Figure 11.**

*Illustration of the occurring deformation of an exemplary salmon aquaculture.*

**Figure 12.** *Temporal variation of the occurring deformation in an aquaculture.*

seawater and the tribological stress occurring at the contact points between the individual wires, the result is a highly complex system in which several damage mechanisms interact.

Before remedial measures can be defined, it is necessary to differentiate the damage patterns and the causes of damage as far as possible. As an example, the damages in **Figures 8** and **13** can be compared. Basically, both damage patterns show a reduction in cross-section in the area of the wire contact. However, tribocorrosion in the contact point, see **Figure 8**, leads to increased local corrosion of the surface, while mechanical overloading leaves a comparatively smooth contact point. The wear marks shown in **Figure 13** do not reveal any discernible corrosive attack. It should be noted that the wire breakage that occurs as a result of the load distribution is not directly present at the generated groove. Due to the geometry of the half mesh or the meshes used, the maximum stress occurs directly next to it.

More in-depth investigations on a laboratory scale and simulations based on these confirm that a simple substitution of plastic nets by steel nets in aquaculture with strong mechanical loads due to waves, wind and tides does not yet produce the fully desired increase in the targeted service life when using steel nets compared to plastic nets. As shown by the study of a wire break **Figure 14** of a net element tested for continuous load, violent bridging in the form of sliding or honeycomb fractures occurs as a result of the bending load.

However, the studies carried out and the first applications in practice also show that there is considerable potential in the technology described here. In order to do justice to this potential in the growth market of aquaculture, selected development approaches are listed in the following chapter.

*New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

**Figure 13.**

*Fracture pattern at a half mesh in the area of the wire contact of two different mesh patterns due to mechanical overload.*

#### **Figure 14.**

*SEM images of the fracture surface of a wire at different magnifications; (a) overview image showing presumed area of origin for the fracture; (b) and (c) detail images showing characteristic honeycombs of a transcrystalline honeycomb fracture.*

In order to round off the comparison between plastic nets and steel nets, the input of plastic/microplastic into the waters must be taken into account in addition to the influence of the antifouling strategy applied. Similar to steel nets, the mechanical stresses that occur between the individual ropes and strands lead to wear. This wear occurs when plastic nets are used for both conventional fishing and aquaculture. It must be pointed out, however, that this input into the world's oceans is comparatively small compared with other sources. Nevertheless, from a current perspective, it is particularly important to reduce the generation of microplastics in the direct vicinity of our food sources [13]. While plastic nets must be disposed of as hazardous waste at the end of their service life due to contamination with heavy metals and biomass, steel nets can be recycled very easily as high-quality steel scrap.

#### **4. Outlook and ongoing developments**

Based on the above experience, selected changes have been made which should make a significant contribution to the possible widespread use of steel nets in aquaculture. The most significant change in the manufacture of nets is the use of the more corrosion-resistant material B, as already explained above. In addition, the structure and overall suspension of the cage were designed to minimize friction in the nodes and to have the most homogeneous load distribution possible within the net. After these modifications, no systematic signs of corrosion or tribocorrosion were observed. Another starting point is the structural adaptation of the fastening of the nets, the load application and the connection of the nets to each other. A project underway for this purpose is now in a phase in which long-term operation is being observed and a continuous improvement process is being carried out in actual use. A long-term tested, technically reliable solution for anti-predator networks should be available by mid-2022.

In parallel to the above mentioned tests, a development project for a sizing tool for farms to be equipped with stainless steel netting is ongoing. Since local influences differ greatly depending on the location of the aquacultures (prevailing currents, wave action, tidal variation and the factors listed above that affect corrosion), it is necessary to evaluate each aquaculture individually. Depending on the stress factors encountered, other design measures can then be taken. The tool will allow statements on the design of the network and its expected service life based on local conditions. This project should be completed by 2025.

Initial tests and simulation on this indicate that a successful use of high-strength stainless steels as network material for aquaculture is possible. However, due to the damage mechanisms that occur, holistic approaches must be pursued, which are aimed in particular at a possible reduction of the acting loads and their uniform distribution. The following approaches can be considered as examples.

By gradually reducing the wire diameter used in steel nets, the weight force occurring in aquaculture can be reduced as much as possible. Calculations show that the use of thinner wire diameters in the lower part of the aquaculture results in a reduction of up to 30% of the mechanical loads in the area of the upper attachment points in steel nets, without any restriction of the function.

In the case of plastic nets, whose density is only slightly higher than that of seawater even when impregnated, the use of weights and attachments to the seabed is necessary to minimize their movement due to currents and tides. These measures can and must be dispensed with wherever possible in the case of steel nets. In this way, the mechanical stress on the aquaculture can be further reduced. At the same time, the higher density of steel nets ensures less deformation of the cages.

The use of flexible connecting elements between the mesh panels allows a more even load distribution, which reduces stress peaks. These stress peaks, which can lead to fatigue failure especially with a high number of load cycles, are thus defused as critical points and a much more homogeneous load distribution is achieved. As can be seen from the example in **Figure 15**, constructive approaches can be used in the design of the network paths. Although this requires a corresponding know-how in the manufacturing and processing technology of rope systems, the simulations carried out as well as the first field tests show a significant reduction of the occurring stress peaks.

Another measure is the application of a hybrid structure of aquaculture. As already mentioned above, it must always be taken into account when selecting materials that materials from different groups in particular differ in a variety of *New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

**Figure 15.** *Exemplary presentation of constructive design possibilities in the application of steel nets.*

**Figure 16.** *Installed predator nets for salmon aquaculture in Chile.*

properties. A hybrid structure provides for the use of elements made of plastic and elements made of steel. The elements made of plastic, which are used to transfer the load between the floats and the steel nets, allow the load to be distributed as evenly as possible. If the bottom of the aquaculture is also designed as a plastic net, it remains correspondingly flexible or easily deformable. If the side elements are made of steel nets, they continue to offer appropriate protection against predators and are easy to clean and do not require any further AF coating (**Figure 16**).

#### **5. Conclusion**

The use of nets made of high-strength and corrosion-resistant steel offers corresponding advantages over conventional nets made of plastic, particularly from an ecological point of view, and subsequently also from an economic point of view after a sufficient service life. The scientific studies carried out show that the challenges of corrosion and tribocorrosion in seawater, biofouling and the mechanical stresses that occur can be met by selecting a suitable stainless steel material. However, it should be remembered that the exclusive use of steel nets for large-scale conventional aquaculture is not possible at present. This challenge can be met in the future with the approaches taken for further development and adjustments in the connection and force application of the nets, according to the available data of these challenges.

#### *Salmon Aquaculture*

Regarding the common aquacultures in the near shore area (near shore), two trends are clearly visible in the past years: off shore and on shore. It is self-evident that for most on shore aquacultures do not require a corresponding network infrastructure. For offshore aquacultures, on the other hand, new challenges and opportunities arise through the application of the high-strength steel nets presented here. The comparatively high tensile strengths of the materials used, up to 2000 MPa, make it possible to operate aquacultures safely even under comparatively harsh conditions if the overall design is adapted.

#### **Thanks**

Special thanks to the Swiss Agency for the Promotion of Innovation - Innosuisse for the financial support of the research work carried out.

#### **Author details**

Paul Gümpel1 , Urs Dornbierer2 , Arnulf Hörtnagl3 \* and Torsten Bogatzky3

1 Konstanz University of Applied Sciences, Konstanz, Germany

2 Geobrugg AG, Romanshorn, Switzerland

3 Institute for Materials Systems Technologies Thurgau, Tägerwilen, Switzerland

\*Address all correspondence to: a.hoertnagl@witg.ch

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*New Development: High-Strength Stainless Steel as a Sustainable Material for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.98680*

#### **References**

[1] Food Outlook – Biannual Report on Global Food Markets. FAO 2020.

[2] Wang CM. Moving offshore for fish farming 2019.

[3] Holmer M. Environmental issues of fish farming in offshore waters: perspectives, concerns and research needs. Aquacult. Environ. Interact. 2010; 1(1): 57-70. DOI: 10.3354/aei00007

[4] Nikolaou M, Neofitou N, Skordas K, Castritsi-Catharios I, Tziantziou L. Fish farming and anti-fouling paints: a potential source of Cu and Zn in farmed fish. Aquaculture Environment Interactions 2014; 5(2): 163-71. DOI: 10.3354/aei00101

[5] Børufsen Solberg C, Sæthre L, Julshamn K. The effect of coppertreated net pens on farmed salmon (*Salmo salar*) and other marine organisms and sediments. Marine Pollution Bulletin 2002; 45(1-12): 126-32. DOI: 10.1016/S0025-326X (01)00296-X

[6] Dean RJ, Shimmield TM, Black KD. Copper, zinc and cadmium in marine cage fish farm sediments: an extensive survey. Environmental Pollution 2007; 145(1): 84-95. DOI: 10.1016/j. envpol.2006.03.050

[7] OSPAR COMMISION, editor. Assessment of Impacts of Mariculture: Biodiversity Series; 2009.

[8] Hanno Schnars CB. Biozid-freier Bewuchsschutze für die Marikultur: no copper. Projektendbericht zum Projekt az29456, Institut für Marine Ressourcen Mai 2014.

[9] M. Baum. Umweltfreundliche Netze für die Fischzucht. Kiel: Forschungsschwerpunkt Kiel Nano Surface and Interface Sciences; 2020 Jul 21.

[10] Global Aquaculture Alliance. Fish farmers seek non-lethal deterrence for persistent predators « Global Aquaculture Advocate; 2021 [cited 2021 May 26] Available from: URL: https:// www.aquaculturealliance.org/advocate/ fish-farmers-seek-non-lethaldeterrence-persistent-predators/.

[11] Seals and Tasmanian Aquaculture. Tasmanian Conservation Trust 2015 Jul 10.

[12] Gümpel P, Hörtnagl A, Sorg M. High Tensile Stainless Steel as a Sustainable Material for Aquaculture. Procedia Manufacturing 2019; 30: 315-22. DOI: 10.1016/j. promfg.2019.02.045

[13] Microplastics in fisheries and aquaculture: status of knowledge on their occurrence and implications for aquatic organisms and food safety 2017.

#### **Chapter 2**

## Integrated Culture of *Oncorhynchus mykiss* (Rainbow Trout) in Pre-Cordilleran Sector under a Recirculation System in Northern Chile

*Renzo Pepe-Victoriano, Héctor Aravena-Ambrosetti and Piera Pepe-Vargas*

#### **Abstract**

An experience of integral farming of *Oncorhynchus mykiss* (rainbow trout) is carried out in Copaquilla, 90 kilometers inland from the city of Arica at 3,000 mamsl. The system used was the Recirculating Aquaculture System (RAS), which had six ponds of 40 mt3 each, two decanters with a capacity of 3.5 mt<sup>3</sup> and a biofilter of 3.5 mt3 with substrate for the fixation of ammonium and nitrite transforming bacteria. The three latter ponds were buried below the lowest level of the fattening ponds. Three pumps, two running and one 1.5 hp. backup, plus a 1 hp. blower, were the water and air equipment utilized in the system. Each pump had a flow capacity of 450 lt min−1. This water was sucked from the biofilter and transferred to the accumulator tank with a capacity of 10 mt<sup>3</sup> . From there it was distributed by gravity to the fattening ponds. In addition, the juvenile system had a particular SAR with a 0.5 hp. pump, a small 0.2 hp. blower and an 80 watt UV lamp. The grow-out SAR received 6,000 trout with an average weight of 15 grams. The group reached approximately 1,200 grams over a year. Thirty fish were selected for reproduction. Eggs were obtained, followed by fry, juveniles and adults. This initiative demonstrated the effectiveness of producing trout in the foothills of the interior city of Arica, Chile.

**Keywords:** indigenous communities, water quality, rainbow trout transport, spawning, rainbow trout eggs and larvae

#### **1. Introduction**

The lack of opportunities, technological improvements and diversification are issues to be resolved in mountain and foothill sectors and the reason for the insertion of new productive and sustainable alternatives. The unbeatable environmental conditions that many of the sectors of this territory present, such as the existence of good quality water sources, availability of space and microclimatic environments, are clear elements of potential. In such conditions, aquaculture shows favorable prospects for development.

#### *Salmon Aquaculture*

Aquaculture, as indicated by Pepe-Victoriano et al. [1], is presented as a real productive alternative, by identifying crops of freshwater species of commercial value as a way to take advantage of the capacities installed in the agricultural activity of the area. Storage ponds for irrigation, greenhouses, hydraulic systems, among others, are some examples of facilities that add to the possibility of reusing water as many times as necessary before being derived as a final destination for irrigation of plants and vegetables.

The utilization of technology such as recirculation in aquaculture systems is presented as a powerful alternative for pre-cordillera areas [2]. A recirculation system allows more precise control of the main environmental parameters [3]. Water temperature, to name one, is a critical parameter for most poikilothermic organisms, such as fish, and can be controlled much more economically in a recirculating system than in an open flow one. Controlling this and other environmental parameters allows for faster growth and more efficient use of feed due to reduced stress on the farmed organisms.

The quality of the water in Copaquilla, as the main variable for trout farming, is within the optimal range for the development of this initiative. In previous studies to the three existing springs that would feed the farm, elements such as arsenic, copper, zinc, iron, and manganese are within the limits allowed by Chilean standards for drinking water consumption and therefore for freshwater crops.

Moreover, it is worth mentioning that trout production in recirculation systems would make it possible to increase the supply of good quality fish for local and regional population. Per capita fish consumption in Chile averages 13 kilos per year, which keeps us quite far from the world average of 20 kilos established by the Food and Agriculture Organization of the United Nations [4]. It is estimated that the unmet demand for fresh fish for human consumption in our region is around 1,000 tons per year. In addition to this, there is a growing consumption with clear preferences for salmonid fish species over red meat and poultry, mainly in the young segment of the population.

The implementation of this fish farm generates clear potential and economic benefits for the communities in the foothills of the Andes that might develop such initiative further into the future. This development is mainly projected as a diversification alternative, adding value to the aquifer resource, and improving the income of the territory by including other economic activities such as tourism and gastronomy, among others. The rich landscape and heritage of the area would also be benefited. In addition, these farming provides useful information to be used as a basis for other farming initiatives in the region. Aquaculture is expected to be boosted and to become one of the main strategic axes of regional development in the short term.

The main objective of this initiative was to develop the integral culture of *Oncorhynchus mykiss* under recirculation conditions, as a productive development alternative for communities in the pre-cordillera of the Arica and Parinacota Region.

#### **2. Why grow trout at 3,000 meters above mean sea level?**

The productive development of the communities located in the foothills of the Arica and Parinacota Region, especially in the Copaquilla sector (**Figure 1**), is based mainly on agriculture, livestock and to a lesser extent, tourism. Most recently, activities such as agriculture and livestock have suffered a clear deterioration and abandonment, mainly due to the lack of diversifying alternatives that encourage and avoid one of the main problems of the territory; the migration of young population to the city.

*Integrated Culture of* Oncorhynchus mykiss *(Rainbow Trout) in Pre-Cordilleran Sector under… DOI: http://dx.doi.org/10.5772/intechopen.98920*

**Figure 1.** *Geographical location of the Pukara de Copaquilla Cultivation Center (CCPC).*

#### **3. Biological characteristics of trout**

The rainbow trout (**Figure 2**) is a teleost fish belonging to the salmonid family (Salmonidae), whose distribution range covers cold waters of North America, Asia and Europe. It tolerates temperatures going from 0 to 25° C, with an optimum range of 10 to 14° C to remain healthy. In order to ensure excellent growth, however, temperatures between 15 and 20° C are preferred [6] in good water quality conditions.

The life cycle of rainbow trout is highly variable in terms of migratory patterns. Out in the wild, they spawn in rivers or streams, and many complete their life cycle in freshwater. Some varieties, nevertheless, migrate and spend their adult life in the ocean. They only return to the river where they were born to spawn, completing the cycle. This behavior is known as anadromous reproduction [7]. Anadromous forms migrate to the sea as juveniles and can travel long distances in the ocean. Freshwater forms (non-anadromous) move between affluent and main river, between river and lake, or spend their entire lives in a particular stream or river [7]. The growth and sexual maturation of these organisms can occur in freshwater or seawater. This last phase can last between 1.5 and 3 years of the fish's life. Spawning generally occurs in water flows of both rivers or affluents of the main channel, on gravel beds of rivers or lake shores, where water seeps through it. The gravel provides protection for the eggs until they emerge as fry ready to eat and migrate [7].

Rainbow trout are highly adaptable to their environment, which is why they have achieved a wide distribution [8]. According to studies carried out by other authors, trout born from the same progeny can adapt to totally different habitats. Some can grow, reproduce and live in a small stream, with a few centimeters of water above their bodies. Meanwhile, others can travel many kilometers to the ocean to feed and grow much larger than the first [9]. This is why resident trout, migratory trout from rivers and lakes, or anadromous trout can be found in the same watercourse [9].

**Figure 2.** *Adult specimen of Oncorhynchus mykiss (Rainbow trout). Image extracted from Cornejo-Ponce et al. [5].*

#### **4. Justification for the usage of a recirculation system**

Recirculating Aquaculture Systems (RAS) are one of the emerging production technologies that are being applied in the national and global aquaculture sector. This is especially true when considering the concept of efficient improvement in production by minimizing the usage of water resources and increasing environmental responsibility. Thus, concentrating and treating the waste generated during the production process.

This technology is ideal for utilization in production systems that involve fish farming and the implementation of wastewater for agricultural irrigation, since the latter improves productivity and profitability in economic terms. In Chile, this technology is mainly used in the early stages of the salmon production process and is associated with fish farming centers where the incubation phase is carried out until smoltification. In the northern zone of Chile, there are no experiences or farms in operation that involve the application of recirculation technology in production processes with trout or other freshwater species.

The implementation of this type of technology (SAR) in aquaculture has enormous potential, especially in desert areas such as the Arica and Parinacota Region, where water resources are scarce. It has been demonstrated that 90 to 98 percent of water can be reused, compared to traditional open flow farming systems. It also has other advantages, such as: energy savings, maximization of production under water and space limitations, minimization of effluent problems by reducing waste discharges to the environment and controlling and regulating water quality parameters of the crop, among others. On the one hand, experiences in farming demonstrate that these systems can intensively produce up to 25–30 kg/m3 of rainbow trout and about 80 kg/m3 of salmon. On the other hand, in semi-closed aquaculture systems with water recirculation, rainbow trout productions of 6,257 kg per year (120 kg per week) have been reported, with a maximum biomass of 66 to 74.6 kg/m3 .

In this innovative technology, some advantages stand out: a) flexibility in the selection of the farming site related to the possibility of using a small amount of water; retaining waste; manipulating the farm medium (especially temperature) and avoiding the entry of organisms from the natural environment or their exit to it, b) biosafety, since it avoids the entry and exit of pathogenic organisms or the exit of specimens of the cultivated species to the natural environment. Moreover, by having direct control over the growing conditions, optimal growing conditions can be kept independent of environmental variations, thus reducing the risk of diseases and ensuring productive yields, c) expedite treatment of certain diseases as it is simpler to handle and minimize the amount of product needed for treatments by immersion, in conditions of relatively high farm density, d) reduction in water consumption, e) reduction in the amount of waste and the possibility of treating it, thus avoiding possible impacts on the environment, f) scaling and replicability in other sectors of the foothills of the region and the rest of the northern side of the country, and g) possibility of integrating renewable energy systems. As of today, there are no viable renewable energy systems due to the volumes and consumption required, but which are perfectly compatible, such as solar energy.

#### **5. Proposed recirculation system for trout farming in the pre-cordillera sector**

The closed water recirculation system installed at the CCPC (**Figure 3**) consists of 6 circular Australian-type ponds for intensive trout production. It is provided with a central drainage system and hydraulic connections for water, air and oxygen *Integrated Culture of* Oncorhynchus mykiss *(Rainbow Trout) in Pre-Cordilleran Sector under… DOI: http://dx.doi.org/10.5772/intechopen.98920*

#### **Figure 3.** *Schematic of the recirculation system employed in the Rainbow trout farming at the CCPC.*

supply; a system of fiberglass ponds located below ground level, including two sedimentation ponds and a pond with biofilters. In addition, two 1.5 hp. water suction pumps, two 1 hp. high pressure blower and an emergency oxygen generator.

The ponds (Australian type) for intensive trout production are made of corrugated galvanized steel with a diameter of 5.4 m and a nominal height of 1.76 m, with a maximum water volume of 40 m3 . The water reaches each pond through a central distribution pipe and lateral outlets, which supply water to each farming unit. The flow is controlled by PVC valves, which, due to their arrangement, generate a circular water movement.

Each pond is internally covered with a 1.0 mm thick black non-toxic liner or geomembrane, which acts as a waterproofing layer. In the center of each pond there is a drain with a 110 mm diameter outlet and a drainage system with 5 mm diameter openings.

In addition, there is an air distribution pipeline that, by means of a blower, allows continuous aeration and oxygenation of the water column to ensure high levels of dissolved oxygen.

Furthermore, there is an oxygen pipeline distributed to all the ponds and that, by means of an oxygen generator will supply a high concentration of this gas, in case of emergency.

The water leaving the six ponds passes through a PVC pipe below ground level until it reaches the sedimentation ponds. They feature an internal division to facilitate the sedimentation of suspended solids.

Waste accumulated at the bottom of the sedimentation ponds is removed periodically through a submersible pump. There, fresh water is added daily to maintain the volume of the pond, which represents approximately 1 to 2% of the total volume of the system. In addition, it has the purpose of removing the final product of the nitrification process (nitrate). Thus, it maintains the water level and compensates for evaporation and handling losses.

Water coming from settling ponds passes into a fluidized submerged biofilter, which contains an ample spectrum plastic substrate for the attachment of nitrifying bacteria. Such bacteria convert the ammonia nitrogen discarded by the fish to nitrites and subsequently to nitrates; molecules that are less harmful to aquatic organisms and, conversely, the main nutrient for most plants.

The nitrification process requires the addition of dissolved oxygen. Reason why this system takes advantage of the volume of air generated by the blower. The diffusion hoses are arranged in such a way that they allow a uniform movement of the biofiltration substrates. By doing so, ammonia nitrogen and oxygen are distributed over the entire surface of the biofilter avoiding the accumulation of solids in the biofilter.

The water from this pond will finally pass to the water accumulator pond, which has a volume of 10 m3 . In this pond the water is aerated before being sent by gravity to the six production ponds.

All power for the recirculation system (two 1.5 hp. pumps, one 1 hp. blower, one 0.5 hp. pump for the juvenile system) was provided by a 7 kw/hour photovoltaic plant, which had 28 photovoltaic panels of 250 watts each, two inverters and 24 batteries. The batteries operated 18 to 20 hours per day, occupying the oxygen generator the rest of the hours.

### **6. Water quality**

The chemical compounds dissolved in the water, as well as other physical factors that affect the water, merge together to form what is known as "water quality". In aquaculture systems, changes in water characteristics that improve the production of a crop should be considered as improvements in water quality,


#### **Table 1.**

*Physical and chemical parameters of farming water, before and after trout were introduced.*

*Integrated Culture of* Oncorhynchus mykiss *(Rainbow Trout) in Pre-Cordilleran Sector under… DOI: http://dx.doi.org/10.5772/intechopen.98920*

while those changes that reduce production are a consequence of a degradation of said water quality.

This is given by the combination of physical and chemical properties and their interaction with living beings. With respect to the farming of aquatic organisms, any water characteristic that affects in one way or another the behavior, reproduction, growth, yields per unit area, primary productivity and management of aquatic species is a water quality variable.

Since one of the main objectives of aquaculture is to obtain the best yields possible, it is necessary to be thoroughly aware of ecological conditions in the ponds and the processes carried out there.

Within pisciculture parameters, water quality is of upmost importance. It must have adequate characteristics in terms of quantity (flow) and quality (physical, chemical and biological factors). Physical properties, such as temperature, pH, oxygen, transparency, turbidity, among others, may be subject to sudden variations due to the influence of external factors —mainly atmospheric and climatic changes. Chemical properties, however, are much more stable and their variations are minimal. Only in exceptional cases contamination can produce irreversible effects. From a biological stand, water quality is conditioned by the absence or presence of living organisms in the aquatic ecosystem, as well as by the greater or lesser presence of pathogenic agents.

Water quality in the Copaquilla trout farm (**Table 1**) is within normal ranges. Exceptional were some parameters in specific conditions, outside the optimal range, with no result in harming the crop itself.

In general, water used for trout farming complies within Chilean standard NCh1333.Of78 Mod. 1987, regarding the maximum limits allowed for water used in aquatic life farming.

#### **7. Acquisition and transport of rainbow trout specimens**

Trout were purchased at Rio Blanco fish farm, a department of Universidad Católica de Valparaíso, located in the city of Los Andes, Valparaíso Region.

The truck used to transport the trout was equipped with a thermos that facilitated the regulation of the internal environmental temperature during transport. A support vehicle was guarding it in case of emergency. These vehicles were disinfected (**Figure 4**) before entering Rio Blanco fish farm, in accordance with the fish transport procedures established by Servicio Nacional de Pesca y Acuicultura (SERNAPESCA) in Chile (or National Fisheries and Aquaculture Service).

Once the truck arrived at Rio Blanco fish farm, 8 ponds of 1 m3 each inside of it were filled with water (**Figure 5**). The ponds housed the fish during the transfer, being loaded at a rate of 625 fish per tank. Once the loading was completed, the outlet temperature and oxygen were recorded, and the ponds were carefully closed.

Parameters mentioned previously were taken (**Figure 6**) during the first 6 hours of the trip and visual evaluation was carried out by a technical team in charge of the transport. Behavior of the animals was evaluated, mainly swimming and opercular movement. During the following 12 hours, parameters were measured, and visual evaluation was carried out every two hours. Twelve more hours later, parameters were taken every three hours. For the last 15 hours they were measured every 5 hours. Thus, completing a total of 45 hours of travel.

Once the fish arrived at the top of Copaquilla, they were transferred to 800-liter water ponds on a pickup truck that carry them to the farm. Eight trips were made to empty the eight ponds. Once all the fish were in the farming center, they were distributed in three 40 m3 ponds in equivalent number of fish.

#### **Figure 4.**

*Disinfection of truck and support vehicle, for rainbow trout transport.*

**Figure 5.** *Filling of water and fish to be transported into the ponds.*

**Figure 6.** *Measurement of parameters during the transport of rainbow trout.*

### **8. Development of the trout farming system**

#### **8.1 Trout fattening**

A large part of the productive efficiency of a commercial fish farm is determined by feed management. For it to be successful, it is essential that farms develop a database where the indispensable parameters for a correct management *Integrated Culture of* Oncorhynchus mykiss *(Rainbow Trout) in Pre-Cordilleran Sector under… DOI: http://dx.doi.org/10.5772/intechopen.98920*

**Figure 7.** *Rainbow trout growth in the CCPC.*

of feeding are recorded. Among these it is worth mentioning; number of individuals corresponding to each pond, mortality, average body weight, growth evolution, feed supply and water temperature. Once the numbers are in, it is possible to calculate the ration to be supplied to each pond as well as the feeding efficiency of each one.

The fish were sampled every 15 days. Between 5 and 7% of the total fish per pond were sampled, in this way, it is possible to correct the variables used to calculate the feed ration to be supplied month by month. The number of fish in the ponds was calculated as the difference between the initial number of the crop minus the number of dead individuals. Mortality was recorded daily to keep numerical control of the fish. With the updated number of individuals per pond and the average body weight records, the total biomass of the crop could be determined (**Figure 7**). After several samplings, the growth rate of the fish could be calculated by the difference in average weights, allowing for definition of trout harvest.

For an optimized determination of the feed ration supplied to the trout, the biomass, water temperature and average individual body weight in the pond must be known. Initially, the commonly disseminated feeding tables for the species can be used as a guide. However, we elaborate our own feeding table under specific conditions of the crop. Under this concept the fish were fed four times a day at a rate of 3 to 4% of the fish biomass in the pond. The conversion factor fluctuated between 1.2 and 1.35.

According to general results obtained in the present study, it is not recommended to apply any type of food restriction when the trout are growing within the optimum temperature range for the species, i.e., they were fed *ad-libitum.*

#### **8.2 Conditioning of breeding specimens at CCPC**

After one year of farming, 30 specimens were selected out of the 6,000 15-gram fish that initially arrived. These were separated in a 40 m3 pond as potential broodstock. A density of 1 kilo of fish per m3 was considered, feeding was supplied three times per week, twice with trout broodstock pellet and once with normal fattening pellet. Water flow was 120 liters per minute.

Fish were selected mainly for their phenotypic appearance, rapid growth rate and presenting no deformities. After one year they had an average weight ranging from 1,300 to 1,500 grams.

#### **8.3 First trout spawning and hatching at the CCPC**

In August 2017, the trout had their first spawning. This process was carried out through an abdominal massage on the female, which expelled her oocytes over a dry stainless-steel container. Already having the females spawn, the males were similarly massaged (two to three males per female). Once the gametes were in the container, they were activated by introducing clean water, which was also used as a mean to wash the eggs. This process was carried out several times until the water turned completely transparent. The eggs were then placed in trays adapted for incubation (**Figure 8A**) in raceway-type ponds of approximately 550 liters. After the first 48 hours, the eggs in the trays were cleaned, removing dead eggs in the process (**Figure 8B**) and finally leaving them unhandled until the eyed stage [10].

About 15,000 eggs were fertilized and hatched, reaching approximately 10,500 eggs to larvae (**Figure 9**), 6,500 to juveniles and 2,400 to 160-gram adults as of June 2018. It should be noted that a large percentage of animals were removed at each stage due to the limited space available at the CCPC.

Water temperature in the hatchery trays varied between 7 and 10 degrees at night and between 12 and 15 degrees during the day. The water flow in the racewaytype ponds was 5 liters per minute. For each pond there were four trays that held the eggs. Each tray was provided with a wire mesh in front of the water flow and at the bottom of the tray, which allowed water circulation and oxygenation.

Once the eggs hatched in the trays and the larvae absorbed the yolk sac, they were transferred directly to the raceway ponds, where they were kept for 20 to 25 days (**Figure 10**).

**Figure 8.** *Trout eggs (A), extraction of dead eggs (B).*

**Figure 9.** *Eggs in hatchery trays (A), rainbow trout larvae in the CCPC (B).*

*Integrated Culture of* Oncorhynchus mykiss *(Rainbow Trout) in Pre-Cordilleran Sector under… DOI: http://dx.doi.org/10.5772/intechopen.98920*

**Figure 10.** *Raceway pond, hatchery trays A, juveniles B.*

**Figure 11.** *Trout fry ponds.*

**Figure 12.** *Trout fattening ponds of 40 mt3 .*

Temperature and water parameters were maintained during the time juveniles were in these ponds. Once the stage in the raceway ponds was completed, juveniles were transferred to the 450-liter fry tanks (**Figure 11**). They remained there for a month, approximately.

This system had a completely independent SAR, with a 0.5 hp. pump, 0.2 hp. blower and 80 w UV lamp. Temperature parameters fluctuated between 9 and 12°C at night and 12 and 16°C during the day. Water flow was 8 to 10 lt m−1. After thirty days, the fish were transferred to a 40 m3 tank (**Figure 12**). They were to remain there until they reached 160 grams, at which point the study was completed and splitting and grading were performed.

#### **9. Problems addressed in trout farming**

Trout farming in the Copaquilla sector was not exempt from problems inherent to fish farming and the climatic conditions of a sector located at 3,000 mamls.

Immature biofilter, massive proliferation of microalgae and accumulation of organic matter at the bottom of the water storage pond, were recurrent problems at the beginning of the culture.

They led to rapid responses in order to ensure the survival of the fish. One of the most worrying natural factors were summer rains, known as "the altiplanic winter". The natural phenomenon causes rains and cloudy days that affect the efficient operation of the solar panels, a system on which the energetic exercise of the equipment (pumps, blower, oxygen generator, etc.) depends.

#### **10. Prospects for viability and sustainability of the Copaquilla Pukara farming center**

The implementation of an aquaculture system in the pre-Cordilleran region made it possible to disseminate knowledge about it and to predict its viability under the surrounding conditions (quality of the water). In order to implement this type of technology on a large scale in the future, both for trout farming systems and for other species, the first step had to be made. Therefore, it is a means by which companies and entrepreneurs can obtain investment resources that allow for the beginning of new companies. This is based off the need to diversify aquaculture and the region's need to promote it; a strategic axis for the region of Arica and Parinacota. In addition, this aquaculture technology is highly suitable for coupling it with solar energy, as it has been demonstrated at the CCPC.

This initiative also poses a potential for entrepreneurship and innovation through the implementation of aquaculture and training of personnel in the region. Newly trained personnel would be capable of operating the crops, analyzing water quality, applying growth rates, among other activities. This, in turn, will allow new studies to be carried out, complementing the ones related to economic activities in which aquaculture plays an important role. The implemented equipment will allow the follow through continuum of the procedures, generating positive externalities such as internships for high school and university students.

Finally, the CCPC is an important business opportunity for small companies in the region that consider aquaculture in surrounding communities and for the sustainability and permanent operation of aquaculture production. In general, the CCPC will generate a preponderant added value that will help to promote and strengthen small businesses. Regarding environmental mitigation measures and risk prevention, the project is entirely sustainable from an environmental stand,

*Integrated Culture of* Oncorhynchus mykiss *(Rainbow Trout) in Pre-Cordilleran Sector under… DOI: http://dx.doi.org/10.5772/intechopen.98920*

as it allows water to be continually reused. Furthermore, this productive activity is intended to prevent the migration of young people from their native villages, providing them with a work alternative and specialization in aquaculture. Lastly, the implementation of the farm and other equipment does not create a significant visual impact as it is installed in a small area similar to local structures.

### **Author details**

Renzo Pepe-Victoriano1 \*, Héctor Aravena-Ambrosetti1 and Piera Pepe-Vargas2

1 Área de Biología Marina y Acuicultura, Facultad de Recursos Naturales Renovables, Universidad Arturo Prat, Arica, Chile

2 Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Concepción, Chile

\*Address all correspondence to: rpepev@unap.cl

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Pepe-Victoriano, R. M. Araya, C. Wurmann, J. Mery, A. Vélez, P. Oxa, C. León, S. Fuentealba & R. Canales. 2015. Estrategia para el desarrollo de la Acuicultura en la región de Arica y Parinacota, 2015-2024. Informe Final CORFO. 453 pp.

[2] Pepe-Victoriano, R & H. Aravena-Ambrosetti. 2018. Primer Centro de Cultivos de Truchas arcoíris bajo un sistema de Recirculación en el sector de Copaquilla, Región de Arica y Parinacota. Rev. Versión Diferente 15: 58-62.

[3] Timmons, M. J. Ebeling, R. Piedrahita. 2009. Acuicultura en Sistemas de Recirculación. NRACE Publication Nº 101-209 Spanish. 959 pp

[4] Food and Agriculture Organization (FAO), 2016. El estado mundial de la pesca y la acuicultura. Organización de las Naciones unidas para la Agricultura y la Alimentación, Departamento de Pesca y Acuicultura, Rome.

[5] Cornejo-Ponce, L., Vilca-Salinas, P., Lienqueo-Aburto H., Arenas, M., Pepe-Victoriano, R., Carpio, E., and Rodriguez J. 2020. Integrated Aquaculture Recirculation System (IARS) Supported by Solar Energy as a Circular Economy Alternative for Resilient Communities in Arid/Semi-Arid Zones in Southern South America: A Case Study in the Camarones Town. Water. MDPI. 12, 3469; doi:10.3390/ w12123469

[6] Gall G & Crandell P (1992) The rainbow trout. Aquaculture 100, 1-10

[7] Groot C (1996) Chapter 3 Salmonid life histories. Developments in Aquaculture and Fisheries Science 29, 97-230.

[8] Laird L & Needham T (1988) The farmed salmonids. In: L.M. Laird and T. Needham (Editors), Salmon and Trout Farming. Ellis Horwood, Chichester 15-31.

[9] Espinós A (1995) El alimento - presa que encontraron las truchas en la Argentina Irene Wais. Boletín mosquero AAPM invierno de 1995. Los salmónidos (enfoque científico) APMN Cornell University - Department of Natural Resources Novascotia - Canada - Agriculture and Fisheries

[10] Blanco, M. 1995. La Trucha, Cría Industrial. Ediciones Mundi-Prensa, Madrid-España. 503. pp.

#### **Chapter 3**

## Developments in Probiotic Use in the Aquaculture of *Salmo* Spp.

*Alexander Dindial*

#### **Abstract**

While interest in probiotic use in aquaculture is not a new phenomenon, the past few years have seen great developments in probiotic research in *Salmo* spp.. This review examines the corpus of literature surrounding the use of probiotics in some of the species of *Salmo* most important to modern aquaculture, including *Salmo salar*, *S. coruhensis*, *S. trutta,* and *S. trutta caspius,* with a particular emphasis on the most recent research. The use of many of these probiotics is associated with such host benefits as enhanced growth, nutrition, and immunity. These benefits and the potential applicability of these probiotics to the modern aquaculture of *Salmo* are reviewed herein.

**Keywords:** Salmo, Atlantic salmon, brown trout, Coruh River trout, Caspian brown trout, *S. salar*, *S. trutta caspius*, *S. coruhensis*, *S. trutta*, fish probiotics, aquaculture probiotics

#### **1. Introduction**

As in other animals, the gut microbiome of fish is a dynamic, complex, organlike system that may contain trillions of microorganisms. It is implicated in a number of functions critical to the animal's health, including digestion, immunity, and nutrient absorption, which may in turn contribute to such things as development, growth and metabolism [1].

There are a few ways by which these salubrious phenomena are known to occur. In terms of immunity, some beneficial bacteria may produce such things as bacteriocins, peroxides, and acids in order to inhibit the growth of pathogenic microorganisms, and have further been observed to mitigate damage caused to the intestines by pathogens [2, 3]. Furthermore, some bacteria have been known to promote the host fish's immunity through such means as the enhanced infiltration of epithelial leukocytes, the modulation of cytokine and chemokine expression, enhanced leukocyte activity, and enhanced lysozyme activity in the cells of the mucosa [4–8]. On the other hand, some bacteria have been known to enhance fish digestion and nutrient absorption through such things as increasing the length of microvilli, enhancing the fold length of the mucosa, secreting various digestive enzymes, and by fermenting non-digestible compounds (e.g., complex carbohydrates) to make them usable [4, 9–11].

In order to take advantage of some of these beneficial health effects, it is critically important that fish reared in aquaculture maintain healthy gut microbiota. This may help ensure the success of aquaculture operations by preventing the spread of disease and promoting the healthy growth of fish to market size. To this end, there has been a considerable amount of research on the fish microbiome and the use of probiotics in fish significant to the field of aquaculture. In general, the term "probiotic" typically refers to a sample of live microorganisms that is used in order to confer some kind of health benefit [12]. In many cases, probiotics are consumed in order to allow the microorganisms to enter the organism's gut, from which point they can exert their salubrious effects. In some cases, probiotics are combined with prebiotics, which are compounds designed to bolster the growth of beneficial bacteria. In many cases, these oligosaccharides can be digested by the bacteria, but not the host. The combination of probiotics and prebiotics is sometimes referred to as synbiotics [12].

Of the aquaculturally significant fish in which probiotics have been studied, genus *Salmo* is perhaps among the most important. This genus contains a number of species relevant in modern aquaculture, including *Salmo salar* (the Atlantic salmon), *Salmo trutta* (the brown trout), *S. trutta caspius* (the Caspian trout/salmon) and *S. coruhensis* (the Coruh River trout), *inter alias*. Of these species, *S. salar* is perhaps the most important to global aquaculture. According to the Food and Agriculture Organization of the United Nations, farmed *S. salar* comprises greater than 90% of all farmed salmon and greater than half of all salmon production worldwide [13].

Underscoring the importance of *Salmo* in modern aquaculture, there is a rapidly growing corpus of literature surrounding the use of probiotics in this genus. The benefits to fish immunity and growth that stem from this research may have the potential to greatly benefit the salmon aquaculture industry, thus necessitating further review of the recent pertinent literature.

#### **1.1 Overview of the gut microbiota of** *Salmo spp.*

To understand the use of probiotics in *Salmo*, it is important to first critically examine this genus' gut microbiota. The intestinal mucosa of *S. salar* can harbor trillions of microorganisms, the majority of which are bacteria. Typically, the identity of these microorganisms may vary considerably depending on several variables, including diet, the bacteria present, biogeography and environmental factors, stress, captivity, disease, the host's species, and life cycle stage [14–19]. To further complicate matters, not all microorganisms in the salmon gut are permanent residents. While many bacteria are indeed capable of colonizing the intestinal mucosa in the long-term, the presence of others may only be transient [20]. Such transience may be due to a number of factors, including the inability to compete with bacteria that have already colonized the gut for such things as nutrients or space, direct inhibition by pre-existing bacteria (e.g., via the secretion of antimicrobial peptides), or even just the general inability to colonize the intestinal tract [2, 21].

Regardless of this dynamism and complexity, researchers have identified a diverse array of bacteria that may inhabit the digestive system of *Salmo*. In one study, the mid and distal intestinal mucosa of *S. salar* kept in seawater were found to contain bacteria from a number of phyla, including Proteobacteria (which comprised ~90% of all mucosal bacteria), Actinobacteria, Armatimonadetes, Spirochaetes, Bacteroidetes, and Firmicutes [20]. Furthermore, in the digesta of *S. salar*, Proteobacteria, Firmicutes, Fusobacteria, and Bacteroidetes (among other less abundant bacteria) were documented [20]. In another study, the digesta of *S. salar* parr reared in a freshwater loch or a recirculating aquaculture system (RAS) were also found to contain representatives of other additional phyla, including Tenericutes and Acidobacteria, among others [22]. In a different study featuring wild *S. salar* at different life cycle stages, Nitrospirae were also found to be abundant in the digestive tracts of parrs, smolts, and adults. This study also found that

*Developments in Probiotic Use in the Aquaculture of* Salmo *Spp. DOI: http://dx.doi.org/10.5772/intechopen.99467*

Firmicutes, Actinobacteria, and Bacteroidetes occur at far lower levels in marine adults than in freshwater life stages [18]. Overall, these phyla are represented by dozens of genera in the *S. salar* microbiome, notably including *Carnobacterium*, *Lactobacillus*, *Pediococcus, Lactococcus*, *Vibrio*, *Pseudomonas*, *Aeromonas*, *Yersinia*, and *Mycoplasma*, many of which will be discussed further in this review [18, 20].

#### **2. Probiotic use in** *Salmo*

In general, colonization is critically important to the establishment of probiotic bacteria within a fish's digestive tract. There are a few main considerations behind this probiotic colonization. Firstly, the bacteria must not pose any danger to the host fish or be otherwise pathogenic. Instead, they should exhibit salubrious effects as described previously. Secondly, it is critical that the bacteria are able to reproduce successfully within the fish's gut, such that their rate of multiplication exceeds the rate at which they are expelled [2]. Thirdly, the bacteria must be able to adhere to the intestinal mucosa. There are a few additional considerations to this third point. For example, the bacteria must be able to successfully compete with other bacteria for adhesion space. Some bacteria (including some lactic acid bacteria) are known to have specific adhesion sites on the intestinal epithelium, while others are known to adhere non-specifically [2, 23, 24]. These considerations are critically important to all potential fish probiotics.

*Salmo* **species Bacterial genus Bacterial species Citation** *Salmo trutta caspius Bacillus B. subtilis & B. licheniformis\** [25–27] *Pediococcus P. acidilactici\*\** [28–30] *Lactobacillus L. plantarum* [31] *Salmo salar Carnobacterium Carnobacterium* spp. [9] *C. divergens* [3, 32] *Pediococcus P. acidilactici\*\** [11, 33] *Lactobacillus L. delbrueckii* [34]

**Table 1** provides an overview of the bacterial genera that have been investigated as probiotics in genus *Salmo*:

*Asterisks denote the use of commercial probiotic formulations: one asterisk denotes BetaPlus ® and two asterisks denote Bactocell ®. These products are discussed in greater detail below. See Table 2 for an overview of other probiotic formulations.*

#### **Table 1.**

*An overview of different bacterial species investigated as probiotics in genus Salmo.*

#### **2.1** *Carnobacterium*

*Carnobacterium* is a genus of Gram-positive lactic acid bacteria (LAB) within the phylum Firmicutes. They are ubiquitous in nature and can survive low temperatures and anaerobic conditions with elevated concentrations of carbon dioxide [35]. In terms of fish, some *Carnobacteria* (such as some strains of *Carnobacterium maltaromaticum*) have been known to be pathogenic in certain salmonids (e.g., *Oncorhynchus mykiss*), while others have been found to exhibit beneficial effects within the gut microbiome [9, 32, 35, 36]. Perhaps one of the most important benefits of probiotic *Carnobacteria* is their ability to inhibit the growth of pathogenic bacteria within the *Salmo* gut. This ability is most likely due to *Carnobacteria's* ability to produce antimicrobial bacteriocins, which may serve to both inhibit pathogenic bacteria and help the *Carnobacteria* to survive within the competitive environment of the gut microbiome [37]. However, the ability of probiotic *Carnobacterium* to inhibit different pathogenic species is known to vary by strain [32, 37]. As a type of LAB, *Carnobacteria* are also capable of lactic acid production, which may also have an inhibitory effect on pathogens. Further recent research has also suggested that the presence of *Carnobacterium* in the pyloric caeca of *S. salar* is associated with enhanced flesh color. While it is hypothesized that this may be related to the production of carotenoids by the *Carnobacteria* as well as their proimmune effects, the true reason for this phenomenon remains unclear [38].

In an early study conducted in 2000, it was revealed that *Carnobacterium* strains isolated from the intestine of *S. salar* could inhibit a number of pathogenic bacteria, including *Aeromonas hydrophila, Aeromonas salmonicida, Vibrio ordalli, Vibrio anguillarum, Streptococcus milleri, Photobacterium damselae piscicida,* and *Flavobacterium psychrophilium in vitro* [9]. In this study, it was also found that the administration of these *Carnobacteria* for at least fourteen days was able to promote the survival of *O. mykiss* in the context of infection by *A. salmonicida, Yersinia ruckerii,* and *V. ordalli.* This study also found that it took 28 days of probiotic administration to achieve the maximum intestinal *Carnobacterium* levels, and that the cessation of probiotic administration in fry and fingerlings resulted in the bacteria becoming undetectable in the gut in ten days or less [9]. It was further confirmed in another study the coincubation of *C. divergens* strain 6251 with the pathogenic *A. salmonicida* and *V. anguillarum* was able to prevent (but not alleviate) damage to *S. salar* microvilli [3]. A later study demonstrated that the administration of a commercial prebiotic (namely EWOS prebiosal) to help promote probiotic bacterial growth vastly enhanced the ability of *C. divergens* to adhere to the epithelia and mucosa of the proximal (but not distal) intestine of *S. salar* [32].

Overall, certain strains of genus *Carnobacterium* (such as the aforementioned *C. divergens* strain 6251) may have great potential as a probiotic for *S. salar*, with the ability to bolster the fish's immunity and perhaps even flesh color. While it is possible that the probiotic may need to be frequently readministered, the use of a commercially available prebiotic may be beneficial in enhancing bacterial adhesion to the salmon gut.

#### **2.2** *Pediococcus*

*Pediococcus* is a genus of Gram-positive LAB within the phylum Firmicutes. It is an acidophilic, facultative anaerobe with well-established probiotic properties, even in humans. Like *Carnobacterium*, *Pediococcus acidilactici* is known to produce bacteriocins and lactic acid, which are potentially useful in inhibiting the propagation of pathogens within the digestive tract [39].

*P. acidilactici* has been investigated as potential probiotic in both *S. salar* and *S. trutta caspius.* In both fish, much of this research has been conducted using Bactocell ®, a commercially available strain of *P. acidilactici* (strain MA 18/5 M) that is also used in other animals of agricultural significance [28–30]. It is one of the only aquacultural probiotics approved in the European Union [33]. Overall, Bactocell ® seems to exhibit some promise in promoting the immunity and growth of *S. trutta caspius*. In one study, Bactocell ® was found to significantly decrease the feed conversion ratio in *S. trutta caspius* following five treatments with the probiotic. Furthermore, white blood cell concentrations were noted to have increased, suggesting pro-immune effects related to the presence of the probiotic. Curiously, red blood cell counts were also found to be lowered relative to the control group that did not receive the Bactocell ® treatment [30]. In another study in which Bactocell

#### *Developments in Probiotic Use in the Aquaculture of* Salmo *Spp. DOI: http://dx.doi.org/10.5772/intechopen.99467*

® was co-administered with iron, parameters like body weight gain and specific growth rate were also found to be enhanced relative to the control group [28].

This same strain of *P. acidilactici* (MA 18/5 M) has also been investigated as a probiotic in *S. salar.* In one such study, saltwater Atlantic salmon were administered *P. acidilactici* MA 18/5 M and short chain fructooligosaccharides (as a prebiotic) twice a day for 63 days. Among other things, the researchers noted that this synbiotic (a combination of a probiotic and prebiotic) modulated both local and systemic immunity. For example, it was found that synbiotic administration was associated with the increased expression of the pro-inflammatory cytokines interleukin-1β, tumor necrosis factor α, and interleukin in the intestinal tissue, in addition to increased expression of the antiviral molecules toll-like receptor 3 and myxovirusresistant protein 1. Furthermore, the researchers observed increased epithelial leukocyte infiltration in the intestines and elevated serum lysozyme levels. Notably, the fish that received the synbiotic also exhibited greater villus length relative to the control and decreased but recoverable intestinal bacterial loads (without adverse health consequences) [11]. In another study, it was found that Bactocell ® was capable of modulating several parameters related to distal intestine inflammation, which overall helped to counteract the inflammation [40]. A later study further studied the administration of *P. acidilacti* MA 18/5 M to *S. salar*, in addition to also considering the effects on the gut microbiome that occur with the transition from the freshwater and saltwater stages. Overall, *Pediococcus* was found to significantly impact the Atlantic salmon microbiota, as in the previous study. Notably, among the fish that received the probiotic treatment, *Pediococcus* was present in greater abundance in both the digesta and mucosa in the freshwater salmon than in the saltwater salmon. In spite of this, the effect of the probiotic treatment on the composition of the gut microbiota was found to be the greatest among the saltwater fish [33].

In summary, there is some evidence to suggest that *P. acidilactici* MA 18/5 M may exhibit positive effects on the growth of *Salmo* spp., as well as significant immunomodulatory effects. Furthermore, its availability on the market may make this strain an enticing choice for those interested in using effective probiotics in their aquaculture systems.

#### **2.3** *Bacillus*

*Bacillus* is a diverse genus of Gram-positive bacteria within the phylum Firmicutes. There are three main species of *Bacillus* that have been investigated as probiotics for *Salmo*, the two most notable of which are *B. subtilis* and *B. licheniformis*. While *Bacillus* species are not LAB, they have nevertheless been successfully implemented as probiotic agents in aquaculture, exhibiting the ability to enhance growth and immunity in a few species [41, 42].

In terms of *Salmo*, *B. subtilis* DSM 5749 and *B. licheniformis* DSM 5750 have been investigated as probiotics in *S. trutta caspius* in the form of the commercially available product known as BetaPlus ®. In one study, Caspian salmon fingerlings were administered a synbiotic composed of BetaPlus ® and galacto-oligosaccharides. In comparison to the control (which did not receive the synbiotic), the group that received the synbiotic exhibited superior performance in parameters relevant to growth and immunity (among other things). In terms of growth, this includes a lower feed conversion ratio and higher weight gain and protein efficiency ratios. As for immunity, the fish that received the probiotic exhibited increased serum levels of lysozyme, immunoglobulins, bactericidal peptides, agglutinins, lectins, and albumin [42]. In another, similar study with the same species, BetaPlus ® was used in conjunction with isomaltooligosaccharides as the prebiotic. In addition to some similar findings to the previous study, the synbiotic group was found to exhibit greater levels

#### *Salmon Aquaculture*

of monocytes, leukocytes, and neutrophils compared to the control, which is further suggestive of enhanced immunity in the context of synbiotic usage [25].

Overall, these findings suggest that the administration of BetaPlus® in conjunction with a prebiotic may be able to significantly enhance both growth and systemic immunity in *S. trutta caspius*. For those involved in the commercial aquaculture of *Salmo* spp., it may be beneficial to consider the use of BetaPlus® or similar probiotic formulations.

#### **2.4** *Lactobacillus*

*Lactobacillus* is a genus of Gram-positive LAB within the phylum Firmicutes. Two species in this genus have been investigated as probiotics for species within *Salmo*, namely *Lactobacillus delbrueckii* in *S. salar* and *L. plantarum* in *S. trutta caspius.*

The use of *Lactobacillus delbrueckii lactis* as an aquacultural probiotic has been known to enhance the innate immune response in other fish, such as *Sparus aurata* [43]. In terms of the Atlantic salmon, one study examined this bacterium's ability to remain on the surface of the intestines in an *in vitro* model containing intestinal tissue from an Atlantic salmon, as well as its ability to prevent damage in the context of an *Aeromonas salmoncida salmoncida* infection. Overall, it was found that the *Lactobacillus* was able to persist on the surface of the intestine, and further caused no damage to the tissue (in contrast to the *Aeromonas*). When the *in vitro* intestine model was co-incubated with both the *Lactobacillus* and *Aeromonas*, the former prevented damage to the tissue caused by the latter [34]. Overall, this is likely suggestive of the ability of *L. delbruecki lactis* to contribute to host innate immunity in *S. salar.*

Further work in *S. trutta caspius* provides some evidence for the ability of *L. plantarum*-based synbiotics to promote both host growth and immunity. In one study, Caspian salmon were assigned into eight groups, which featured combinations including the fishes' basal diet, *L. plantarum*, and the prebiotics beta-glucan and mannan oligosaccharide. All groups featuring probiotics and/or prebiotics exhibited decreased feed conversion ratios and feed intake, as well as increased weight gain and protein efficiency ratios. These same groups also exhibited enhanced parameters relevant to immunity, including elevated levels of immunoglobulin M, and lysozyme (*inter alia*). It is also important to note that with the exception of the beta-glucan group, the groups featuring *L. plantarum* exhibited lower cortisol and glucose levels than the other experimental group, suggesting yet another physiological benefit of the use of *L. plantarum* as a probiotic [31].

*In toto*, while there are not many available studies focusing on the use of *Lactobacillus* spp. as a probiotic in *Salmo* on their own, the promising findings related to growth, immunity, and decreased feed intake associated with this bacterial genus may warrant further investigation as a probiotic in *Salmo.*

#### **2.5 Other probiotics**

In addition to the probiotics discussed above, there are some other probiotics that have been investigated in *Salmo*, including those with multi-genus or variable composition:

#### *2.5.1 Kefir*

There has been some research on the use of kefir as a probiotic for *S. coruhensis*. Kefir is a fermented, dairy-based beverage with origins in the North Caucasus and

*Developments in Probiotic Use in the Aquaculture of* Salmo *Spp. DOI: http://dx.doi.org/10.5772/intechopen.99467*

has been used as a probiotic in humans. Kefir is known to contain a variety of different microorganisms, including representatives of *Lactobacillus, Lactococcus,* and *Leuconostoc*, as well as other LAB, acetic acid bacteria, and even yeasts [44–46]. Overall, the use of kefir as a probiotic in this species seems to offer some promise. In one study, it was demonstrated that groups of Coruh trout administered kefir exhibited decreased activity of catalase, an enzyme with antioxidant activity, as well as decreased levels of the highly reactive compound malondialdehyde in hepatic tissue. This may suggest that the probiotics associated with kefir may have antioxidant activity [44]. In another study with the same species, kefir administration at a dose of ten or twenty grams per kilogram was associated with elevated immunoglobulin, which may suggest that kefir's probiotics may also exhibit immunomodulatory effects in the Coruh trout. However, this same study did not find any difference in growth or survival rates between the control and experimental groups [45]. Finally, in another study, it was found that Coruh trout in the groups administered kefir also exhibited changes in digestive and hepatic enzyme expression and glucose levels relative to the control group. For example, it was found that serum amylase and lipase levels, as well as serum glucose decreased, suggesting that kefir administration may modulate digestion in *S. coruhensis*. Notably, glucose levels were also found to be lower in the kefir groups, which may be suggestive of decreased stress or decreased carbohydrate absorption in the intestines [46].

Overall, kefir may show some promise as a probiotic for *S. coruhensis*, especially considering the relative ease by which it may be acquired and its antioxidant and immunomodulatory properties. However, its inability to affect host growth or survival rates (unlike other previously discussed probiotics) may warrant careful consideration when choosing it as a probiotic for *Salmo* spp..

#### *2.5.2 Other multi-species probiotic formulations*

Other probiotic formulations featuring multiple species of microorganisms have also been investigated. One notable example of such a formulation is Bio-aqua ®, an aquacultural probiotic that is commercially available in Iran. In addition to the species enumerated in **Table 2**, Bio-aqua® further contains yeast extract and fructooligosaccharides as prebiotics. In the sole English language publication investigating this formulation, it was found that Bio-aqua did not produce any significant effects on the growth performance, activity of digestive enzymes, or intestinal histomorphology in juvenile *S. trutta caspius* when administered at a dose of 0.2 grams per kilogram at feeding times [50].

In another study, four experimental groups of juvenile *S. salar* reared in a RAS were administered combinations of *Rhodotorula mucilaginosa* CGMCC 1013 and *Bacillus velezensis* V4 CGMCC 10149 over a 62-day period. Relative to the control group, the experimental group fish exhibited significantly decreased feed conversion ratios and mortality, as well as increased weight gain ratios and specific growth rates. Furthermore, immunological and antioxidant parameters were suggestive of an enhanced immune response and antioxidant activity in the experimental groups in comparison with the control group. Curiously, however, cortisol levels were found to be elevated in the experimental groups relative to the control. Finally, in a challenge trial conducted with *Aeromonas salmonicida*, the experimental group fish exhibited far lower mortality rates than the control group, perhaps due to a combination of the enhanced immune response and the direct inhibition of the pathogen by the probiotic bacteria in the gut [51].

Overall, in spite of the lack of observed benefits in the study featuring Bioaqua ®, the promising findings of the second study featuring *R. mucilaginosa* and


*microorganism. Three asterisks denote a hypothetical probiotic source.*

#### **Table 2.**

*An overview of probiotic formulations featuring multiple bacterial species that have been investigated for use in Salmo.*

*B. velezensis* suggest that the utilization of multiple species of bacteria in probiotics has the potential to be highly productive.

#### *2.5.3 Fermented soybean meal*

There has also been considerable research on the use of fermented plant meals (such as soybean meal) in the aquaculture of *Salmo,* including *S. salar, S. trutta,* and *S. trutta caspius* [47–49]. However, it is important to note that this usage of plant meal fermentation is often implemented to enhance the bioavailability of nutrients and energy in plant products that are not natural components of the salmonid diet [52]. Regardless, it is possible that some fermented plant meal products may exhibit probiotic or prebiotic effects in *Salmo.* In one study, the provision of fermented soybean meal to *S. salar* was found to be associated with an increase in the abundance of intestinal LAB (including *Lactobacillus, Pediococcus,* and *Lactococcus*) when compared with a group that was fed a fishmeal-based diet and another that received non-fermented soybean meal [21]. While it is unclear from the results of the study whether this increase in LAB abundance was due to prebiotic or probiotic effects, it is well-established that certain fermented foods (such as miso, which is also soybean-based) are associated with probiotic LAB populations [53, 54]. Therefore, it is plausible that at least some fermented soybean feeds for *Salmo* may exhibit probiotic effects. However, further research may be needed in order to investigate this hypothesis.

#### **3. Conclusions**

In general, the use of probiotics in the aquaculture of species within the genus *Salmo* has the potential to be highly productive, with the ability to promote fish immunity, nutrition, and growth as well as to decrease the likelihood of mortality. These beneficial effects are more important now than ever, with the increasing popularity of intensive aquaculture systems like RAS. In such crowded conditions, fish are constantly exposed to pathogens, underscoring the need for enhanced

#### *Developments in Probiotic Use in the Aquaculture of* Salmo *Spp. DOI: http://dx.doi.org/10.5772/intechopen.99467*

immunity. Furthermore, the enhanced growth and decreased mortality from certain diseases associated with the use of some probiotics may help the operators of salmon aquaculture systems to increase their profits and minimize the unnecessary loss of fish. The current market availability of such tested probiotic formulations as BetaPlus ® and Bactocell ® may also be instrumental in helping salmon aquaculturalists to achieve these outcomes.

In spite of the benefits that can be understood from the current corpus of literature pertaining to the use of probiotics in *Salmo*, many questions remain unanswered. Some of these questions are quite simple, and could consider such things as which probiotic species and strains (or perhaps combinations thereof) are the best at achieving particular outcomes in particular species of *Salmo* (e.g., which species and strains result in optimal growth rates in *S. salar*). The answers to such questions would likely necessitate the conduction of relatively large studies featuring multiple experimental groups that each receive different probiotics. Other questions concern the biochemical activity of probiotic bacteria within the host, including such things as how or why certain probiotics influence the host's immune system (e.g., how does BetaPlus® cause elevated white blood cell levels in *S. salar* or, why does the administration of certain probiotics result in elevated immunoglobulin levels). It further remains unclear as to why some probiotic species and strains differ from others in certain effects on the host (e.g., why were host cortisol levels decreased relative to the control when *Lactobacillus plantarum* was used in *S. trutta caspius,* but elevated when *Bacillus velezensis* and *Rhodotorula mucilaginosa* were used in *S. salar*). The elucidation of answers to these (and related) questions may help us better understand the relationship between the salmon gut microbiota and fish health, as well as potentially inform future efforts to optimize the salmon gut microbiome with biotechnology.

Overall, while much remains to be explored in the use of probiotics in *Salmo*, recent findings have strongly indicated that they are associated with remarkable potential benefits that should warrant any *Salmo* aquaculturalist to consider their use.

#### **Author details**

Alexander Dindial Zanvyl Krieger School of Arts and Sciences, Johns Hopkins University, Baltimore, MD, USA

\*Address all correspondence to: adindia1@jh.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### *Salmon Aquaculture*

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Section 2
