Beneficial bacteria for aquaculture: nutrition, bacteriostasis and immunoregulation
Despite being the fastest growing sector, the modern aquaculture industry faces serious challenges such as the lack of protein source in feed, the susceptibility to pathogens, and deterioration in quality during culture and storage. Bacterial biomass is considered as a proper protein source for feed, and the beneficial bacterial species protect aquatic animals from infection or reduce spoilage of products. In this review, we summarized the application of beneficial bacteria to aquatic products, focusing mainly on the nutritional, anti-pathogenic, anti-spoilage and immunoregulatory functions of these bacteria. We then discussed the relationship between beneficial bacteria, intestinal microbiota and host immunity, and the recent progress and drawbacks of the technology.
With the increasing demand for seafood and the rapid development of culture technology, the aquaculture industry has been growing rapidly in recent years. In fact, the growth of aquaculture sector is faster than any other animal food-producing sectors (Zorriehzahra et al. 2016), and it can be predicted that aquatic organisms will represent the predominant sources of high-quality protein in the near future (Gamboa-Delgado and Marquez-Reyes 2018). In contrast, the fast-developing aquaculture industry has been facing serious challenges. The increase in cultivation calls for the reliable source of raw material as feed, but the conventional feed sources are either limited (e.g. fish meal as a source of protein) or costly. These limit the growth of the aquaculture industry, and therefore, novel feeds with sufficient supply and low cost are required (Olsen and Hasan 2012). Additionally, the modern high-density aquaculture system facilitates the outbreak of disease induced by pathogen infection, which usually results in mass mortality of aquatic animals, the decline in quality of products and subsequent enormous economic loss (Carrias et al. 2012).
Bacteria are usually rich in proteins and easy to large-scale production, making them possible to be an alternative source of protein for feeds. A large percentage of bacteria are considered to be beneficial or termed probiotic that do not cause any harm to host health but show some beneficial biological activities to facilitate host growth. Consequently, particular beneficial bacteria might be used as anti-pathogenic reagents for given host species with specific disease or as anti-spoilage reagents during product storage.
Thus, beneficial bacteria are a promising supplement with multiple functions for aquaculture. This review detailed the applications of beneficial bacteria and discussed the possible mechanisms underlying the benefits of using bacteria in aquaculture, thus lending support for further mechanistic investigations and novel applications.
Bacteria-derived nutrients for aquaculture
With the rapid growth of aquaculture industry, the requirement of aquaculture feeds has also increased. In fact, the availability of ingredients for aquaculture feeds has become limited in the aquaculture industry (Olsen and Hasan 2012). Furthermore, the increase in traditional fish meal production may increase economic and environmental concerns. Thus, alternative feed sources are needed and this has attracted great efforts in both research and applications; among this, micro-organisms are considered as promising candidates for aquaculture feed. In contrast, some beneficial bacteria can provide micronutrients such as vitamins, fatty acids and essential amino acids in addition to macronutrients to support the healthy growth of aquatic animals. In addition, bacteria may also regulate the host digestion process by producing extracellular enzymes.
Bacteria as the alternative aquaculture feeds
Aquatic animals are not only ideal source of protein for humans due to their high protein content, but they also require abundant protein in their feed. Micro-organisms, especially bacteria, are usually rich in proteins which can make up to 65% of the total dry weight. In addition, bacteria contain more nucleotides compared with traditional fish meal. Dietary supplementation with bacteria can be beneficial for the growth, immunity and stress response of aquatic animals, and can also improve the diet palatability (Gamboa-Delgado and Marquez-Reyes 2018).
Several studies have investigated the possibility of partially or even totally replacing fish meal by bacterial biomass and encouraging results were obtained. In a 10-week growth trial in juvenile Florida pompano (Trachinotus carolinus), partially replacing fish meal with up to 12·82% of dried fermented biomass on an isonitrogenous and isolipidic basis did not significantly change the final weight, survival, percent weight gain, food conversion ratio and thermal-unit growth coefficient (Rhodes et al. 2015). Similarly, partially replacing fish meal with bacterial single-cell protein did not influence the growth rates, feed consumption and absorption efficiency of rainbow trout (Oncorhynchus mykiss) (Perera et al. 1995). In addition to common bacteria that take complex organics as the carbon source, methanotroph bacteria that grow on natural gas have also been studied as a substitute for fish meal. In Atlantic salmon (Salmo salar) culture, experiments that partially replace fish diet with methanotroph bacteria, especially Methylococcus capsulatus, demonstrated that bacterial protein meal could be an alternative protein source to fish meal. Intake of the bacteria protein meal does not largely change the fish growth rate or induce health problems and can relieve soybean meal-induced enteritis (Aas et al. 2006; Romarheim et al. 2011). Furthermore, compared with finfish culture, in shellfish culture, the application of bacterial biomass not only supported animal growth as traditional feeds but also improved culture efficiency in some aspects. In black tiger prawn culture, the application of bacterial biomass offset the growth losses result from the absence of fish meal or oil (Glencross et al. 2014), and additional bacterial biomass in feeds further improved shrimp growth (Arnold et al. 2016).
Moreover, the bacteria can further form flocculated material which not only provides an additional food source but also improves the water quality and thus raises the breeding density (De Schryver et al. 2008). In the Pacific white shrimp (Litopenaeus vannamei), dry bioflocculated material collected from aquaculture can be used to partially replace fish meal, and replacing over 20% of fish meal with biofloc meal may actually improve shrimp growth (Dantas et al. 2016). Similar studies and applications performed in other aquatic animals, such as flatfish (Paralichthys olivaceus) and sea cucumber (Apostichopus japonicus), also showed improved growth performance (Chen et al. 2018; Kim et al. 2018).
However, it should be noted that some studies also found that the overuse of bacterial biomass may be counterproductive, resulting in the low digestibility of nutrition and the reduction in growth, which could be due to the decrease in N absorption and the increase in urea excretion (Perera et al. 1995). Moreover, even though the reproductive performance of domesticated broodstock was not influenced, a high percentage of bacterial biomass may reduce the egg hatching of black tiger prawn (Goodall et al. 2016).
Micronutrients produced by beneficial bacteria
In addition to the basic macronutrients that are usually provided by feeds, aquatic animals also need various micronutrients such as vitamins, fatty acids and essential amino acids to support the growth and normal physiological functions, which may be insufficient from some feeds. A large number of bacteria produce various kinds of micronutrients; the application of some of the strains of these bacteria has been widely reported in human (LeBlanc et al. 2017) and the utilization of these micronutrients to support the growth of aquatic animal has also been investigated.
Vitamin B-12 is one of the important vitamins produced by various probiotic strains, which is a cofactor in DNA synthesis and participates in both fatty acid and amino acid metabolism. A study on isolated intestinal bacteria from carp showed that a large percentage of the commensal bacteria can produce vitamin B-12 (Teshima and Kashiwada 1967). Furthermore, these bacteria can provide vitamin B-12 for supporting fish growth even in the absence of diet vitamin B-12 (Kashiwada et al. 1970). Similarly, another study showed that dietary supplementation of vitamin B-12 was not necessary for the normal growth of channel catfish because intestinal bacteria synthesize approximately 1·4 ng of vitamin B-12 per gram of body weight per day, which can be absorbed directly from the digestive tract into the blood. Consequently, the dysbiosis induced by antibiotic could reduce the rate of intestinal synthesis and liver stores of vitamin B-12 (Limsuwan and Lovell 1982). In addition, other kinds of vitamins, such as other B-group vitamins and vitamin K, synthesized by human gut microbiome have also been reported (LeBlanc et al. 2013, 2017); however, relevant studies in aquatic animals are limited and further investigations are needed.
The plant protein source is considered as a viable alternative to fish meal due to its sustainable availability and reasonable price. However, because of the deficiencies in essential amino acids like tryptophan, lysine and sulphur-containing amino acids, the use of plant protein sources may result in the reduction of growth performance in shrimp. To overcome the disadvantage, Jannathulla and colleagues treated plant protein sources with bacterial, fungal and yeast fermentation methods and found that the essential amino acid contents were greatly increased after the treatment. Moreover, the anti-nutrients such as trypsin inhibitor, phytic acid, saponin, tannin, glucosinolate and guar gum in plant protein sources were also reduced after the fermentation. The application of micro-organism in treating plant protein sources paves way for a higher ratio of replacement for fish meal in aquaculture feed formulations (Jannathulla et al. 2017).
Recent studies have revealed that commensal and probiotic bacteria can produce short-chain fatty acids (SCFAs) by fermentation of certain fibres in the intestine, which participate in host energy metabolism and immunity (LeBlanc et al. 2017). A feeding trial using dietary probiotic Clostridium butyricum in kuruma shrimp (Marsupenaeus japonicas) demonstrated that the administration of the strain increased the content of intestine SCFAs including propionic acid and butyric acid, which together with several other beneficial effects promoted M. japonicus growth and elevated body crude protein content (Duan et al. 2018). Similarly, in grass carp (Ctenopharyngodon idellus), the diet type can modify the hindgut microbiota structure, which is tightly correlated with SCFAs concentration (Hao et al. 2017).
Bacteria regulate host digestion process
Besides directly providing nutrition to aquatic animals, some beneficial bacterial strains improve host intestinal digestion process by secreting extracellular enzymes such as proteases, lipases and growth-promoting factors. For example, using axenic algae (Isochrysis galbana) as the feed led to the poor growth of axenic oyster (Crassostrea gigas) larvae; however, when marine bacterial strain CA2 was used, the growth returned to normal, and the survival and growth rates also increased greatly. Since the strain CA2 did not influence the algal growth and food availability, it was suggested that it makes a nutritional contribution to the growth of oyster larvae (Douillet and Langdon 1993).
More detailed studies have provided direct evidence for the production of extracellular enzymes by commensal bacteria. The isolation and identification of different strains of micro-organisms from the gut of adult penaeid shrimp (Penaeus chinensis) revealed the composition of gut microbiota, and several bacterial strains were found from the foregut, midgut and hindgut that can produce digestive enzymes including amylase, chitinase, lipase, gelatinase and caseinase; among these, most digestive enzyme-producing bacteria were settled in the foregut (Wang et al. 2000). Bacillus cereus isolated from the intestine of fish Mugil cephalus showed a strong ability to produce extracellular protease in vitro, and the enzyme activity could reach more than 300 U/mL under optimized conditions, especially in the presence of defatted fish meat and galactose (Esakkiraj et al. 2009). Similarly, the fish pathogen Yersinia ruckeri was also reported to produce extracellular protease, lipase and haemolysin. However, these enzymes led to the appearance of symptoms associated with pathogenicity, indicating the significant effect of intestinal bacterial extracellular enzymes on the host physiology and the two sides of this effect (Romalde and Toranzo 1993; Secades and Guijarro 1999). Sugita and colleagues analysed the amylase-producing ability by intestinal microflora in different cultured freshwater fish, and found that anaerobes were more likely to produce amylase than aerobes. Specifically, the strains of Aeromonas, Bacteroidaceae and Clostridium produced amylase effectively while the strains of Acinetobacter, coryneforms, Enterobacteriaceae, Moraxella, Plesiomonas and Streptococcus did not. These results suggested that amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent (Sugita et al. 1997).
In a 56-day feeding trial in kuruma shrimp (M. japonicas), the application of dietary probiotic C. butyricum significantly increased the activities of intestine pepsin, 5-hydroxytryptamine, amylase and lipase but not alanine aminotransferase or aspartate aminotransferase in a dose-dependent manner. Moreover, the contents of intestinal SCFAs and body crude protein also improved after application of C. butyricum, indicating the beneficial effect of the strain in promoting shrimp digestive ability and improving host nutrition and growth (Duan et al. 2018).
Inhibition of pathogenic or spoilage micro-organisms by beneficial bacteria in the seafood chain
Environmental micro-organisms always exist throughout the seafood chain; despite the fact that many of them are harmless, a sizeable percentage of them can cause harm to the system. Particularly, the aquatic pathogens such as bacteria and viruses may impair animal growth or even result in death, and spoilage bacteria corrupt the aquatic products during storage. These effects subsequently decrease aquaculture yield and reduce product quality, eventually leading to economic loss and damaging consumer health. Application of beneficial bacteria is a promising biocontrol approach for suppressing harmful micro-organisms by multiple mechanisms (Fig. 1), and hence has broad application prospects from aquaculture to aquatic products.
Anti-pathogenic effect of beneficial bacteria
Due to the wide application of high-density aquaculture, infectious diseases caused by pathogens such as bacteria, viruses, fungi, protozoa and parasites are extremely detrimental to the global farming of aquatic organisms, resulting in enormous economic losses and potential threats to consumer health (Carrias et al. 2012). Vaccines and antibiotics are major therapeutic approaches for bacterial pathogens and have played an essential role in protecting aquatic animals from infection and reducing disease-related losses. However, these methods cannot cover all the diseases or hosts, and also show some drawbacks such as high cost, drug resistance, and the potential harm to environment and consumers (Carrias et al. 2012). Alternatively, several beneficial bacterial (or probiotics) strains have been developed to treat bacterial pathogen-induced diseases, and the effectiveness of this method has been proved. In general, there are three major approaches for beneficial bacteria to suppress aquaculture pathogens: competing for nutrients and spaces, producing inhibitory compounds and stimulating the host immunity.
Several bacterial strains have been reported to compete with pathogens for iron, which is essential for micro-organism growth (Verschuere et al. 2000). Among 30 Vibrio anguillarum strains, only one strain that yielded a significantly higher amount of siderophore (a ferric ion-specific chelating agent facilitate iron availability) was capable of inhibiting fish pathogen Vibrio ordalii. Notably, the inhibitory effect was blocked in the presence of additional iron salt, implying the anti-pathogenic effect was mediated by the competition of iron (Pybus et al. 1994). Smith and Davey have isolated a Pseudomonas fluorescens strain F19/3, which inhibits Aeromonas salmonicida by competing for free iron, and thus protects against stress-induced furunculosis on external locations (Smith and Davey 1993).
By screening 12 lactic acid bacteria isolated from fish and sediments, Sica and colleagues found that most of these strains can attach to rainbow trout skin mucus and thus competing with and excluding two salmonid pathogens, Y. ruckeri and A. salmonicida, suggesting them to be potential anti-infective agents for use in rainbow trout culture (Sica et al. 2012). Similarly, the candidate probiotic C. butyricum CB2 exhibited strong adhesion property and antagonistic activity to two fish pathogens Aeromonas hydrophila and V. anguillarum both on agar plate and fish intestinal cell model (Pan et al. 2008). With the aid of radioactive isotopes, Vine and colleagues investigated the function of several candidate probiotics isolated from the intestinal tract of the common clownfish in vivo and found that these strains could displace the fish pathogens A. hydrophila and Vibrio alginolyticus from the intestine. However, the effect could only be observed when the probiotics were applied after, but not before pathogen colonization, indicating that the anti-pathogenic activities of these probiotics were therapeutic rather than preventative (Vine et al. 2004).
Moreover, some beneficial bacteria can produce inhibitory compounds to suppress or even kill pathogens. Bacteriocins are small peptides that disrupt the integrity of bacterial cell membranes produced by various kinds of bacteria. Different bacteriocins may vary in their inhibitory strength, and some of which are active against different bacteria (Teplitski et al. 2009). Lactococcus lactis TW34 isolated from marine fish can produce the bacteriocin nisin Z, which can inhibit the growth of the fish pathogen Lactococcus garvieae at 5 AU ml−1, and is thus considered as an alternative in the prevention of the global aquaculture disease lactococcosis (Sequeiros et al. 2015). By screening bacteria isolated from deep sea shark (Centroscyllium fabricii) gut for antagonistic activity, Bindiya et al. (2015) found that the strain BTSS-3 was most likely related to Bacillus amyloliquefaciens and it showed antimicrobial activity against pathogenic bacteria including Salmonella Typhimurium, Proteus vulgaris, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Bacillus circulans, Bacillus macerans and Bacillus pumilus by producing a specific bacteriocin. However, it should be noted that bacteriocins are often more effective against Gram-positive pathogens, but most of the pathogens associated with aquatic animals are Gram-negative gammaproteobacteria, so the effect of bacteriocin in treating aquaculture pathogen can be uncertain (Teplitski et al. 2009). Fortunately, several other kinds of anti-pathogenic compounds produced by beneficial bacteria have been found. For example, in a study, several roseobacter strains isolated from turbot hatchery showed the ability to reduce or even completely kill the fish pathogen V. anguillarum, which was associated with the tropodithietic acid production, as a tropodithietic acid-negative mutant lost the anti-pathogenic activity (D’Alvise et al. 2010). By studying the response of four common pathogens of turbot (Scophthalmus maximus) to nine potential probiotics, Vázquez and colleagues conclude that the effect of lactic acid bacteria on pathogens can be either stimulatory or inhibitory dependent on the strain of pathogens. In detail, it is evident that the stimulatory effect is due to the fermentative metabolism (proteins and sugars) and the inhibitory effect to the lactic or acetic acids produced by the lactic acid bacteria (Vázquez et al. 2005).
In addition to producing compounds that directly suppress pathogens, some beneficial bacteria can modulate pathogen growth and activity by regulating signal transduction, especially through the quorum-sensing system. By growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL, a class of quorum-sensing signalling molecules), several bacterial enrichment cultures that could degrade AHL molecules were isolated from the digestive tract of Pacific white shrimp (P. vannamei). These bacteria could further degrade the quorum-sensing signal molecules (i.e. HAI-1) produced by pathogen Vibrio harveyi in vivo, neutralizing the AHL-mediated negative effect of V. harveyi (Tinh et al. 2007). Practically, these kinds of bacteria could protect giant freshwater prawn (Macrobrachium rosenbergii) larvae from V. harveyi infection, and thus promoted larval survival and larval quality (Nhan et al. 2010). Similarly, a quorum-quenching enzyme producing probiotic Bacillus sp. QSI-1 was found to affect the composition of fish gut microbiota and decrease the percentage of AHL-producing fish pathogen A. hydrophila in vivo (Zhou et al. 2016). In contrast, several Gram-negative strains isolated from various freshwater Chilean salmonid farms were shown to inhibit the growth of the fish pathogens A. hydrophila, V. anguillarum and Flavobacterium psychrophilum. They also could block the quorum-sensing system as well as produce homoserine lactone molecules at the same time, indicating that in addition to directly degrading quorum-sensing molecules, their extra production may also disrupt quorum-sensing signalling and thus suppress the corresponding pathogens. However the detailed mechanisms required further investigations (de la Fuente et al. 2015).
Anti-spoilage effect of beneficial bacteria
Due to high water and protein contents, aquatic products not only have good taste and high nutrition values, but also are susceptible to spoilage. The spoilage of aquatic products results in huge economic losses and consumer health risks every year. Therefore, various kinds of chemical preservative have been developed to preserve aquatic products during storage; however, the usage of these compounds may have secondary safety issues to both the consumer health and the environment, and the application is also restricted to product types and storage methods. By using similar mechanisms, beneficial bacteria can also inhibit spoilage bacteria and aquatic animal pathogens. Therefore, it can be used as a supplement to the traditional preservative. However, due to the distinct environment of in vivo gut and in vitro storage condition, the relevant bacteria can be totally different; therefore, the detailed bacteriostatic approaches are also distinct.
Fermentation is a traditional and common processing method for food including aquatic products, which can prevent the food from spoilage and produce specific flavour. Fermentation generally suppresses the growth of most spoilage bacteria because of hypertonic or anaerobic environment. In addition, the beneficial bacteria involved in fermentation also take part in the anti-spoilage effect. By oxidizing carbohydrates during metabolism, these bacteria generate a range of products mainly organic acids, alcohol and CO2, which dramatically change the whole environment and also have a preservative effect by limiting the growth of spoilage and/or pathogenic flora in the food product (Ray and Montet, 2015).
In addition to completely changing the environment, some beneficial bacteria can produce more delicate products to inhibit the activity of spoilage bacteria, such as bacteriocin and quorum-quenching enzymes.
Nisin is one of the bacteriocins approved by Food and Drug Administration as a food preservative due to its ability to kill a broad spectrum of Gram-positive pathogens and food spoilage bacteria (Lubelski et al. 2008). For example, both the cell pellet and cell-free supernatant of nisin Z-producing L. lactis subsp. lactis KT2W2L isolated from brackish water were demonstrated to inhibit a broad spectrum of food-spoilage bacteria and food-borne pathogens. In addition, the application of the strain inhibited the growth of spoilage bacteria and also extended the shelf-life of cooked, peeled and ionized tropical shrimps during storage at 8°C under modified atmosphere packaging (Hwanhlem et al. 2015). Similarly, the application of nisin enhanced the quality and extended the shelf-life of seer fish (Scomberomorous guttatus) steaks and low-temperature storage large yellow croaker (Pseudosciaena crocea), especially when combined with chitosan, glazing and radiation processing (Guohua et al. 2016; Kakatkar et al. 2017). In addition to nisin, other bacteriocins such as AMPNT-6, Coagulin L1208 and DY4-2 from various bacterial species have also been proved to suppress seafood spoilage bacteria growth and hence ensure food quality as well as extend shelf-life (Deng et al. 2017; Lv et al. 2018; Fu et al. 2018a).
As in the case of pathogens, the quorum-sensing system also regulates the spoilage potential of bacteria during the storage of aquatic products. As a result, disrupting quorum-sensing signalling may suppress the activity of spoilage bacteria and thus preserve the food. For instance, AHL lactonase AiiA (AI96) produced by Bacillus quenched the quorum-sensing system of Aeromonas veronii LP-11, a specific spoilage organism of sturgeon. AiiA (AI96) treatment significantly reduced the protease and motility activities of A. veronii LP-11 but did not show any effect on the cell growth. In practice, AiiA (AI96) application inhibited the spoilage progress of vacuum-packaged sturgeon stored at 4°C (Gui et al. 2017).
Regulation of immune system by beneficial bacteria in aquatic animals
Due to high concerns and heavy investments on human health as well as the rapid development of microbiomics in recent years, it has been well demonstrated that the commensal microbiota is tightly involved in human immune homeostasis, and the administration of probiotics can regulate the microbiota composition and thus maintain immune homeostasis (Azad et al. 2018; Fu et al. 2018b). On the contrary, relevant studies on aquatic animals are limited. Nevertheless, researchers have taken efforts to investigate the point in recent years, and valuable results have been obtained.
Commensal microbiota in maintaining host immune homeostasis
There are sufficient observations that healthy fish has a higher diverse microbial community than diseased ones, indicating the close correlation between commensal microbiota and body homeostasis. For example, compared with healthy crucian carps (Carassius auratus), those individuals affected by ‘red-operculum’ disease have less diverse and stable gut bacterial communities, which were also affected by the environmental factors especially water temperature and ammonia concentration. In contrast, some specific bacterial species, including Vibrio, Aeromonas and Shewanella, were relatively abundant in diseased fish, suggesting them to be the potential pathogens (Li et al. 2017). Similarly, results were obtained in largemouth bronze gudgeon (Coreius guichenoti) affected by furunculosis, where pathogenic A. salmonicida was identified as the critical species responsible for the disease (Li et al. 2016). In ayu (Plecoglossus altivelis) exposed to V. anguillarum, reduced dynamics of gut bacterial diversity and evenness, as well as less complex, fewer connected and lower cooperative gut bacterial interspecies interaction, were observed in infected subjects, which was significantly associated with the immune responses including the expression of TNF-α and IL-1β. The results imply that there is an interaction between gut microbiota and host immune responses to pathogen infection from an ecological perspective (Nie et al. 2017).
In addition to the correlation, the change in gut microbiota could be a genuine cause of immune diseases in aquatic animals. Low-dose antibiotics have been used as growth promoters in fish, which was suspected to cause disease outbreaks in aquaculture. When exposed to antibiotics oxytetracycline and sulfamethoxazole, Nile tilapia (Oreochromis niloticus) showed retarded growth performance accompanied by reduced nutrients digestibility, feed efficiency, organ indices and lipid body composition in treated fish. Moreover, antibiotics also induced microbiota dysbiosis, suppressed innate immunity and stimulated inflammatory. Furthermore, subsequent human health risks were also observed (Limbu et al. 2018). To reveal the underlying mechanisms, He and colleagues applied antibiotic olaquindox to zebrafish together with pathogen A. hydrophila infection. The results showed that both intestinal microbiota and innate immune responses of zebrafish were compromised by olaquindox. Additionally, microbiota transfer assay proved that increased pathogen susceptibility was driven by olaquindox-induced dysbiosis rather than directly by the antibiotic effect (He et al. 2017). This result strongly demonstrated that the host immune system could be affected by microbiota.
Probiotics regulate commensal microbiota, maintain immune homeostasis and promote growth performance in aquatic animals
Probiotics are safe and some may already exist in the digestive tract, which makes it possible to apply them to regulate the host commensal microbiota and immune system. The application of probiotics in human has been intensively studied. A wide range of beneficial effects, such as the prevention of degenerative diseases and the regulation of immune responses, have been observed (Azad et al. 2018). In recent years, the roles of probiotics in aquatic animals have attracted great research interests, and some results with potential scientific novelty and practical value have been obtained.
To investigate the beneficial effects of probiotics, three host-associated probiotics (Bacillus sp. AHG22, Alcaligenes sp. AFG22 and Shewanella sp. AFG21) were isolated from the gastrointestinal tract of Malaysian Mahseer (Tor tambroides) and applied to the same fish species. The intake of probiotics drastically modified the gut microbiota composition by increasing the species richness as well as some particular species such as lipolytic, proteolytic and cellulolytic bacteria. Moreover, the production of acetate and total volatile SCFA were also increased after probiotic application. This study demonstrated that host-derived probiotics could enhance the nutrients utilization and metabolism; however, the effects on host immune system and disease resistance remained to be revealed (Asaduzzaman et al. 2018). In contrast, Van Doan and colleagues applied host-associated probiotics (Lactobacillus plantarum N11 and Bacillus velezensis H3.1) to Nile tilapia (O. niloticus), and assessed the subsequent change in several innate-immune parameters, disease resistance and growth performance. The results showed that both the probiotics increased skin mucus lysozyme and peroxidase activities as well as the serum immune responses, and finally, promoted growth performance. Unfortunately, the change in intestinal microbiota was not measured in the study by Van Doan et al. (2018). Similar results were obtained in shrimp culture, where the application of probiotics decreased pathogen loading, improved growth performance and maintained immune homeostasis (Zhang et al. 2009; Madani et al. 2018). Another study showed that probiotics might reduce the viable culturable heterotrophic bacteria count, but the particular bacterial composition was not assessed (Jatoba et al. 2011).
Furthermore, several studies have established the linkage between probiotics, host immunity and commensal microbiota in aquaculture. When treated with either viable or heat-inactivated probiotic B. pumilus SE5, the grouper (Epinephelus coioides) exhibited an altered immune system, with the upregulation of immune gene (such as TLRs, cytokines and antibacterial epinecidin-1) expression. In addition, the microbiota was also affected, as several potentially pathogenic bacterial species were suppressed (Yang et al. 2014). Another study in shrimp also showed that with the increasing doses of probiotic Bacillus OJ in diets, the shrimp survivals and immune parameters generally increased whereas the counts of total viable bacteria and Vibrio decreased (Li et al. 2009). Interestingly, another study showed that when the rainbow trout (O. mykiss) was fed the probiotic Bacillus cereus var. toyoi, the fish formed a well-defined cluster composed of one super clade; however, the diversity of the total gut microbiota was decreased. Along with this, fish fed the probiotic showed rapid and high homogeneous growth rate, and the level of leucocyte infiltration in the lamina propria of the intestinal mucosa, the number of goblet cells as well as the villi height, were also increased (Gisbert et al. 2013).
In conclusion, the results showed that the application of probiotics could increase the survival rate and growth speed of aquatic animals, leading to the increase in output of products. The effect of probiotic on the host gut microbiota and immune system can be different depending on the probiotic strains as well as the host species. In most cases, the diversity of microbiota is negatively correlated with the host immune responses, which is consistent with the observation in the clinical studies of human beings (Azad et al. 2018). However, it should be noted that although the probiotic-induced biostatic, immune homeostasis and growth improvement showed close correlation, the causal association between them were unclear and required more detailed and direct investigations.
Probiotics improved quality of aquatic products by regulating immunity
Several studies have shown that maintaining immune homeostasis not only promotes the growth rate but also improves the flesh quality of aquatic products. For example, astaxanthin or carotenoid supplementation in striped satfish (Pangasianodon hypophthalmus) culture can improve fillet appearance and antioxidant status and it also enhances the non-specific immune response. In detail, in the presence of astaxanthin or carotenoid, the fillet colour exhibited a significantly high intensity of redness or yellowness, the texture parameters of the fillets were slightly changed, and the total erythrocyte count, haemoglobin, respiratory burst and lysozyme activities were also enhanced (Gopan et al. 2018). In another similar study, the supplement of Ulva sp. in Nile tilapia culture significantly increased the complement activity (ACH50), an important component of the innate immune system in fish, meanwhile the muscle colour was modified, and the sour parameter of the fillets was decreased (Valente et al. 2016).
In contrast, it has been demonstrated that pathogen infection not only impairs the yield of aquaculture but also deteriorates the quality of products (Lerfall et al. 2012). For example, Ingerslev and colleagues showed that even previous infections can influence product quality parameters in fish. In detail, fillets from rainbow trout (O. mykiss) were subjected to sensory evaluation for more than a year after recovery following infection by the bacterial pathogens Y. ruckeri and V. anguillarum. The results showed that the previously infected fish was less flaky, had a lower oiliness and a higher toughness and fibrousness compared with control fish. Moreover, the vaccination that suppressed the pathogen infection prevented the product from quality deterioration (Ingerslev et al. 2012).
Consequently, the application of beneficial bacteria is able to improve the aquatic product quality by maintaining host immune homeostasis and suppressing pathogen infection. In an 11-week feeding trial in juvenile Porthole livebearer (Poeciliopsis gracilis) with the use of Lactobacillus casei as a probiotic, only limited growth performance and survival rate improvement were observed. However, high values of protein and lipid contents were observed in the groups fed with the probiotic, indicating the effect of Lactobacillus casei in improving product quality (Hernandez et al. 2010). Nevertheless, relevant studies in this field are rare, and further studies are also required for the full elucidation of the beneficial bacteria effect on host immunity and product quality.
Conclusion and perspectives
Novel applications of beneficial bacteria in aquaculture
Due to the tremendous helpful effects of beneficial bacteria in aquatic animals described above, the usage of these bacteria in aquaculture has been intensively investigated and widely applied, and several novel applications were also developed.
Biofloc technology is an advanced technology based on the principle of waste nutrients recycling into microbial biomass. By steering the C/N ratio in water, heterotrophic microbiota is stimulated to grow, which can be used in situ by the cultured animals or be harvested and processed into feed ingredients (Bossier and Ekasari 2017). Although under further development, this technology has been applied in several aquaculture systems such as Pacific white shrimp (Dantas et al. 2016), flatfish (Kim et al. 2018) and sea cucumber (Chen et al. 2018). The biofloc technology improves aquaculture mainly by two approaches: first, providing a nutritious food source and improving feed utilization efficiency, and second, reducing water utilization and waste generation (Bossier and Ekasari 2017). Moreover, recent studies also demonstrated that biofloc was able to improve the immunological status of aquatic animals, and it also contributed to the improvement of the larvae robustness against diseases and environmental stress (Ekasari et al. 2015, 2016).
Another novel application is the heterogeneous expression of functional genes in bacteria and administrating them in aquaculture. For example, Jia and colleagues established a heterocystous cyanobacterium Anabaena sp. PCC 7120 expressing the major envelope protein VP28 of white spot syndrome virus as an oral vaccine. The administration of the recombinant probiotic significantly increased the survival of shrimp with the viral infection, indicating the effectiveness of the method (Jia et al. 2016).
Perspectives of beneficial bacteria in aquaculture
Nevertheless, it should be noted that the application of the so-called ‘beneficial’ bacteria may result in some side effects, and thus cannot be widely used before fully investigated and tested. The excess amount of limited bacteria in the culture system may compete with other essential micro-organisms and thus destroys the ecological balance. In contrast, overgrown bacteria may also compete for nutrients and oxygen with the cultured aquatic animal, or produce toxic metabolites such as biogenic amine that may be harmful to the animals. Recent studies showed that probiotics impaired the gut mucosal microbiome reconstitution in human beings (Suez et al. 2018). Similar phenomenon was also observed in aquaculture, where the application of probiotic decreased the total gut microbiota and increased intestinal mucosal immune responses in rainbow trout; however, virtual damage to fish health was not observed (Gisbert et al. 2013). Consequently, reliable and adequate investigations on the potential side or even toxic effects of each strain in each application should be performed to guarantee safety before large-scale industrial applications.
As described above, various bacterial strains have been proved to be beneficial to both aquatic animals and human beings. Whether a strain applied in aquaculture can directly or indirectly promote consumer health through the aquatic products or not is still unknown. In contrast, although numerous studies have demonstrated the beneficial effect of diverse bacterial strains in improving aquaculture, practical applications are still limited. Consequently, the prospects of beneficial bacteria in improving aquaculture efficiency and quality, as well as in developing healthy aquatic products are extremely broad; therefore, the related basic researches and industrial trials are valuable.
In recent years, the aquaculture industry has enjoyed rapid growth due to the increasing requirement of seafood and the development of culture technologies. However, the current aquaculture industry, especially high-density aquaculture, is facing several serious challenges, such as the lack of ample and cheap protein source for feeds, the enormous loss due to infection by pathogens and the deterioration in quality during culture and storage. Due to the relatively low cost, high protein content and various biological activities, the application of bacteria can be a possible solution to these challenges (Fig. 2). In reality, various beneficial bacterial species have been investigated and applied to aquaculture for several purposes. Nevertheless, to better improve the current methods and to minimize potential risks, novel applications should be developed and relevant mechanistic investigations are warranted in the future.
This study was financially supported by the National Natural Science Foundation of China [grant number 31571913 and 31772050] and the Zhejiang Provincial Natural Science Foundation of China [grant number LZ15C200001].
Conflict of Interest
The authors declare that they have no conflict of interest.
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