During the past two decades, the use of biological agents, particularly in feed and as water additives, as an alternative to the use of antibiotics and chemicals has shown to be promising in aquaculture, particularly in fish and shellfish larviculture [43]. The concept of biological agents has been traditionally associated with the use of beneficial microorganisms to restore the microbial balance in the gastro-intestinal tract of the host and to treat or prevent diseases and/or disorders [44]. Biological agents are emerging as a significant microbial supplements in the field of prophylaxis [36]. Many studies to date have revealed the potential of these beneficial organisms to combat disease in an aquaculture environment [5, 45-51].

In aquatic ecosystems there is an intimate relationship between microorganisms and other biota in the environment [47]. Apart from the aquatic animal being surrounded by water, there is also a constant flow of water through the digestive tract of the aquatic animal. This conse‐ quently affects the synergistic balance of indigenous microflora associated with the cultured animal. The classical definition of a probiotic being that of microbes added to food, has become modified with respect to aquaculture, whereby a biological agent is used as a wider term and is defined as "a live microbial adjunct which has a beneficial effect on the host by modifying the host-association or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment" [47]. Some studies have shown that as a result of intensification of aquaculture farms a negative impact has been conferred on the composition of the different protective microbial flora interacting with the host [5]. This occurrence results in an increase in susceptibility of the host to diseases. It has become evident that augmentation of aquaculture systems with biological agents can lead to growth of beneficial bacteria thus improving overall health of the culture system and the host [5].

acids, amino acids and vitamins [63,64]. Some isolates also have the ability to break down potentially indigestible components of the feed thus reducing toxicity and improving feed conversion efficiency [23,63]. Abalones are in most instances, fed a diet consisting mainly of kelp, which is a complex macroalgal polysaccharide deficient in many essential nutrients [64]. It is therefore imperative that enteric bacteria in the abalone gut are present in sufficient amounts which will adequately facilitate digestion by supplying highly effective polysacchar‐ olytic enzymes [23]. Many bacteria displaying these properties have been found to exist throughout the digestive tract of *H. midae* [23,40]. Some findings indicated that enteric bacteria isolated from the gastrointestinal tract of abalone were capable of degrading agar, carrageenan, laminarin, and alginate. It was also shown that 70 - 90% of the enzyme activity was extracellular suggesting that bacterial enzymes were secreted into the lumen of the gut where they were

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Related studies have indicated that *Debaryomyces hansenii* HF1; isolated from larvae of European bass (*Dicentrarchus labrax*) demonstrated high levels of amylase and trypsin; which aided in the digestion of feed [65]. Similar studies on a combination of 3 potential probiotic strains (*Agarivorans albus* F1-UMA, *Vibrio sp*. C21-UMA and *Vibrio sp*. F15-UMA) showed significant increases in growth of abalone over a 210 day period [58]. An average monthly improvement in growth of 9.58% of length and 15.94% in weight was observed in relevant test systems. Probiotic organisms persisted in the gut up to a concentration of 106 CFU.g-1 and also remained present for 16 to 19 days in juvenile and adult abalone after cessation of feeding with a probiotic supplemented diet. Authors, [40] and [66] also reported that when probiotics were applied to a host, a higher growth rate was observed, as isolated gut bacteria produced

enzymes that were able to aid in digestion thus improving the health of abalone.

An inaugural application of probiotics in abalone aquaculture was demonstrated by [23]. They reported that microbes isolated from the gastrointestinal tract of *H. midae* demonstrated an ability to improve digestion, growth and immunity of abalone. From their study it was discovered that *D. hansenii*, *Cryptococcus sp., V. midae, and Pseudoalteromonas sp.* reside in the intestinal tract of *H. midae* and have the ability to improve the nutritional status of the abalone feed. Further research demonstrated that these probionts were able to breakdown complex proteins and starches, hence making the subsequent assimilation by abalone easier. Studies conducted by [23] indicated that abalones that had been supplemented with probiotics had a survival rate of 62% compared to 25% of untreated abalones; in challenge trials against bacterium *V. anguillarum*. They later formulated a mixture of probiotics using two yeasts and one bacterial strain (SS1, AY1 and SY9) respectively for abalone. The probiont cocktail was

(20 mm) improved by 8% and large abalone (60 mm) increased by 34%. In addition, increases in intestinal proteolytic and amylolytic activity were observed, in probiotic fed abalone when

Lactic acid bacteria (LAB) from different sources and evaluated potential probiotic effects in abalones *in-vitro*, *Lactobacillus sp*. strain a3 and *Enterococcus sp*. strain s6, was isolated by [56], and were shown to inhibit the growth of three abalone pathogens *viz.*, *Listonella anguillarum*, *V. carchariae* and *V. harveyi*. Furthermore these organisms were able to colonize the gut of

cells.g-1. The growth rate of small abalone

able to hydrolyse complex algal polysaccharides [40].

added to dry feed to a final concentration of 1×107

compared to abalone fed the standard feed devoid of probiotics [30].

The use of biological agents in disease control and improvement of aquaculture is important as demand for environmentally friendly aquaculture practices is on the rise. Biological agents that may be applied in aquaculture comprise of isolates belonging to a wide range of yeast, bacteria and even phytoplankton species [52]. In abalone aquaculture, potential probionts listed to date include, *Vibrio* spp., [23,53-55] *Debaryomyces sp*., *Cryptococcus sp*., and *Pseudoal‐ teromonas sp*., [23,24,30], *Lactobacillus* and *Enterococcus sp*., [56]; *Pediococcus* sp. Strain Ab1 [57], *Agarivorans albus* F1-UMA [58] and *Shewanella sp* [51].

Biological agents have been found to confer beneficial effects on the host by various modes of action. These may occur as a singular or combined effect, and thus far the following have been reported; (1) the production of antimicrobial products; (2) competitive exclusion; (3) coloni‐ sation of the gut and improving microbial balance; (4) enhancement of the host immune response; (5) detoxification of harmful compounds; (6) improved growth rate of the host;(7) antiviral effects, (8) provision of nutrients and enzymatic functions; and (9) improved water quality. Further reports by [24] stated that the addition of probiotics to the diet of farmed abalone, could possibly lead to a boost in abalone growth by a number of potential strategies. Some of which include (1) increasing the nutrients accessible to the abalone for absorption in the gut, (2) increasing the pool of secreted digestive enzymes in the gut of abalone, and (3) use of bacterial supplements as an additional nutrient source.

In many instances, pathogen inhibition and/or disease control has been observed as a conse‐ quence of the release of chemical substances with bactericidal effects by probiotic bacteria [47]. The production of antibiotics, bacteriocins, enzymes, hydrogen peroxide, siderophores and the altering of the pH levels due to the generation of organic acids are all traits displayed by biological agents [47,59-61]. In addition, these biological agents compete with pathogens based on intrinsic growth rate and spacial attachment. Microbial colonisation is characterised by the attachment of the biological agent to the mucosal surface and epithelial cells of the host. This prevents the proliferation of opportunistic pathogens thereby preventing infection [62]. It is common knowledge that for a pathogen to be active and replicate in a host system, it requires attachment to these surfaces [62]. When probiotics are administered over a long period, they successfully colonize the gastrointestinal tract, even after cessation of feed supplemented with probiotics. This occurs since the multiplication rate of these probiotics is higher than the rate at which they are removed, thus a build-up in the intestinal mucosa of the host is observed [62].

Host nutrition is improved, as the applied probionts secrete high levels of hydrolytic enzymes such as amylases, proteases and lipases; as well as the provision of growth factors such as fatty acids, amino acids and vitamins [63,64]. Some isolates also have the ability to break down potentially indigestible components of the feed thus reducing toxicity and improving feed conversion efficiency [23,63]. Abalones are in most instances, fed a diet consisting mainly of kelp, which is a complex macroalgal polysaccharide deficient in many essential nutrients [64]. It is therefore imperative that enteric bacteria in the abalone gut are present in sufficient amounts which will adequately facilitate digestion by supplying highly effective polysacchar‐ olytic enzymes [23]. Many bacteria displaying these properties have been found to exist throughout the digestive tract of *H. midae* [23,40]. Some findings indicated that enteric bacteria isolated from the gastrointestinal tract of abalone were capable of degrading agar, carrageenan, laminarin, and alginate. It was also shown that 70 - 90% of the enzyme activity was extracellular suggesting that bacterial enzymes were secreted into the lumen of the gut where they were able to hydrolyse complex algal polysaccharides [40].

is defined as "a live microbial adjunct which has a beneficial effect on the host by modifying the host-association or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment" [47]. Some studies have shown that as a result of intensification of aquaculture farms a negative impact has been conferred on the composition of the different protective microbial flora interacting with the host [5]. This occurrence results in an increase in susceptibility of the host to diseases. It has become evident that augmentation of aquaculture systems with biological agents can lead to growth of beneficial bacteria thus improving overall health of the culture system and the host [5].

The use of biological agents in disease control and improvement of aquaculture is important as demand for environmentally friendly aquaculture practices is on the rise. Biological agents that may be applied in aquaculture comprise of isolates belonging to a wide range of yeast, bacteria and even phytoplankton species [52]. In abalone aquaculture, potential probionts listed to date include, *Vibrio* spp., [23,53-55] *Debaryomyces sp*., *Cryptococcus sp*., and *Pseudoal‐ teromonas sp*., [23,24,30], *Lactobacillus* and *Enterococcus sp*., [56]; *Pediococcus* sp. Strain Ab1 [57],

Biological agents have been found to confer beneficial effects on the host by various modes of action. These may occur as a singular or combined effect, and thus far the following have been reported; (1) the production of antimicrobial products; (2) competitive exclusion; (3) coloni‐ sation of the gut and improving microbial balance; (4) enhancement of the host immune response; (5) detoxification of harmful compounds; (6) improved growth rate of the host;(7) antiviral effects, (8) provision of nutrients and enzymatic functions; and (9) improved water quality. Further reports by [24] stated that the addition of probiotics to the diet of farmed abalone, could possibly lead to a boost in abalone growth by a number of potential strategies. Some of which include (1) increasing the nutrients accessible to the abalone for absorption in the gut, (2) increasing the pool of secreted digestive enzymes in the gut of abalone, and (3) use

In many instances, pathogen inhibition and/or disease control has been observed as a conse‐ quence of the release of chemical substances with bactericidal effects by probiotic bacteria [47]. The production of antibiotics, bacteriocins, enzymes, hydrogen peroxide, siderophores and the altering of the pH levels due to the generation of organic acids are all traits displayed by biological agents [47,59-61]. In addition, these biological agents compete with pathogens based on intrinsic growth rate and spacial attachment. Microbial colonisation is characterised by the attachment of the biological agent to the mucosal surface and epithelial cells of the host. This prevents the proliferation of opportunistic pathogens thereby preventing infection [62]. It is common knowledge that for a pathogen to be active and replicate in a host system, it requires attachment to these surfaces [62]. When probiotics are administered over a long period, they successfully colonize the gastrointestinal tract, even after cessation of feed supplemented with probiotics. This occurs since the multiplication rate of these probiotics is higher than the rate at which they are removed, thus a build-up in the intestinal mucosa of the host is observed [62]. Host nutrition is improved, as the applied probionts secrete high levels of hydrolytic enzymes such as amylases, proteases and lipases; as well as the provision of growth factors such as fatty

*Agarivorans albus* F1-UMA [58] and *Shewanella sp* [51].

214 Sustainable Aquaculture Techniques

of bacterial supplements as an additional nutrient source.

Related studies have indicated that *Debaryomyces hansenii* HF1; isolated from larvae of European bass (*Dicentrarchus labrax*) demonstrated high levels of amylase and trypsin; which aided in the digestion of feed [65]. Similar studies on a combination of 3 potential probiotic strains (*Agarivorans albus* F1-UMA, *Vibrio sp*. C21-UMA and *Vibrio sp*. F15-UMA) showed significant increases in growth of abalone over a 210 day period [58]. An average monthly improvement in growth of 9.58% of length and 15.94% in weight was observed in relevant test systems. Probiotic organisms persisted in the gut up to a concentration of 106 CFU.g-1 and also remained present for 16 to 19 days in juvenile and adult abalone after cessation of feeding with a probiotic supplemented diet. Authors, [40] and [66] also reported that when probiotics were applied to a host, a higher growth rate was observed, as isolated gut bacteria produced enzymes that were able to aid in digestion thus improving the health of abalone.

An inaugural application of probiotics in abalone aquaculture was demonstrated by [23]. They reported that microbes isolated from the gastrointestinal tract of *H. midae* demonstrated an ability to improve digestion, growth and immunity of abalone. From their study it was discovered that *D. hansenii*, *Cryptococcus sp., V. midae, and Pseudoalteromonas sp.* reside in the intestinal tract of *H. midae* and have the ability to improve the nutritional status of the abalone feed. Further research demonstrated that these probionts were able to breakdown complex proteins and starches, hence making the subsequent assimilation by abalone easier. Studies conducted by [23] indicated that abalones that had been supplemented with probiotics had a survival rate of 62% compared to 25% of untreated abalones; in challenge trials against bacterium *V. anguillarum*. They later formulated a mixture of probiotics using two yeasts and one bacterial strain (SS1, AY1 and SY9) respectively for abalone. The probiont cocktail was added to dry feed to a final concentration of 1×107 cells.g-1. The growth rate of small abalone (20 mm) improved by 8% and large abalone (60 mm) increased by 34%. In addition, increases in intestinal proteolytic and amylolytic activity were observed, in probiotic fed abalone when compared to abalone fed the standard feed devoid of probiotics [30].

Lactic acid bacteria (LAB) from different sources and evaluated potential probiotic effects in abalones *in-vitro*, *Lactobacillus sp*. strain a3 and *Enterococcus sp*. strain s6, was isolated by [56], and were shown to inhibit the growth of three abalone pathogens *viz.*, *Listonella anguillarum*, *V. carchariae* and *V. harveyi*. Furthermore these organisms were able to colonize the gut of *Haliotis gigantea* thus enhancing the production of volatile short chain fatty acids (VSCFA) such as acetic acid. They later showed that by supplementing commercially available abalone feed with a potential probiotic organism, *Pediococcus sp*. Ab 1, a change in host intestinal flora was observed. In addition, higher levels of alginate lyase activity and VSCFAs were recorded. All of these factors led to a combined impact by enhancing the growth of the abalone, *H. gigan‐ tea* [57].

economic competitiveness of the bioprocess technology [74]. Nutrient sources generally play a dominant role in the productivity of the production process since they supply nutrient and growth factors that are directly linked with the formation of biomass and metabolites [75]. It has been suggested that economical and commercially available medium options be investi‐ gated in order to reduce production costs [76,77]. The growth medium used can be either a defined or undefined medium. A defined medium has known quantities of all the ingredients that constitute the formulation. An undefined medium contains complex ingredients such corn steep liquor (CSL), which consist of a mixture of chemicals in unknown quantities that vary according to supplier and production batches. The undefined medium option is usually

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Yeast extract is a commonly used growth medium component, and has been used extensively in many production processes. It is an important nitrogen and nutrient source as it contains an array of amino acids, vitamins and other growth factors required for microbial growth [78-80]. Several studies have indicated that high cell yields and productivities have been obtained with the use of yeast extract in various production processes [81-83]. However, the disadvantage of using this nutrient source is that it results in high-priced production processes due to its associated cost [78]. The use of yeast extract in a production process is therefore regarded as a major technical hurdle that should be overcome in order to successfully minimize production costs [79]. Other nutrient sources that have been used include casamino acids and peptone, which are produced via the enzymatic digestion of meat. The use of these products results in expensive production processes; even though these have been shown to be highly effective nutrient sources. Furthermore, these nutrients sources have negative market accept‐ ance as they are animal by-products [80]. Regulations have also exerted significant pressure on the use of these animal by-products, which have limited their availability. It is therefore imperative that cheaper, safer and readily accessible nutrient sources, capable of supporting production of biological agents, be used in order to ensure that a production process is

CSL has been identified as a lower cost, more effective growth medium that can be used in production, in comparison to conventional nutrient substrates such as yeast extract, peptone and casamino acids [50]. It is produced by immersing corn into dilute sulphur oxide during the starch-manufacturing processes and is a major by-product of the corn-starch processing industry [84]. It has also been shown to be a supplementary source of vitamins and nitrogen to the culture medium [85,86]. The use of CSL has had numerous successes in diverse industrial fermentation processes [76] with high cell yields and productivities being major benefits [87]. Other than the assessment of a suitable nitrogen source, alternative carbohydrate sources also need to be reviewed as they play a dominant role in the productivity of a production process. These nutrient sources are directly linked with energy provision for the formation of biomass and metabolites [75]. Different microbes utilise carbohydrate sources in varying ways. Glucose is a relatively expensive carbohydrate source, and its use in large scale process is limited as a result of high subsequent production costs [88]. When developing efficient bioprocesses, attempts are made to obtain economical and commercially available carbohydrate sources such that the production costs are minimised [74,76,77,89,90]. High test molasses (HTM) is a

applied in industrial processes based on its low cost [74].

economically attractive.

Studies conducted by [51] revealed that within a week of supplementing the feed of *Haliotis discus hannai Ino* with two probiotic organisms, *Shewanella colwelliana* WA64 and *Shewanella olleyana* WA65, increases in cellular and humoral immune response, higher haemocytes, respiratory burst activity, serum lysozyme activity and total levels of protein were observed. It was therefore concluded that both strains may be used as a dietary probiotic supplement to improve innate immunity and disease resistance in abalone.

Studies on feed probiotics for abalone aquaculture show much promise, however the use of water bioremediation bacteria has been neglected. With intensification of abalone culture activities, increased energy costs of pumping sea water and stricter regulation on environ‐ mental pollution, the need for such biological agents will become obvious in the near future. A study based on carp was done by [67] where a consortium of three *Bacillus* isolates demon‐ strated the ability to reduce diseases and improve water quality. Additionally, studies revealed that when a three organism consortium was added to a culture environment, a decrease in the prevalence of pathogenic bacteria was observed. Moreover it was found that nitrate, nitrite and ammonium concentrations were significantly lower as compared to the control treatments and that the applied treatment did not alter the health, growth and oxygen sufficiency of the test systems negatively. The attractive nature of *Bacillus* spp. as biological agents should be considered for application in abalone mariculture.

#### **4. Rationale used for the production of probiotics and biological agents**

The use of biological agents in aquaculture has over the years gained momentum. It is thus, imperative that these micro-organisms be commercially produced in order to meet market needs. A comprehensive production process needs to be developed and optimised for each biological agent. This will facilitate the commercial roll-out of probiotic products of this nature, but will be largely dependent on (1) the efficiency of the production process and (2) the ability to produce large quantities of the probiotic in a suitable form with practical shelf stability [69]. Important criteria influencing the commercial use of biological products are cost, efficacy, shelf life and convenience to the end user [70,71]. The cultivation of microorganisms at a large scale is influenced by various factors such as the composition of the media, physical and chemical variables, substrate feed, oxygen availability and many others [72]; each of which have to be optimized to ensure a cost effective production process.

The growth medium that is used to support high productivities in commercial bioprocesses is predominantly formulated with inexpensive nutrient sources [73]. The choice of medium to be used in production is an essential aspect of process development as it influences the economic competitiveness of the bioprocess technology [74]. Nutrient sources generally play a dominant role in the productivity of the production process since they supply nutrient and growth factors that are directly linked with the formation of biomass and metabolites [75]. It has been suggested that economical and commercially available medium options be investi‐ gated in order to reduce production costs [76,77]. The growth medium used can be either a defined or undefined medium. A defined medium has known quantities of all the ingredients that constitute the formulation. An undefined medium contains complex ingredients such corn steep liquor (CSL), which consist of a mixture of chemicals in unknown quantities that vary according to supplier and production batches. The undefined medium option is usually applied in industrial processes based on its low cost [74].

*Haliotis gigantea* thus enhancing the production of volatile short chain fatty acids (VSCFA) such as acetic acid. They later showed that by supplementing commercially available abalone feed with a potential probiotic organism, *Pediococcus sp*. Ab 1, a change in host intestinal flora was observed. In addition, higher levels of alginate lyase activity and VSCFAs were recorded. All of these factors led to a combined impact by enhancing the growth of the abalone, *H. gigan‐*

Studies conducted by [51] revealed that within a week of supplementing the feed of *Haliotis discus hannai Ino* with two probiotic organisms, *Shewanella colwelliana* WA64 and *Shewanella olleyana* WA65, increases in cellular and humoral immune response, higher haemocytes, respiratory burst activity, serum lysozyme activity and total levels of protein were observed. It was therefore concluded that both strains may be used as a dietary probiotic supplement to

Studies on feed probiotics for abalone aquaculture show much promise, however the use of water bioremediation bacteria has been neglected. With intensification of abalone culture activities, increased energy costs of pumping sea water and stricter regulation on environ‐ mental pollution, the need for such biological agents will become obvious in the near future. A study based on carp was done by [67] where a consortium of three *Bacillus* isolates demon‐ strated the ability to reduce diseases and improve water quality. Additionally, studies revealed that when a three organism consortium was added to a culture environment, a decrease in the prevalence of pathogenic bacteria was observed. Moreover it was found that nitrate, nitrite and ammonium concentrations were significantly lower as compared to the control treatments and that the applied treatment did not alter the health, growth and oxygen sufficiency of the test systems negatively. The attractive nature of *Bacillus* spp. as biological agents should be

**4. Rationale used for the production of probiotics and biological agents**

The use of biological agents in aquaculture has over the years gained momentum. It is thus, imperative that these micro-organisms be commercially produced in order to meet market needs. A comprehensive production process needs to be developed and optimised for each biological agent. This will facilitate the commercial roll-out of probiotic products of this nature, but will be largely dependent on (1) the efficiency of the production process and (2) the ability to produce large quantities of the probiotic in a suitable form with practical shelf stability [69]. Important criteria influencing the commercial use of biological products are cost, efficacy, shelf life and convenience to the end user [70,71]. The cultivation of microorganisms at a large scale is influenced by various factors such as the composition of the media, physical and chemical variables, substrate feed, oxygen availability and many others [72]; each of which have to be

The growth medium that is used to support high productivities in commercial bioprocesses is predominantly formulated with inexpensive nutrient sources [73]. The choice of medium to be used in production is an essential aspect of process development as it influences the

improve innate immunity and disease resistance in abalone.

considered for application in abalone mariculture.

optimized to ensure a cost effective production process.

*tea* [57].

216 Sustainable Aquaculture Techniques

Yeast extract is a commonly used growth medium component, and has been used extensively in many production processes. It is an important nitrogen and nutrient source as it contains an array of amino acids, vitamins and other growth factors required for microbial growth [78-80]. Several studies have indicated that high cell yields and productivities have been obtained with the use of yeast extract in various production processes [81-83]. However, the disadvantage of using this nutrient source is that it results in high-priced production processes due to its associated cost [78]. The use of yeast extract in a production process is therefore regarded as a major technical hurdle that should be overcome in order to successfully minimize production costs [79]. Other nutrient sources that have been used include casamino acids and peptone, which are produced via the enzymatic digestion of meat. The use of these products results in expensive production processes; even though these have been shown to be highly effective nutrient sources. Furthermore, these nutrients sources have negative market accept‐ ance as they are animal by-products [80]. Regulations have also exerted significant pressure on the use of these animal by-products, which have limited their availability. It is therefore imperative that cheaper, safer and readily accessible nutrient sources, capable of supporting production of biological agents, be used in order to ensure that a production process is economically attractive.

CSL has been identified as a lower cost, more effective growth medium that can be used in production, in comparison to conventional nutrient substrates such as yeast extract, peptone and casamino acids [50]. It is produced by immersing corn into dilute sulphur oxide during the starch-manufacturing processes and is a major by-product of the corn-starch processing industry [84]. It has also been shown to be a supplementary source of vitamins and nitrogen to the culture medium [85,86]. The use of CSL has had numerous successes in diverse industrial fermentation processes [76] with high cell yields and productivities being major benefits [87]. Other than the assessment of a suitable nitrogen source, alternative carbohydrate sources also need to be reviewed as they play a dominant role in the productivity of a production process. These nutrient sources are directly linked with energy provision for the formation of biomass and metabolites [75]. Different microbes utilise carbohydrate sources in varying ways. Glucose is a relatively expensive carbohydrate source, and its use in large scale process is limited as a result of high subsequent production costs [88]. When developing efficient bioprocesses, attempts are made to obtain economical and commercially available carbohydrate sources such that the production costs are minimised [74,76,77,89,90]. High test molasses (HTM) is a valuable carbohydrate used commercially due to its local availability and cost competitiveness. It has been applied extensively as an alternative carbohydrate source in various production processes [74,76,77,80,90,91]. HTM, unlike conventional molasses, is a purer product form that enhances mass transfer and reduces impurities in a production process. HTM has been used as a carbohydrate source because it consists of glucose, fructose and sucrose. Inverted HTM is also readily accessible, which contains mainly glucose and fructose in equal proportions with a small amount of residual sucrose. Other than being a carbohydrate source, HTM also provides abundant vitamins and other growth factors required for microbial growth [80,92].

between the optimum and the maximum pH value [107]. It is well known that pH is important in controlling initiation of growth by microorganisms [108]. The effect of pH on growth include: (1) affecting the production and activity of enzyme systems controlling growth and division, (2) altering the solubility of essential nutrients, (3) modifying the permeability of cells to substances essential for growth, (4) changing the nature of cell surfaces of envelope materials and cell morphology, and (5) modifying the composition of the cultivation medium [108-111]. Oxygen sufficiency is an additional factor to be considered in the design of an optimum bioprocess strategy. In high-cell density cultivations, oxygen limitation can be very challeng‐ ing, and prevents attainment of high cell titres [112]. The method of oxygenation must be given a high degree of consideration, as excessive rates of agitation and sparging will encourage foam formation and initiate cell sheer. On the contrary, inadequate aeration causes oxygen limitation, and has been reported to be highly detrimental to process productivity, in terms of

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These factors have an impact on the improved yield and productivity of a process and as a result the overall cost of the production process. In addition they also confer information of the functionality of the probiotic once it enters the host environment [50,90]. Therefore, bioprocesses are designed such that the overall process has increased cell yields, productivities and a lowered cost, which ultimately results in a feasible and economically attractive produc‐ tion process. It is essential that these requirements are met to ensure that biological agents can

Once the relevant biological agents have been successfully cultivated at a large scale, the resultant fermentation broth needs to be recovered efficiently to be utilized in subsequent processing and formulation steps [113,114]. The downstream process has a major influence on product commercialization as major constraints in most processes are embedded in harvesting and formulation costs [115,116]. This includes key aspects such as maximising recovery and preservation of viability, which are essential, in terms of applying an effective biological agent, especially in aquatic systems [117]. In addition, it is also vital to ensure that the final product to be administered to the host aligns with end user requirements such as stability, consistency, easy application, efficacy and affordability [115,118,119]. As a consequence; robust costeffective choices of process steps and ingredients, dictated by the end product characteristics, are necessary to improve the commercial success of newly developed biological agents [115].

The main objective for downstream processing is to minimise the number of unit operations involved in the process, thus reducing overall process and validation costs, while also simplifying ease and economy of process automation [115]. An additional consideration is the final anticipated form of the end product which has implications on the choice of process options while meeting customer expectations (Figure 2) [120]. The downstream process unit operations, completes the process chain; from the upstream fermentation to the end product. It is therefore considered to be an extremely important prerequisite for commercialization of

growth rates and product formation as well as cell viability [96,112]

**5. Processing of probiotics into market acceptable products**

be affordably adopted for use in abalone aquaculture.

In some instances, microorganisms may require vitamins to be present in the cultivation medium which can be found in the supplemented complex nutrient sources, whereas others can be cultivated in a medium devoid of vitamins [93]. Vitamins are growth factors required by most microorganisms for the production of enzyme cofactors [74,94]. Other than vitamins, microorganisms also require trace elements for their growth. Trace elements form part of enzymes and co-factors and they aid in the catalysis of reactions and maintenance of protein structures [74,95]. Supplementation of exotic trace elements and vitamins can be costly and are therefore avoided if cheaper nutrient sources can satisfy the essential requirements for growth of biological agents in production processes.

Other than an influence on growth, the type of growth medium used in a production process also influences physical parameters such as mass transfer and the formation of foam. Growth media rich in nitrogen sources usually result in increased foam formation [96]. In addition the sparging of gas through the growth medium and agitation at high speeds results in excess foam formation, in oxygen intensive processes. This reduces the efficiency of gaseous exchange at the surface of the culture, as a barrier is formed between the culture and the gases present in the headspace of the vessel [97,98]. Additionally, cells and the culture medium can be lost in the foam phase in the event of vessel overflow. The sensitivity of microorganisms to antifoam toxicity is an important factor that must be considered during the development of production processes; as it can result in a significant decrease of the process performance [98].

Once a suitable fermentation medium has been developed, optimization of physiological growth conditions such as temperature, pH and oxygen sufficiency are imperative, in order to successfully produce biological agents on a large scale. Temperature and pH have been reported to be amongst the most important environmental parameters which control the activities and growth rates of many microorganisms as it governs all the physiological processes. The impact of temperature has been observed at the cellular level, and can either increase or decrease the catalytic activity of pertinent metabolic and digestive enzymes [99-102]. It has been reported that the alteration of growth conditions to an unsuitable range can significantly increase the lag phase of a wide range of micro-organisms, which is highly undesirable when designing an efficient bioprocess strategy [104]. Since temperature affects microbial growth rate, it also affects the growth yield of a culture because the relative energy requirements for cell maintenance increases; when growth rates are reduced [105]. pH homeostasis is another important factor that needs to be considered during the growth of microorganisms [106]. For most microorganisms, there is an increase in growth rate between the minimum and the optimum pH levels and a corresponding decrease in growth rate between the optimum and the maximum pH value [107]. It is well known that pH is important in controlling initiation of growth by microorganisms [108]. The effect of pH on growth include: (1) affecting the production and activity of enzyme systems controlling growth and division, (2) altering the solubility of essential nutrients, (3) modifying the permeability of cells to substances essential for growth, (4) changing the nature of cell surfaces of envelope materials and cell morphology, and (5) modifying the composition of the cultivation medium [108-111]. Oxygen sufficiency is an additional factor to be considered in the design of an optimum bioprocess strategy. In high-cell density cultivations, oxygen limitation can be very challeng‐ ing, and prevents attainment of high cell titres [112]. The method of oxygenation must be given a high degree of consideration, as excessive rates of agitation and sparging will encourage foam formation and initiate cell sheer. On the contrary, inadequate aeration causes oxygen limitation, and has been reported to be highly detrimental to process productivity, in terms of growth rates and product formation as well as cell viability [96,112]

valuable carbohydrate used commercially due to its local availability and cost competitiveness. It has been applied extensively as an alternative carbohydrate source in various production processes [74,76,77,80,90,91]. HTM, unlike conventional molasses, is a purer product form that enhances mass transfer and reduces impurities in a production process. HTM has been used as a carbohydrate source because it consists of glucose, fructose and sucrose. Inverted HTM is also readily accessible, which contains mainly glucose and fructose in equal proportions with a small amount of residual sucrose. Other than being a carbohydrate source, HTM also provides abundant vitamins and other growth factors required for microbial growth [80,92].

In some instances, microorganisms may require vitamins to be present in the cultivation medium which can be found in the supplemented complex nutrient sources, whereas others can be cultivated in a medium devoid of vitamins [93]. Vitamins are growth factors required by most microorganisms for the production of enzyme cofactors [74,94]. Other than vitamins, microorganisms also require trace elements for their growth. Trace elements form part of enzymes and co-factors and they aid in the catalysis of reactions and maintenance of protein structures [74,95]. Supplementation of exotic trace elements and vitamins can be costly and are therefore avoided if cheaper nutrient sources can satisfy the essential requirements for

Other than an influence on growth, the type of growth medium used in a production process also influences physical parameters such as mass transfer and the formation of foam. Growth media rich in nitrogen sources usually result in increased foam formation [96]. In addition the sparging of gas through the growth medium and agitation at high speeds results in excess foam formation, in oxygen intensive processes. This reduces the efficiency of gaseous exchange at the surface of the culture, as a barrier is formed between the culture and the gases present in the headspace of the vessel [97,98]. Additionally, cells and the culture medium can be lost in the foam phase in the event of vessel overflow. The sensitivity of microorganisms to antifoam toxicity is an important factor that must be considered during the development of production

processes; as it can result in a significant decrease of the process performance [98].

Once a suitable fermentation medium has been developed, optimization of physiological growth conditions such as temperature, pH and oxygen sufficiency are imperative, in order to successfully produce biological agents on a large scale. Temperature and pH have been reported to be amongst the most important environmental parameters which control the activities and growth rates of many microorganisms as it governs all the physiological processes. The impact of temperature has been observed at the cellular level, and can either increase or decrease the catalytic activity of pertinent metabolic and digestive enzymes [99-102]. It has been reported that the alteration of growth conditions to an unsuitable range can significantly increase the lag phase of a wide range of micro-organisms, which is highly undesirable when designing an efficient bioprocess strategy [104]. Since temperature affects microbial growth rate, it also affects the growth yield of a culture because the relative energy requirements for cell maintenance increases; when growth rates are reduced [105]. pH homeostasis is another important factor that needs to be considered during the growth of microorganisms [106]. For most microorganisms, there is an increase in growth rate between the minimum and the optimum pH levels and a corresponding decrease in growth rate

growth of biological agents in production processes.

218 Sustainable Aquaculture Techniques

These factors have an impact on the improved yield and productivity of a process and as a result the overall cost of the production process. In addition they also confer information of the functionality of the probiotic once it enters the host environment [50,90]. Therefore, bioprocesses are designed such that the overall process has increased cell yields, productivities and a lowered cost, which ultimately results in a feasible and economically attractive produc‐ tion process. It is essential that these requirements are met to ensure that biological agents can be affordably adopted for use in abalone aquaculture.

#### **5. Processing of probiotics into market acceptable products**

Once the relevant biological agents have been successfully cultivated at a large scale, the resultant fermentation broth needs to be recovered efficiently to be utilized in subsequent processing and formulation steps [113,114]. The downstream process has a major influence on product commercialization as major constraints in most processes are embedded in harvesting and formulation costs [115,116]. This includes key aspects such as maximising recovery and preservation of viability, which are essential, in terms of applying an effective biological agent, especially in aquatic systems [117]. In addition, it is also vital to ensure that the final product to be administered to the host aligns with end user requirements such as stability, consistency, easy application, efficacy and affordability [115,118,119]. As a consequence; robust costeffective choices of process steps and ingredients, dictated by the end product characteristics, are necessary to improve the commercial success of newly developed biological agents [115].

The main objective for downstream processing is to minimise the number of unit operations involved in the process, thus reducing overall process and validation costs, while also simplifying ease and economy of process automation [115]. An additional consideration is the final anticipated form of the end product which has implications on the choice of process options while meeting customer expectations (Figure 2) [120]. The downstream process unit operations, completes the process chain; from the upstream fermentation to the end product. It is therefore considered to be an extremely important prerequisite for commercialization of probiotic technologies. Regrettably, published literature regarding downstream processing and formulation for commercially available products is very limited [115,119].

should seamlessly integrate into standard food production and farming activities [115,129]. Formulations of biological agents can be broadly classified into either dry solids in various forms, or liquid suspensions or emulsions [130]. The inclusion of additives that enhance process-ability eco-friendliness and customer acceptance of the final product are also impor‐ tant considerations [115]. Any potential impact to the host, environment and even end product

Application of Biological Agents in Abalone Aquaculture: A South African Perspective

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221

In the case of a probiotic product, the formulation needs to encompass ingredients that aid viability and growth of the cells in its intended application. Sugars and proteins are normally the key nutrients that support the stability of cell preparation. It also further provides a protective layer for the cells, preventing death and assists in the recovery of injured cells during processing [115,132,133]. The addition of nutrients was also shown to improve storage of a *Pseudomonas fluorescens* F113 strain [134] and a *Bacillus megaterium* [135] for use in biocontrol applications. Appropriate formulations can facilitate easier processing and influence the

Processing options for abalone biological agents, will include, both dry and liquid product forms (Figure 2). Due to the intended use of the selected isolates as a living cell preparation, product options with a high stability are considered to be most appealing. The application of fresh cells that need to be routinely produced is not attractive as there is significant risk in ensuring consistent inclusion into the abalone feed [135]. This complicates the processing segment of the technology to a large extent, as innovative ways of ensuring and maintaining cell viabilities are required. Potential options for commercial processes to stabilize these probiotic products include refrigeration, freezing, freeze drying, spray drying and low temperature fluidised bed agglomeration. Refrigerated and frozen cultures occupy large storage volumes and demand higher storage and shipment costs in contrast to dry cultures which are an economic and practical alternative; however, some microorganisms are highly vulnerable to death when any form of drying is carried out [117,136,137]. An alternate approach is to concentrate the product into a convenient dosage quantity and form that reduces the bulk logistics burden for products that are not amenable to drying. Low temperature drying processes such as freeze drying are suitable for higher value, heat labile bio-products, but is costly, time consuming and discontinuous for bulk production compared with moderate temperature drying processes [131,138]. Spray drying processes are widely used for large scale drying of products; however, higher drying temperatures decrease the viability of microbes faster than lower drying temperatures [131,139]. Spray drying requires high temperatures to facilitate water evaporation, which can cause irreversible changes to structural and functional integrity of the intended biological product and reduce viability and activity of the organisms itself [140,141]. Spray drying also has a high energy demand requiring 2500 to 10 000 J.g-1 of evaporated water and is therefore not the most attractive process option for drying of abalone

There are several reports on the use of agglomeration as a commercially viable process option for moderate temperature drying of biological material, mainly due to excellent mass and heat transfer characteristics [133,139,142]. During agglomeration, a mixture is atomised to form

C) which results in coating of the probiotic cells

consumer, must be thoroughly investigated prior to application [131].

stability and appeal of an end product in large scale production [70].

probiotics [122].

droplets at lower temperature (typically 30-40o

**Figure 2.** Schematic illustration of potential downstream process unit options [121].

The harvesting efficacy of a unit operation governs the marketability of a product, as it affects the potency and aids in any potential further processing during formulation and product development. The goal of the recovery process is to produce a product of acceptable quality, in compliance with any regulatory and safety requirements, at an acceptable cost [120]. Process options for cell harvesting from fermentation broth include microfiltration, sedimentation, flocculation and ultrafiltration (Figure 2) [116,122]. Flocculation and flotation using surface action or electrical charge have been reported to be inefficient in the separation of bacterial cells [120]. Although there have been some positive reports for harvesting using ultra-filtration [116], the most widely used process remains centrifugation [123-125]. Among all options, centrifugation appears to be the most viable alternative for cell harvesting resulting in recoveries of ~ 99% [115,126]. Usually, product intermediates are anticipated to be a high cell concentration paste containing the biological agent. Tube centrifugation has been considered to be a useful process that can yield a lower moisture paste thus minimizing the energy required in later stage drying steps if necessary [127,128].

Subsequent to cell separation, formulation generates a crucial link between production and application of probiotics (Figure 2). This crucial step dictates process-ability, economy, shelf life, and efficacy as well as ease of application and provision of a product form that commands customer appeal. Intelligent formulations will allow innovation in application techniques using unique combinations of active ingredients, adjuvants or inerts and the end product should seamlessly integrate into standard food production and farming activities [115,129]. Formulations of biological agents can be broadly classified into either dry solids in various forms, or liquid suspensions or emulsions [130]. The inclusion of additives that enhance process-ability eco-friendliness and customer acceptance of the final product are also impor‐ tant considerations [115]. Any potential impact to the host, environment and even end product consumer, must be thoroughly investigated prior to application [131].

probiotic technologies. Regrettably, published literature regarding downstream processing

and formulation for commercially available products is very limited [115,119].

• Centrifugation • Flocculation/Flotation

• Formulation • Addition of protectants

• Spray drying • Freeze drying

Dry product form

Drying

• Tablet • Powder • Pellet

Liquid product form

• Fluidised bed agglomeration

**Figure 2.** Schematic illustration of potential downstream process unit options [121].

required in later stage drying steps if necessary [127,128].

The harvesting efficacy of a unit operation governs the marketability of a product, as it affects the potency and aids in any potential further processing during formulation and product development. The goal of the recovery process is to produce a product of acceptable quality, in compliance with any regulatory and safety requirements, at an acceptable cost [120]. Process options for cell harvesting from fermentation broth include microfiltration, sedimentation, flocculation and ultrafiltration (Figure 2) [116,122]. Flocculation and flotation using surface action or electrical charge have been reported to be inefficient in the separation of bacterial cells [120]. Although there have been some positive reports for harvesting using ultra-filtration [116], the most widely used process remains centrifugation [123-125]. Among all options, centrifugation appears to be the most viable alternative for cell harvesting resulting in recoveries of ~ 99% [115,126]. Usually, product intermediates are anticipated to be a high cell concentration paste containing the biological agent. Tube centrifugation has been considered to be a useful process that can yield a lower moisture paste thus minimizing the energy

Subsequent to cell separation, formulation generates a crucial link between production and application of probiotics (Figure 2). This crucial step dictates process-ability, economy, shelf life, and efficacy as well as ease of application and provision of a product form that commands customer appeal. Intelligent formulations will allow innovation in application techniques using unique combinations of active ingredients, adjuvants or inerts and the end product

Cell separation

• Filtration

220 Sustainable Aquaculture Techniques

In the case of a probiotic product, the formulation needs to encompass ingredients that aid viability and growth of the cells in its intended application. Sugars and proteins are normally the key nutrients that support the stability of cell preparation. It also further provides a protective layer for the cells, preventing death and assists in the recovery of injured cells during processing [115,132,133]. The addition of nutrients was also shown to improve storage of a *Pseudomonas fluorescens* F113 strain [134] and a *Bacillus megaterium* [135] for use in biocontrol applications. Appropriate formulations can facilitate easier processing and influence the stability and appeal of an end product in large scale production [70].

Processing options for abalone biological agents, will include, both dry and liquid product forms (Figure 2). Due to the intended use of the selected isolates as a living cell preparation, product options with a high stability are considered to be most appealing. The application of fresh cells that need to be routinely produced is not attractive as there is significant risk in ensuring consistent inclusion into the abalone feed [135]. This complicates the processing segment of the technology to a large extent, as innovative ways of ensuring and maintaining cell viabilities are required. Potential options for commercial processes to stabilize these probiotic products include refrigeration, freezing, freeze drying, spray drying and low temperature fluidised bed agglomeration. Refrigerated and frozen cultures occupy large storage volumes and demand higher storage and shipment costs in contrast to dry cultures which are an economic and practical alternative; however, some microorganisms are highly vulnerable to death when any form of drying is carried out [117,136,137]. An alternate approach is to concentrate the product into a convenient dosage quantity and form that reduces the bulk logistics burden for products that are not amenable to drying. Low temperature drying processes such as freeze drying are suitable for higher value, heat labile bio-products, but is costly, time consuming and discontinuous for bulk production compared with moderate temperature drying processes [131,138]. Spray drying processes are widely used for large scale drying of products; however, higher drying temperatures decrease the viability of microbes faster than lower drying temperatures [131,139]. Spray drying requires high temperatures to facilitate water evaporation, which can cause irreversible changes to structural and functional integrity of the intended biological product and reduce viability and activity of the organisms itself [140,141]. Spray drying also has a high energy demand requiring 2500 to 10 000 J.g-1 of evaporated water and is therefore not the most attractive process option for drying of abalone probiotics [122].

There are several reports on the use of agglomeration as a commercially viable process option for moderate temperature drying of biological material, mainly due to excellent mass and heat transfer characteristics [133,139,142]. During agglomeration, a mixture is atomised to form droplets at lower temperature (typically 30-40o C) which results in coating of the probiotic cells on the surface of suitable carrier particles. Probiotic cell cultures are subjected to evaporative cooling during the warming up and constant-rate drying periods and therefore have a substantially lower temperature than the drying air, resulting in increased viability [139.]. Advantages of fluidised bed drying over freeze and spray drying include lower investment and maintenance costs, ease of large scale continuous production, rapid exchange of heat, minimising heat damage, rapid mixing providing near isothermal conditions and uniform end product [122,139,143]. Due to these reasons fluidized bed drying has become an accepted method for large scale production of heat labile biological materials, however, viability losses have still been reported [142].

[117]. The soaking method uses preformed feed pellets, which are soaked in a saline broth containing the probiotic organism at a desired concentration [57,149]. Soaked pellets are then dried and stored appropriately for further use. A modified method of soaking, whereby actual fronds of macroalgal species, *Macrocystis integrifolia* were soaked in preconditioned tanks containing bacterial cells were used by [58]. Upon aeration of these tanks, the bacteria were allowed to colonize the surface of the fronds, and were thereafter fed to test abalone. In other studies, the spraying of feeds with probiotic cells was also carried out [153]. In addition, [154] described methods of spraying dried feeds with cells that were placed onto plastic trays. Lastly, the bathing option of probiotics involving the application of living cells directly to the rearing water has been used [155]. All potential mechanisms of probiotic inclusion into the feed must be suitably ratified in order to maximize the potential of the technology. The method selected, should have the ability to integrate easily into existing feed production process, and should in no way negatively impact on the host or the rearing environment. The journey taken to produce a commercially viable probiotic product is by no means forthright. It encompasses innovative process design, effective cell production and formulation technologies, as well as successful maintenance of cell viability and stability. Once all the identified challenges have been effectively overcome, the uptake of this technology and the associated boom in abalone export

Application of Biological Agents in Abalone Aquaculture: A South African Perspective

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223

**6. Considerations for application of biological agents in abalone**

potential contamination by common food pathogenic bacteria [68].

Over the past two decades, the applicability of probiotics as solutions to various aquaculture related challenges have been widely reported. However, it is still imperative to consider the safety issues associated with the use of these probiotic products [29,52,156]. Safety is the state of being certain that a biological agent used will not have undesirable effects under defined conditions. The production system in which the cell cultivations are conducted must also maintain high levels of sterility to easily facilitate a monoseptic culture, as well as reduce any

Once the selected culture has been accurately identified and deposited into a culture reposi‐ tory, extensive literature searches and relevant scientific experimentation must be carried out in order to obtain information on the biological agent of interest. As the number of isolated probiotic species increase, it is important not to assume biosafety levels and characteristics of each probiotic strain. Furthermore, it is recommended that the exact mode of action of the probiotic organisms be elucidated, in order to achieve the desirable effect when applied to the host system. It has been suggested that prior to incorporation of these organisms in abalone aquaculture, it is important to carefully assess the probiotics for pathogenicity, infectivity, toxicity and their resultant metabolite production for quality assurance [157]. These critical factors have sometimes been overlooked, and have consequently led to the failure of probiotic technologies in some instances [41]. In many case studies, the use of LAB as probiotics have been rendered safe, however, in recent times there have been reports of disease-causing members belonging to *Lactococcus*, *Vagococcus* or *Carnobacterium* families [158]. Additionally,

by means of aquaculture will be inevitable.

**aquaculture**

In addition to production and formulation of user-friendly product through a downstream process, the stability and consistency of product intermediates and the end product itself are crucial requirements for successful commercialization [144]. A loss of bioactivity in a product, that is intended to be applied in a viable state, will definitely incur a great deal of process complications and as a result impart a direct increase in production cost [141]. In a typical production process, the lag time between process operations can vary due to process integra‐ tion and scheduling during manufacture. Thus storage conditions and the addition of specific stabilizers may be required to prevent vegetative growth or the appearance of contamination in the probiotic product or its relevant intermediates [145].

The problems of stability during processing, storage and after application have stalled development of biologically based products [146]. Accelerated aging studies based on the methodology of death rate plots at different temperatures to generate thermal resistance curves have been shown to be a useful technique for predicting stability [147]. Temperature depend‐ ant half-life plots can be generated to predict stability of the probiotic product intermediates as well as the end product. This approach has however only been used to a limited extent [121,123,135].

After addressing the considerations of the actual production process, success of the technology is still not a certainty. It is imperative, that in order to realise the success of using this new technology, the probiotic product must be supplied as a live cell preparation, and must be able to survive not only the feed production process, but also maintain viability in the digestive tract of the host [44,47]. Many probiotics have been successfully applied to land-based animal production practices, however, the aquaculture industry are faced with further limitations as a result of continuous water exposure [148]. The method of incorporation selected must overcome challenges faced in feed production and the mariculture system itself; such as major losses of viability, in order to achieve the desired effect of the probiotic technology.

There have been various methods applied to successfully administer viable probiotics to a host in aquaculture environments. These include mixing, soaking, spraying, vacuum infusion, extrusion and bathing [148]. Incorporation of the probiotic into the feed is almost always the method of choice, except when bioremediation agents are added directly to the water. Mixing is the most commonly used method and involves the incorporation of the probiotic into the dry ingredients of the feed during the feed production process. Many researchers [56,150,151] have successfully used this method; however, probiotics that are susceptible to excessive heating and drying during the feed production process do not show high rates of survival [117]. The soaking method uses preformed feed pellets, which are soaked in a saline broth containing the probiotic organism at a desired concentration [57,149]. Soaked pellets are then dried and stored appropriately for further use. A modified method of soaking, whereby actual fronds of macroalgal species, *Macrocystis integrifolia* were soaked in preconditioned tanks containing bacterial cells were used by [58]. Upon aeration of these tanks, the bacteria were allowed to colonize the surface of the fronds, and were thereafter fed to test abalone. In other studies, the spraying of feeds with probiotic cells was also carried out [153]. In addition, [154] described methods of spraying dried feeds with cells that were placed onto plastic trays. Lastly, the bathing option of probiotics involving the application of living cells directly to the rearing water has been used [155]. All potential mechanisms of probiotic inclusion into the feed must be suitably ratified in order to maximize the potential of the technology. The method selected, should have the ability to integrate easily into existing feed production process, and should in no way negatively impact on the host or the rearing environment. The journey taken to produce a commercially viable probiotic product is by no means forthright. It encompasses innovative process design, effective cell production and formulation technologies, as well as successful maintenance of cell viability and stability. Once all the identified challenges have been effectively overcome, the uptake of this technology and the associated boom in abalone export by means of aquaculture will be inevitable.

on the surface of suitable carrier particles. Probiotic cell cultures are subjected to evaporative cooling during the warming up and constant-rate drying periods and therefore have a substantially lower temperature than the drying air, resulting in increased viability [139.]. Advantages of fluidised bed drying over freeze and spray drying include lower investment and maintenance costs, ease of large scale continuous production, rapid exchange of heat, minimising heat damage, rapid mixing providing near isothermal conditions and uniform end product [122,139,143]. Due to these reasons fluidized bed drying has become an accepted method for large scale production of heat labile biological materials, however, viability losses

In addition to production and formulation of user-friendly product through a downstream process, the stability and consistency of product intermediates and the end product itself are crucial requirements for successful commercialization [144]. A loss of bioactivity in a product, that is intended to be applied in a viable state, will definitely incur a great deal of process complications and as a result impart a direct increase in production cost [141]. In a typical production process, the lag time between process operations can vary due to process integra‐ tion and scheduling during manufacture. Thus storage conditions and the addition of specific stabilizers may be required to prevent vegetative growth or the appearance of contamination

The problems of stability during processing, storage and after application have stalled development of biologically based products [146]. Accelerated aging studies based on the methodology of death rate plots at different temperatures to generate thermal resistance curves have been shown to be a useful technique for predicting stability [147]. Temperature depend‐ ant half-life plots can be generated to predict stability of the probiotic product intermediates as well as the end product. This approach has however only been used to a limited extent

After addressing the considerations of the actual production process, success of the technology is still not a certainty. It is imperative, that in order to realise the success of using this new technology, the probiotic product must be supplied as a live cell preparation, and must be able to survive not only the feed production process, but also maintain viability in the digestive tract of the host [44,47]. Many probiotics have been successfully applied to land-based animal production practices, however, the aquaculture industry are faced with further limitations as a result of continuous water exposure [148]. The method of incorporation selected must overcome challenges faced in feed production and the mariculture system itself; such as major

losses of viability, in order to achieve the desired effect of the probiotic technology.

There have been various methods applied to successfully administer viable probiotics to a host in aquaculture environments. These include mixing, soaking, spraying, vacuum infusion, extrusion and bathing [148]. Incorporation of the probiotic into the feed is almost always the method of choice, except when bioremediation agents are added directly to the water. Mixing is the most commonly used method and involves the incorporation of the probiotic into the dry ingredients of the feed during the feed production process. Many researchers [56,150,151] have successfully used this method; however, probiotics that are susceptible to excessive heating and drying during the feed production process do not show high rates of survival

have still been reported [142].

222 Sustainable Aquaculture Techniques

[121,123,135].

in the probiotic product or its relevant intermediates [145].

### **6. Considerations for application of biological agents in abalone aquaculture**

Over the past two decades, the applicability of probiotics as solutions to various aquaculture related challenges have been widely reported. However, it is still imperative to consider the safety issues associated with the use of these probiotic products [29,52,156]. Safety is the state of being certain that a biological agent used will not have undesirable effects under defined conditions. The production system in which the cell cultivations are conducted must also maintain high levels of sterility to easily facilitate a monoseptic culture, as well as reduce any potential contamination by common food pathogenic bacteria [68].

Once the selected culture has been accurately identified and deposited into a culture reposi‐ tory, extensive literature searches and relevant scientific experimentation must be carried out in order to obtain information on the biological agent of interest. As the number of isolated probiotic species increase, it is important not to assume biosafety levels and characteristics of each probiotic strain. Furthermore, it is recommended that the exact mode of action of the probiotic organisms be elucidated, in order to achieve the desirable effect when applied to the host system. It has been suggested that prior to incorporation of these organisms in abalone aquaculture, it is important to carefully assess the probiotics for pathogenicity, infectivity, toxicity and their resultant metabolite production for quality assurance [157]. These critical factors have sometimes been overlooked, and have consequently led to the failure of probiotic technologies in some instances [41]. In many case studies, the use of LAB as probiotics have been rendered safe, however, in recent times there have been reports of disease-causing members belonging to *Lactococcus*, *Vagococcus* or *Carnobacterium* families [158]. Additionally,

strain testing of potential probiotics should encompass the robustness of the product against process fluctuations under farm conditions and confirmation of non-transmission of drug resistance genes or virulence plasmids [159]. Another barrier preventing the worldwide adoption of this technology, relates to the absence of efficacy data, which as a result casts a shadow of doubt over the technology, thereby hindering its uptake by the aqua-culturists.

**Author details**

**References**

Ghaneshree Moodley1,2\*, Lethabo Mashigo1

1 CSIR Biosciences, Pretoria, South Africa

Technology, Durban, South Africa

2005; 71(8) 4307-4317.

\*Address all correspondence to<EMAIL_ADDRESS>
, Rajesh Lalloo1

2 Department of Biotechnology & Food, Faculty of Applied Sciences, Durban University of

[1] Marques A., Dinh T., Ioakeimidis C., Huys G., Swings J., Verstraete W., Dhont P., Sorgeloos, P. and Bossier, P. Effects Of Bacteria On *Artemia* Cultured In Different Gnotobiotic Environments. Journal of Applied and Environmental Microbiology

[2] Subasinghe RP. Epidemiological Approach to Aquatic Animal Health Management: Opportunities and Challenges for Developing Countries to Increase Aquatic Produc‐

[4] Britz PJ. A study on the Status of Aquaculture Production and Trade in South Africa. Volume 2: Growth Potential of the South African Aquaculture Industry and Recom‐ mendations for Sector Development. A report for the Department of trade and In‐

[5] Olafsen JA. Interactions between Fish Larvae and Bacteria in Marine Aquaculture.

[6] Gordon HR and Cook PA. World Abalone Fisheries and Aquaculture Update: Sup‐

[7] Stanford J. Aqua Culture Ambition: South African Aquaculture Industry Is Heading

[8] Reddy-Lopata K., Auerswald L. and Cook P. Ammonia Toxicity and Its Effect on the Growth of the South African Abalone *Haliotis Midae Linnaeus*. Aquaculture 2006;

[9] Troell M., Robertson-Andersson D., Anderson R.J., Bolton J.J., Maneveldt G., Halling C and Probyn T. Abalone Faming In South Africa: An Overview With Perspectives On Kelp Resources, Abalone Feed, Potential For On-Farm Seaweed Production And

ply and Market Dynamics. Journal of Shellfish Research 2004; 23 935-939.

tion through Aquaculture. Preventative Veterinary Medicine 2005; 67 117-124.

[3] FAO. The State Of World Fisheries and Aquaculture. Rome. 2012; 209pp.

dustry produced by Enviro-fish Africa (Pty.) Ltd. 2007; 24p.

For Export Boom. Engineering News 16-17 August. 2004; 6-12.

Socio- Economic Importance. Aquaculture 2006; 257 266-281.

Aquaculture 2001; 200(1-2) 223-247.

261(2) 678-687.

and Suren Singh2

http://dx.doi.org/10.5772/57197

225

Application of Biological Agents in Abalone Aquaculture: A South African Perspective

### **7. The impact and benefits of the application of biological agents in abalone aquaculture**

Most aquaculture industries are leaning towards the use of probiotic technology as a solution to many of the challenges faced by the industry. The basis for the inclusion of probiotics into the farming environments include higher survival rate of juvenile and adult abalone, improved feed uptake and conversion ratios resulting in faster growth rates, improved resistance to disease and reduced contribution to water pollution [47]. Using probiotics is more environ‐ mentally friendly because the effluent water is cleaner and there are significant improvements in the gut flora thus enhancing the overall immune response of the host and an increase in food assimilation [160]. However other factors such as temperature, enzyme levels, water quality and genetic resistance may have an effect on the success of the technology in the farming facility [29].

Thus far, the uptake of the technology is slow-moving. This is due to the fact that farmers expect the probiotic technology to operate using the same basis as antibiotic treatment technology, in that they require and anticipate fast rapid results [160]. However changes in the microbial ecosystems present in the environment is a gradual one; and requires the continuous addition of beneficial microorganisms to compound the desired effect [158]. In addition, ineffective and costly probiotic products previously offered in aquaculture has negatively tainted the impact of this technology. Some products include *Clostridium spp*., *Pseudomonas putida* and other potential human pathogens, and others consist of cell densities that are too low to deliver any sort of benefit to the host [160].

The commercial aquaculture sector will make a notable difference in terms creating jobs and economic development in most developing countries by embracing this activity. To date, South Africa has validated itself to be a key player in the abalone mariculture arena. With support from government,this industrycouldexperienceafurtherboom,andasaresult,assistinreducingthe high levels of unemployment that exists [27], particularly in coastal areas that can effectively participate in aquaculture practices. Abalone industries not only include direct employment at thefarmlevel,italsoindirectlysupports interlinkedbusinesses suchas theseaweedandabalone processing industries [9]. The challenge is to ensure long term sustainable growth of the abalone mariculture industry. The use of appropriate and safe biological agents in abalone mariculture has excellent potential to meet the new challenges of this important industry.

#### **Author details**

strain testing of potential probiotics should encompass the robustness of the product against process fluctuations under farm conditions and confirmation of non-transmission of drug resistance genes or virulence plasmids [159]. Another barrier preventing the worldwide adoption of this technology, relates to the absence of efficacy data, which as a result casts a shadow of doubt over the technology, thereby hindering its uptake by the aqua-culturists.

**7. The impact and benefits of the application of biological agents in abalone**

Most aquaculture industries are leaning towards the use of probiotic technology as a solution to many of the challenges faced by the industry. The basis for the inclusion of probiotics into the farming environments include higher survival rate of juvenile and adult abalone, improved feed uptake and conversion ratios resulting in faster growth rates, improved resistance to disease and reduced contribution to water pollution [47]. Using probiotics is more environ‐ mentally friendly because the effluent water is cleaner and there are significant improvements in the gut flora thus enhancing the overall immune response of the host and an increase in food assimilation [160]. However other factors such as temperature, enzyme levels, water quality and genetic resistance may have an effect on the success of the technology in the

Thus far, the uptake of the technology is slow-moving. This is due to the fact that farmers expect the probiotic technology to operate using the same basis as antibiotic treatment technology, in that they require and anticipate fast rapid results [160]. However changes in the microbial ecosystems present in the environment is a gradual one; and requires the continuous addition of beneficial microorganisms to compound the desired effect [158]. In addition, ineffective and costly probiotic products previously offered in aquaculture has negatively tainted the impact of this technology. Some products include *Clostridium spp*., *Pseudomonas putida* and other potential human pathogens, and others consist of cell densities that are too

The commercial aquaculture sector will make a notable difference in terms creating jobs and economic development in most developing countries by embracing this activity. To date, South Africa has validated itself to be a key player in the abalone mariculture arena. With support from government,this industrycouldexperienceafurtherboom,andasaresult,assistinreducingthe high levels of unemployment that exists [27], particularly in coastal areas that can effectively participate in aquaculture practices. Abalone industries not only include direct employment at thefarmlevel,italsoindirectlysupports interlinkedbusinesses suchas theseaweedandabalone processing industries [9]. The challenge is to ensure long term sustainable growth of the abalone mariculture industry. The use of appropriate and safe biological agents in abalone mariculture

has excellent potential to meet the new challenges of this important industry.

**aquaculture**

224 Sustainable Aquaculture Techniques

farming facility [29].

low to deliver any sort of benefit to the host [160].

Ghaneshree Moodley1,2\*, Lethabo Mashigo1 , Rajesh Lalloo1 and Suren Singh2

\*Address all correspondence to<EMAIL_ADDRESS>
1 CSIR Biosciences, Pretoria, South Africa

2 Department of Biotechnology & Food, Faculty of Applied Sciences, Durban University of Technology, Durban, South Africa

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**Chapter 8**

**Use of Probiotic Bacteria against Bacterial and Viral**

The term "probiotic" was firstly used to denominate microorganisms that have effects on other microorganisms [1]. Etymologically, the term "probiotic" was originated from the Latin word "pro" which means "for" and the Greek word "bios" which means "life". The best known definition for probiotics was developed by the Food and Agriculture Organization (FAO), that defined them as live microorganisms which when administered in adequate amounts confer a health benefit on the host [2]. According to this description, the potential benefits are varied, and if probiotics were administered to shellfish or fish under intensive culture they could improve their production. It is known that virus and bacterial diseases/infections are one of the most important problems in aquaculture production at present. Probiotics can provide some solutions to this problem through different mechanisms or properties such as the production of inhibitory compounds such as bacteriocins, competition for adhesion sites with opportunistic or pathogen microorganisms, competition for nutrients with other bacteria or an improvement of the immune status (e.g. increase of production of immunoglobulins, acid phosphatase, antimicrobial peptides, improvement of cellular activities, etc.) [3-10]. Several reviews have already documented the benefits of probiotics in shellfish and fish but they mainly focused on their effects in the immune response. Thus, hypothetical and desired results of administering probiotics to shellfish or fish in culture will be improving their antiviral and antibacterial defences, which is the focus of the present review. Firstly, a brief description of probiotics is included, and then a review of the main used probiotics against pathogenic virus and bacteria for shellfish and finally, the same for fish. The novelty of this review is based on the shared ability of probiotics to control both viral and bacterial diseases in shellfish and fish

> © 2014 Cordero et al.; licensee InTech. This is a paper 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.

**Infections in Shellfish and Fish Aquaculture**

Héctor Cordero, María Ángeles Esteban and

Additional information is available at the end of the chapter

often share, which could be the basis for sustainable aquaculture.

Alberto Cuesta

**1. Introduction**

http://dx.doi.org/10.5772/57198

## **Use of Probiotic Bacteria against Bacterial and Viral Infections in Shellfish and Fish Aquaculture**

Héctor Cordero, María Ángeles Esteban and Alberto Cuesta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57198

#### **1. Introduction**

The term "probiotic" was firstly used to denominate microorganisms that have effects on other microorganisms [1]. Etymologically, the term "probiotic" was originated from the Latin word "pro" which means "for" and the Greek word "bios" which means "life". The best known definition for probiotics was developed by the Food and Agriculture Organization (FAO), that defined them as live microorganisms which when administered in adequate amounts confer a health benefit on the host [2]. According to this description, the potential benefits are varied, and if probiotics were administered to shellfish or fish under intensive culture they could improve their production. It is known that virus and bacterial diseases/infections are one of the most important problems in aquaculture production at present. Probiotics can provide some solutions to this problem through different mechanisms or properties such as the production of inhibitory compounds such as bacteriocins, competition for adhesion sites with opportunistic or pathogen microorganisms, competition for nutrients with other bacteria or an improvement of the immune status (e.g. increase of production of immunoglobulins, acid phosphatase, antimicrobial peptides, improvement of cellular activities, etc.) [3-10]. Several reviews have already documented the benefits of probiotics in shellfish and fish but they mainly focused on their effects in the immune response. Thus, hypothetical and desired results of administering probiotics to shellfish or fish in culture will be improving their antiviral and antibacterial defences, which is the focus of the present review. Firstly, a brief description of probiotics is included, and then a review of the main used probiotics against pathogenic virus and bacteria for shellfish and finally, the same for fish. The novelty of this review is based on the shared ability of probiotics to control both viral and bacterial diseases in shellfish and fish often share, which could be the basis for sustainable aquaculture.

© 2014 Cordero et al.; licensee InTech. This is a paper 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.

#### **2. Probiotic bacteria**

There is a great diversity of tested probiotic bacteria, but only few of them have become in commercial probiotics (Table 1). Thus, further studies are mandatory to expand the use of laboratory described microorganisms with probiotic effects to the commercial level and then be used in the aquaculture industry. The procedure to test and market a probiotic is resumed in Figure 1.

Probiotics are usually consisting on bacteria but some other microorganisms such as yeast, microalgae or even some fungi. They are mainly used as living cells but some studies have also shown their benefits when supplied as heat-inactivated cells (also known as heat-killed cells), formalin-killed (FKC), freeze-dried, dead cells or cell-free supernatant (CFS). Among the vast number of probiotic species used most information relies on the use of *Bacillus* sp. and *Lactobacillus* sp. Different administration modes have been checked, as bath, intraperitoneal or intramuscular injection and in diet being the bath and diet those preferred for the use in the aquaculture. Moreover, more recently, for oral dietary administration the probiotics can be encapsulated in different ways. Besides that, *Artemia* and rotifers (two main diets larvae in marine larviculture) are usually enriched with probiotics in order to produce benefits in the

Use of Probiotic Bacteria against Bacterial and Viral Infections in Shellfish and Fish Aquaculture

http://dx.doi.org/10.5772/57198

241

fish/shellfish larvae.

**Figure 1.** Process for making commercial probiotics.

**Table 1.** List of commercial probiotics, including those for shellfish and fish.

Probiotics are usually consisting on bacteria but some other microorganisms such as yeast, microalgae or even some fungi. They are mainly used as living cells but some studies have also shown their benefits when supplied as heat-inactivated cells (also known as heat-killed cells), formalin-killed (FKC), freeze-dried, dead cells or cell-free supernatant (CFS). Among the vast number of probiotic species used most information relies on the use of *Bacillus* sp. and *Lactobacillus* sp. Different administration modes have been checked, as bath, intraperitoneal or intramuscular injection and in diet being the bath and diet those preferred for the use in the aquaculture. Moreover, more recently, for oral dietary administration the probiotics can be encapsulated in different ways. Besides that, *Artemia* and rotifers (two main diets larvae in marine larviculture) are usually enriched with probiotics in order to produce benefits in the fish/shellfish larvae.

**Figure 1.** Process for making commercial probiotics.

**2. Probiotic bacteria**

240 Sustainable Aquaculture Techniques

in Figure 1.

**Bactocell®**

**BioPlus®**

There is a great diversity of tested probiotic bacteria, but only few of them have become in commercial probiotics (Table 1). Thus, further studies are mandatory to expand the use of laboratory described microorganisms with probiotic effects to the commercial level and then be used in the aquaculture industry.  List of commercial probiotics, including those for shellfish and fish.

 **2B** Fish [73]

**Biogrow®** Mammalian Contains *Bacillus subtilis* and *B. licheniformis*

**Biostart®** Fish Contains a mix of *Bacillus* spp. and *Paenobacillus* sp.

**Biosporin®** Human Contains *B. subtilis* and *B. licheniformis*

**Bispan®** Human Contains *Bacillus polyfermenticus*

**Lactopure** Mammalian Contains *Lactobacillus sporogenes*

**Neolactoflorene** Human Contains *Lactobacillus* spp. and *Bacillus* spp.

 **PA 10** Fish [42]

**Alibio®** Fish [30]

**BGY-35** Fish [51]

**Cernivet®** Fish [85]

**Lactobacil** Fish [45]

**Sporolac** Fish [45]

**Toyocerin®** Fish [85]

**Ecomarine®** Shellfish

**Promarine®** Shellfish

#### **3. Probiotics against virus in shellfish**

Viral infections are one of the most important problems in aquaculture production. In the case of shellfish, probiotics might provide a good preventive solution to this problem since they promote the innate immune response, which is the only one attributed to be responsible for the resistance in these animals.

Several studies have been conducted in bivalves. In the case of Pacific oyster larvae (*Crassostrea*

the larvae prolonged their viability up to 144 hours indicating its benefits when used by bath

sp. OY15 for three weeks showed no effect in survival ratio after challenge with *Vibrio s*p. M183 [19]. It has been reported [20] that abalone (*Haliotis discus hannai*) specimens fed supplemented

showed a better survival rate (with mortalities of 27%-50% in WA64, and 30%-43% in WA65 compared with 77%-80% in the control group) when infected with *Vibrio harveyi*. In other research with other abalone specie, *Haliotis midae* specimens fed supplemented diet with a mix

showed a better survival ratio (62%) than control group specimens after intra-mantle injection of *Vibrio anguillarum* [21]. Further studies are still needed to broad the use of probiotics in

Among the shellfish, most of the studies have at this respect focused on shrimps. Thus, western

and *Pseudomonas synxantha* for eighty-four days and afterwards challenged with *V. harveyi*. *P. aeruginosa-*supplemented diet improved the survival rate of the western king prawns more effectively than *P. synxantha-*supplemented diet, and furthermore, administration of both probiotics in combination resulted in better results than when administering separately [22-23].

Most of the studies administering probiotics have been developed in white shrimp (*Litopenaeus vannamei*) at different development stages. For example, *Bacillus subtilis* E20 administered in

respectively, after the injection of pathogenic *V. alginolyticus* [24]. In juvenile specimens,

achieved a mortality of 18% against pathogenic infection of vibrios (including *V. harveyi*, *V. alginolyticus* and *V. parahaemolyticus*) while the control group mortality exceeded of 50% [25]. In other research, juvenile specimens fed supplemented diets containing *V. alginolyticus* UTM 102, *B. subtilis* UTM 126, *Roseobacter gallaeciensis* SLV03 or *Pseudomonas aestumarina* SLV22,

after immersion with *Vibrio parahaemolyticus* PS-017 compared with the control group (33%) [26]. In adult specimens of *L. vannamei* fed supplemented diet with 3×105 cfu of the probiotic *Vibrio gazogenes* per shrimp showed a decrease of mortality after infection with *Vibrio* spp. (including *V. harveyi*, *V. anguillarum* and *V. alginolyticus*) [27]. In addition, the inhibitory effect was also demonstrated in a *in vitro* assay [27]. Other recent work [28] has been carried out with

*Bacillus subtilis* strains) for 2 months, and afterwards each shrimp was injected with 107

*Vibrio harveyi*. Results indicate that cumulative mortality of the control group was 63.3%, whereas in the groups fed probiotics were of 20% and 33.3%, for the group fed BM8 or BM5 strains, respectively. Cumulative mortality also decreased in white shrimp fed a supplemented diet with 1010 cfu kg-1 of *Lactobacillus plantarum* after injection with *V. alginolyticus* [29].

cfu kg-1 increased the survival rates at 13.3%, 16.7% and 20%

cfu g-1 diet for four weeks showed low mortality (between 17%-22%)

cfu g-1 (BM5) and 108

cfu g-1 of *Shewanella colwelliana* WA64 and *Shewanella oyellana* WA65 for four weeks