Exogenous Probiotics on Biofloc based Aquaculture: A Review

The successful entrepreneurship of aqua farming relies on the production of aquatic animals in the cost effective, social and environmental friendly approach. Nevertheless, presently fish farming is suffering from various problems related to these. Biofloc technology and/or application of probiotics provide promising results to aquaculture in terms of improvement in the growth and survival of aquatic animals, along with other benefits such as maintaining water quality without causing pollution to the environment. Biofloc is mainly comprised of various beneficial microbial communities, but the action of some probiotics it contains is unknown. On the other hand, probiotics are single, known live microbial strains and their actions to the animals are well established. Therefore, probiotics are recognized for having the most important constituents in the aquaculture. Although biofloc method and probiotics applications are promised to have positive roles aforementioned, the fish welfare often disturbed as the survival of the animals are always less in the fish farming. These led researchers to try generate a new technique to minimize these concerns. Recently new strategy of integrating both biofloc and probiotics were introduced called the exogenous addition of known probiotic bacteria to the biofloc. The study was demonstrated in the area by keeping biofloc as a control. Results promised that addition of single or combination of known probiotics to the biofloc further improve the growth performance of animals in addition with the maintenance of water quality parameters. Besides they also were promising the highest survival to animals with the reduction of pathogenic microbes. An exogenous root of probiotic bacteria on biofloc based aquaculture is a novel approach; relatively less number of studies has been performed in the area. This review describes the impacts of exogenous probiotics on biofloc based fish culture systems.

Introduction

A goal of aquaculture is to produce a healthy fish to assure the maximum profit. Diseases and unregulated water quality management in the culture systems interferes this.^17,^92,103 Application of antibiotics for the treatment of bacterial diseases was encouraged in the past.¹¹⁸ However, aquaculture depends on antibiotics is now criticized due to the following reasons. An antibiotic kills both the good and bad microbes in the gut of the animals. Also, continual usages of antibiotics lead animals to become resistant to pathogenic bacteria which lower the treatment effect. As using antibiotics in the aquaculture is severely condemned farmers are now looking for alternative methods to replace the use of antibiotics in disease control.^35,69

Basic resource needed for aquaculture is the water and land. In most of the places water and land available for fish culture is very less, thus farmers intend to go for intensive culture,²⁹ but intensification results in deterioration of water quality which causes stress to the fish followed by disease outbreak.¹⁰⁷ Some intensive production techniques such as re-circulatory aquaculture systems (RAS) are generated to solve these concerns; however, they are not economically beneficial to the farmers as using RAS is quite expensive. The generation of a cost efficient technology would overcome these problems.²⁹

Biofloc is a technology followed in the fish and shrimp farming; its outstanding feature is that they contain the mixture of bacteria, algae and other detritus which would be available feed for the fishes of omnivorous feeding habits.^11,29,39 A growing body of literature has recognized the positive influence of the biofloc technology on growth, non-specific immunity and disease prevention in fish.^{37,40,61,134,138} It also helps in the improvement of water quality in fish farming.²⁹ On the other hand, probiotics are specific microbial strains; its beneficial roles are known as they favourably contain all the necessary functions as biofloc does.⁵² Despite biofloc and/or probiotics are adopted by farmers for the practical reasons discussed, an occurrence of certain diseases are still common in fish as they reflect in the form of lower survival rates at the farms.¹²⁶ As diseases acquired by animals are often linked to specific bacteria, the action of specific antagonistic/beneficial bacteria would favorably minimize these problems.¹³⁹ Therefore, it was hypothesized that addition of specific, known probiotic bacteria to the biofloc proliferate the bacterial population either in the water or animals gut in order to suppress the potentially harmful pathogenic strains.^{1,31,62,77,146} Based on the hypothesis, recent studies have been attempted in these area, and reports seems to suggest that addition of probiotics to the biofloc further improve the water quality, animal growth, immunity, and survival of the animals than those of biofoc alone does.^{1, 31,62,77,146} A study on the influences of exogenous probiotics on biofloc based aquaculture system is a novel and integrative approach; but very less explored. Considering the practical merits of the technology mentioned above, significant studies are required to be conducted in the area on commercially cultivable fish species for the economic and environmental friendly sustainable aquaculture.

Biofloc

What is Biofloc Technology?

In general, biofloc is the macro-aggregation of bacteria, algae, detritus and other decomposed components.¹¹ According to Decamp and co-authors, it is the combination of bacteria, diatoms, zooplankton, protozoa, macro-algae, feces, uneaten feed, and exoskeleton from dead organisms.³⁹ As said by Hargreaves and co-authors, it is a group of biotic and abiotic particulate components suspended in the water which includes bacteria, planktons, and other organic materials.⁶³

Principle and Concept

The main principle of this technique is the practice of nutrient recycling.¹⁰⁹ It is originated depends on the maintenance of carbon/ nitrogen supplementation to pond water.¹¹ Initially researchers acquired the knowledge of carbon/ nitrogen for the production of heterotrophic bacteria, which in reverse they feed are for the fish and shrimp.⁸ A ratio of the carbon/ nitrogen (C/N) is managed to stimulate the growth of heterotrophic bacteria to produce microbial biomass.⁹ Supplemented carbon will help to hold the excreted ammonia from the animals,¹¹ and by the proper inclusion of carbon and nitrogen to the system ammonia in the water will be altered into bacterial biomass.¹¹⁹

Reasons to Maintain C/N Ratio

The maintenance of C/N ratio is quite prerequisite for controlling of accumulating organic nitrogen and for the production of microbial communities in the water.⁴⁵ The inorganic nitrogen is converted into organic nitrogen when C: N ratio is sufficient to produce bacterial cells; preferably 5:1.⁸⁸ As carbohydrate is involved in the part of respiration process, during aerobic situations the condition of C: N ratio must be more than bacterial body compositions.⁴⁶ It was found that around 10 mg NH₄⁺-N/L can be completely absorbed when glucose was added as a substrate and when the maintenance of C/N ratio was 10:1¹⁰. To minimize the artificial feed requirement, the practice of increasing C: N of higher than 10:1 by utilizing different low-cost carbon sources which are locally obtainable is common in biofloc waters.²⁸ Apart from reducing the feed cost, utilization of biofloc components will also decrease the amount of protein in the feed.^9,63 It was established that the accumulation of toxic inorganic components including, NH₄⁺ and NO₂^– will be stopped in the water when the maintenance of C/N ratio is high in the biofloc system as the ammonium consumption by the microbial community will increase.^11,84 Earlier findings in the literature attempted to see the production of microbial protein by varying levels of C: N ratio in the feed. The reports show that floc generation was high in the tanks received with low protein in the feed than that of high protein.^13,14 Therefore, biofloc system may not need much protein supplementation and as such this technique can be used without increasing the protein content in the feed as demonstrated by earlier workers.^9,63 Fontenot and co-authors studied the manipulating four levels of C: N ratios (5:1, 10:1, 20:1 and 30:1) for the removal of inorganic nitrogen from waste water from the shrimp pond. It was noticed that the maximum removal of inorganic nitrogen is contributed by C: N ratio of 10:1.⁵⁰ Previous workers have also assessed the impact of C/N ratio for giant fresh water prawn in the periphyton-based aquaculture system; the ponds received with periphyton as a substrate had higher production of heterotrophic bacteria and prawn production.⁷

Different Sources of Carbon

It is suggested that economic use of carbon for biofloc technology depends on locally available industrial by-products. Reports say that cheapest carbon sources such as plant meals (tapioca, wheat, corn, rice, etc.), molasses and glycerol can be applied in the pond water to develop bioflocs.²⁸ In addition to this, the mixture of different pelletized plant meals¹³¹ or low protein ingredients contains high C: N ratio is also be used to enrich the bioflocs.^14,19 Although carbon sources act as a substrate for the microbial protein cell production, the mode of action differs between different carbon sources.⁹ Various authors were used different carbon source for the production of heterotrophic bacteria; molasses from solid fish waste,¹¹⁹ acetate, glycerol and glucose,³¹ tapioca flour,^64,65 wheat flour.¹⁴ Previous study established that every one gram of carbohydrate, the carbon yield is 0.4 gram.⁸⁹ Earlier reports also saying that 20g carbohydrate will be required for the immobilization of on gram of mineral nitrogen.⁹

Importance of Biofloc Technology in Aquaculture

Biofloc technology is reliable for the cost effective, environmental friendly fish production.²⁹ It is a preferable technique for facing the economic, ecological and social issues relevant to current aquaculture.¹¹⁰ The system has an advantage in intensive farming practices.²⁹ The practice of rearing aquatic animals in the biofloc was established in the many countries. Today, this technology is productively successful in the commercial shrimp farming to the countries including Asia, China, America, Italy, Brazil and South Korea.⁴⁶ Besides this, most of the universities, colleges and research centers are currently working on biofloc technology to explore this fundamental technique thoroughly.⁴⁶ An important feature of this technology is ammonia wastes are consumed by bacteria for their growth that increases the microbial biomass yield as well as improves the water quality.^9,93 Besides these, the technology carries a lot of benefits from nursery rearing to the all stages of rearing animals including the augmentation in the growth and immunity of animals, water quality, less cost for feed, reduced water consumption (zero water exchange), supplying sufficient quality nutrients and lesser environmental damage to the culture system.^{9,21,32,71,78,93,102,129,142} These features attract the farmer communities to implement this technique in the farm.^21,26

The following subheading covers the role of biofloc on different fields of aquaculture. Overview of some of the study conducted in fish with reference to biofloc based culture systems are listed in the Table 1.

Water Quality Management

Biofloc technology offers an ample advantage ensuring zero water exchange through minimal consumption of water and less water pollution.⁴⁶ The elimination of pathogen entry is guaranteed by this technique as there is no re-entry of water is needed.¹⁰ Biofloc technology is applied for decreasing the effluent discharge, preventing risks from the disease outbreak, protecting the water free from pathogen entry; thus, ultimately improve the biosecurity at the farm level.²² Regarding the presence of microorganisms, biofloc play a major role in the management of water quality.^84,91 In order to attain more growth, fish fed with a lot of feed. As aqua feeds are rich in protein that contain 65% of nitrogen content, it is considered that most of the uneaten feeds that present in the water damage the pond water and threaten the animals to disease susceptibility.⁵¹ Uneaten feed present in the water columns not only deteriorate the culture water, but it also unnecessarily involves with the wastage of money. Overfeeding is often common practice in the farming which increases the nitrogen content in the feed and when fish utilize this nitrogen through the feed it is excreted as ammonia which is toxic to the fish.⁵¹ It was demonstrated in the earlier findings that adopting biofloc technology would solve the problems concerned with ammonia toxicity as increasing consumption of nitrogen by heterotrophic bacteria rapidly increases the nitrification process, which ensures the reduction in the concentration of ammonium in the culture systems.⁶³ The study also demonstrated that the production rate for heterotrophic bacteria for the utilization of ammonium is 10 times greater by heterotrophic bacteria as compared to that of nitrifying bacteria.⁶³

Feeding, Growth and Metabolism

Generally it is considered that biofloc contributes significant amounts of nutrition to the farm animals.^84,91 Previous authors indicating that the augmentation of animal growth is due to utilization of algae, bacteria which contains adequate nutrients that support the growth.⁴⁶ It is known that aquaculture can’t be sustainable without supplementary feed as it relies on 50 to 60 % of artificial feed which is about 60% of the total operating cost. In order to reduce the feed costs, methods including the addition of live feeds are followed as an alternate to supplementary feeds.⁸¹ Nevertheless, none of the methods can replace the supplementary feed. It is supported by previous authors that implementing biofloc technique farmers can able to minimize the dependence of supplementary feed to the greater as microbial protein from biofloc origin has higher bioavailability than feed protein.⁸ Prior studies reported that animal reared in the biofloc water reduces the FCR and feed costs.⁴⁶ The results from the earlier studies also indicated that L. vannamei can replace the supplementary feed of up to 29 % if biofloc method opts for the culture.²¹ In addition to these, the available reports also show that there was 20% improvement in feed utilization found in tilapia reared in Biofloc.¹¹

Earlier study reported that the bacterial biomass yield per every gram of carbon used as a substrate is 0.5 g.²⁹ It was reported in the early study that production of bioflocs takes place when the microbial concentration reaches 10⁷ CFU/ml.²¹ The group matters (algae, detritus and other decomposed components) in the water including bacteria are attached with one another and forming a floc, which is called as biofloc.⁸ In biofloc technology, the obtainability of biofloc as feed to the animals is available whole the day; therefore, the dependence of artificial feed and the expenditures for feed and feeding will be dramatically reduced for following this method.¹⁰ Biofloc will be a main feed for filter feeders such as tilapia,^10,13,30 shrimp,^21,64,131 sturgeon and snook.¹²³ However, utilization capacity of this fluctuates from animals of other feeding habits (omnivorous and carnivorous); thus, the concept of biofloc as a feed ingredient has been proposed. From this idea, biofloc from the pond water can also be collected and processed for feed supplementation.^{9,10,27,78,79}

Immune Response and Disease Resistance

Literature appears to suggest that biofloc contains the abundant amounts of beneficial bacteria which help in the improvement of immunity to the animals.^37,40,61,95 Further evidence supporting that there was a significant improvement in the non-specific immunity obtained by the animals when animals cultured in the biofloc water.^39,134,138 Asaduzzaman and co-authors found that survival rate was higher with increasing the abundance of the heterotrophic bacteria in periphyton based prawn culture system.⁷ Biofloc bacteria have poly –β- hydroxybutyrate (PHB), which terminate the pathogenic bacterial attack to the farming animals.^40,61 It is speculated that the presence of heterotrophic microbial biomass in the biofloc tends to mitigate the invasion of pathogenic bacteria.⁴⁶ Kim and co-authors claims that decreasing in the mortality rate can be seen when the biofloc treated animals were injected with the potentially harmful bacteria.⁵⁵

Advantage of Biofloc Technology in Fish Culture Systems

This technology is basically of zero water exchange oriented i.e. water exchange is not required in the culture ponds; therefore it required less water input which is not only economical to the farmers, but these will also minimize the pathogenic entry of animals through water and certify for more biosecurity in the fish culture. It also promises the less environmental impacts and footprints.¹⁴²
This technology allows the animals to rear under the higher stocking density with effective feed management.^28,²⁹
The requirement for the feed is considerably less as biofloc itself will be a feed for the cultivable animals, which results in the lower FCR.^2,^43,89 Therefore, application of the technology will reduce the feed cost to the farmers.
Biofloc increase the survival of fish since the beneficial microorganisms dominate in the biofloc act as an antagonism to the pathogenic bacteria which prevent the disease outbreak and expand the percentage of survival during the harvest. This way (beneficial) bacteria present in the biofloc prevent the colonization of any harmful bacteria that ensure the highest survival rate of the fish in the farms.^76,117,102 In nutshell, biofloc act as an immunostimulants to the aquatic animals.³⁰
Biofloc enhance the gene expression of immune related genes such as ProPO1, ProPO2, PPAE, ran, mas and SP1 in shrimps to protect them from the harmful diseases.¹⁸ Therefore, biofloc technology would be a preventive solution to many of the emerging diseases in shrimp farming.
Unlike artificial feeds, biofloc is available whole the day which facilitates animals to eat whenever they feel eating. This certifies the improvement in the body weight of the aquatic animals when reared under this system.^8,10
Biofloc bacteria produce the polyhydroxybutyrate (PHB) which are beneficial in the digestion and metabolism of fatty acid and growth increment to the fish.³⁶
Biofloc waters rich in the heterotrophic bacteria which utilize the toxic nitrogenous matters as a substrate for their growth that helps maintaining the water quality through reducing the organic loads as well as biochemical oxygen demand of the system.^11,21,64,142

Biofloc technology in aquaculture with special attention to Indian authors

Despite the vast growth of biofloc research in outside the country, only little research has been carried out in India. The list of Indian authors who worked in the area is discussed as follows: In the year 2012, Prajith and co-authors reported that giant freshwater prawn reared under the biofloc had better growth, nutrient utilization and further suggested that it can be used in prawn farming to improve the ecological and economical sustainability of prawn farmers.¹⁰⁶ In the year 2015, Mahanand and co-authorsworked in the biofloc reported that when rohu fed fish artificial feed and biofloc in wet form at the ratio of 1:1, the growth parameters were significantly higher.⁸⁵ They concluded that microorganism community such as protozoa grazers, rotifers, bacteria and diatoms were represented in the biofloc would be the reason for the improved growth. Again, the similar results were demonstrated by the same authors in rohu when reared under the biofloc technology.⁸⁶ During the year 2015, Faizullah and co-authorsassessed the impact of biofloc technology on the growth of goldfish young ones and reports suggested that growth and survival was significantly higher when reared under biofloc as compared to the normal water culture system.⁴⁷ The similar results were once again evidenced by same authors in the goldfish fry reared under biofloc.⁴⁸ One more study in the same year (2015) was conducted by Sharma and co-authors in rohu. The results suggested that biofloc can be a feed for this species without inferring the optimal growth as that of normal water systems.¹²⁴ The authors were also noticed that feed cost was reduced up to 50% using this technique. Recent studies on biofloc attempted by Harini and co-authors in Blue morph suggesting that fish reared under the biofloc significantly improved the growth and survival.⁶⁶ Biofloc technology is popularized and practiced in the certain foreign countries. But the technology have not yet popularised in India for fish farming. This is probably due to the deficiency of disseminating this technology and lack of awareness to farmers regarding the merits of the technology over other culture systems. It is value to note that many earlier works have been done in the field, but most of them are outside the country (India) and only few Indian workers were worked in this area as mentioned. But according to the primary information gained by the authors, it come to know that some fish and shrimp farmers of Tamil Nadu, Andhra Pradesh and Haryana state of India were recently established applying the biofloc technology at their farms.

Table 1: Some of the study conducted in fish with reference to biofloc based culture systems.

S. No	Species studied	Duration of study	Results acquired in the study with biofloc
1.	Litopenaeus vannamei	35 days	Significant growth increment and reduced feed cost⁷⁸.
2.	Oreochromis sps.	14 weeks	Improvement in the water quality, fish survival and minimization in the external feed requirement⁴².
3.	Litopenaeus vannamei	30-day	Promoted the animal growth, health, digestion and feed utilization performances¹⁴⁴.
4.	Farfantepenaeus paulensis	15 days	Increased survival and growth rates of shrimp⁴⁴.
5.	Rhamdia quelen	21-day	Increased the larval survival and stress mitigation¹⁰⁴.
6.	Marsupenaeus japonicus	106-day	Comparing with the control group, the ammonium and nitrite concentration was significantly reduced in the bioflocs treatment groups¹⁴⁷.
7.	Labeo rohita	90 days	Reduced the artificial feed reliance and improved the utilisation of bioflocs as feed to 50%¹²⁴.
8.	Oreochromis niloticus	N/A	Fish survival was 100% and results in the utilization of biofloc as food¹³.
9.	Litopenaeus vannamei	2 weeks	Biofloc improved the growth and immune-related gene expression⁷⁵.
10.	Litopenaeus vannamei	34 days	There was a significant increase in the survival rate, in addition to increases in growth¹²⁰.
11.	Litopenaeus vannamei		Effectively improved the water quality, bacterial activities and zooplankton growth; consequently resulted in the better growth performances⁵³.
12.	Litopenaeus vannamei	13 weeks	Affected the nitrogen cycling pathways and de-nitrification process¹⁰⁹.
13.	Penaeus monodon	60-day	Gave the beneficial effects on growth performances and digestive enzyme activities⁶.

Probiotics

Definition and its Debate

Probiotic is a Greek word derivative of pro and bios; “pro means promoting and bio means life”.⁵⁸ The first probiotics discovered is the fermented milk, which contains lactic acid bacteria (LAB). According to Metchnikoff, probiotics are live microbes consumed with the aim to improve the health of the host organisms.⁹⁰ In later year, parker, defined they are bacteria and its constituent which plays a role in the maintenance of healthy intestine.¹⁰¹ Later, Fuller, redefined probiotics are feed supplement which contains live microbes that positively disturb the host intestine results in the improvement of intestinal microbial balance.⁵² As the probiotic effect in the intestinal microbial balance has not been observed in some fish, Tannock redefined that they are live microbial cells utilized as a dietary supplements to improve the host health.¹³⁰As stated by Gatesoupe, probiotics are single or the combination of viable microorganisms which improves the indigenous micro floral property to the host.⁵⁴

The definitions above mentioned are widely accepted for the terrestrial animals; however, still there is a debate for the aquatic animals. As the aquatic animals have very close relationship with water and environment, the definition was reformed that they are live, viable microbial addition, results in the beneficial modification of host by having an association with microbial community or along with feed administration it improves the nutritional value of feed and/ or improve the immunity of host.¹³⁸ At present, the scientific understanding on the probiotics study implies that even non-viable microbial constituents too can be administered for beneficially disturb the host intestine. Hence, Salminen and co-authors redefined that they are either microbial cell preparations or microbial cell components that exerts beneficial impact to the host health.¹¹⁶ Based on above concept, eventually Irianto and Austin redefined that they are entire microbial cell components that exerts beneficial effect to the host health.⁷⁰

Table 2: Some of the study conducted in fish with reference to probiotics supplementation.

S. No	Species studied	Strains used for study	Days of study	Results acquired in the study
1.	Litopenaeus vannamei	B. subtilis	60 days	No improvement on survival, final weight, FCR and water quality⁴⁹.
2.	Oreochromis niloticus	B. subtilis and L. acidophilus	60 days	Improved the disease resistance and growth performance⁵.
3.	Litopenaeus vannamei	B. subtilis	14 days	Improved the larval survival rate, development, stress resistance and immune status⁸².
4.	Litopenaeus vannamei	Bacillus species	N/A	Improved the growth, survival and some water quality parameters such as pH, ammonia and nitrite as compared to controls⁹⁷.
5.	Clarias gariepinus	L. acidophilus	21 days	Significantly improved the haematology parameters and histopathology⁴.
6.	Penaeus vannamei	B. coagulans	N/A	Significantly increase survival rate and digestive enzyme activities¹⁴⁹.
7.	Penaeus vannamei	Bacillus sp	28 days	Positive effects on enzyme activity and resulted in an increase in the growth performances¹⁴⁰.
8.	Clarias gariepinus	Lactobacillus and Bifidobacterium	90 days	Improved the growth performance and blood parameters¹².
9.	Oncorhynchus mykiss	Enterobacter amnigenus	N/A	Improved the health status²⁰.
10.	Litopenaeus vannamei	B. licheniformis, B. megaterium	60 day	Effectively enhanced both digestive enzyme activity and non-specific immunity simultaneously⁸⁰.
11.	Sparus aurata	Lactobacillus spp.	31 days	No effect on growth parameters and digestive enzyme activities¹²⁸.
12.	Paralichthys olivaceus	L. lactis	5 weeks	Enhanced the immune response and effectively controlled bacterial infection⁶⁷.
13.	Penaeus monodon	Bacillus S11 (probiont)	90-days	Enhanced both cellular and humoral immune defense⁹³.
14.	Penaeus monodon	B. subtilis	N/A	The growth of pathogenic V. harveyi was effectively controlled¹³⁷.

The Need of Probiotics?

A significant demand for fishery commodities urges many farmers to go for intensive farming.¹⁵ But, disease outbreaks are very common in the intensive practices.¹⁰³ To prevent the disease, use of antimicrobial drugs are common in the aquaculture.¹¹⁸ However, using these drugs is criticized as they kill both adverse and beneficial microbes.⁶⁹ Also, they establish the resistance to those bacteria during continuous application.³ Also, they transfer some antibiotic resistant genes to the consumers as well.^59,121 The application of chemotherapeutics to the system degrades the aquatic ecosystem as residues stay in the system.¹¹³ At present, governments and many social welfare organizations restricted the usage of antimicrobial drugs.⁸³ Due to these concerns, it is the need for farmers to adopt techniques that eliminate the use of antimicrobial drugs usage.⁶⁹ Defoirdt and co-authors recommend the use of probiotics is potent alternatives to the use of antibiotics in culture systems.⁴⁰ An alternative to the use of anti-microbial drugs are immunostimulants such as vaccines and probiotics.⁹⁹ Both methods use microbes for the action. But vaccines are better than probiotics; however, they can be applied for only single disease and commonly cultured shrimps lack with the adaptive immune system. Thus, the applications of probiotics are important for controlling the disease.¹²⁵

Figure 1: Schematic representation of biofloc technology integrated with exogenous probiotics.
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Figure 2: Experimental set up for rearing animals on biofloc water with supplemented probiotics.
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Characteristics of an Ideal Probiotic

Many authors have been defined the characteristics of an ideal Probiotic.^{73,111,138,141} According to the available works of literature, an ideal probiotic strain must have following characteristics.

It should be non-pathogenic to host.
It taxonomy must have confirmed.
It should have a potential to grow and survive in the host.
It must survive even during unfavorable conditions generated in the digestive tract due to the production of gastric acid and bile juices.
It should have capable enough to produce antimicrobial constituents to kill the invading pathogenic bacteria.
It should modulate the host immune response and offer a health benefit.
It must be genetically stable.
It must survive during processing and storage conditions.
It should be viable even at high centration.
It should have desired organoleptic and technological characteristics to be included for fermentation methods.

Types of Probiotics

Depending on the way in which the probiotic bacteria are introduced to the animal, it differs. There are three types of probiotics such as soil, feed and water probiotics. But, only two types of probiotics (feed and water probiotics) are majority used in aquaculture.¹¹⁴ In feed probiotics, the probiotic strain is introduced to animals via feed.⁵⁴ In water probiotics, the bacterial spores are directly added to the culture water.¹⁴⁸Timmermans and co-authors reported that water probiotics are especially important to the early larval stages and small fishes as they are very less exposure to artificial feed.¹³³ Various authors mentioned that water probiotics have a significant role in the water quality management.^23,91,138 Cabak and co-authors reported gram-positive bacteria are more efficient for the conversion of organic carbon to the bacterial biomass.²³ Verschuere and co-authors reported that gram-positive Bacillus sp. improved the water quality parameters when they added into the rearing water.¹³⁸ Moriarty, indicated that seawater receives with an inoculum containing frozen cells reduced the time taken for nitrification process of about 30 %.⁹¹

Figure 3: Components needed for biofloc inoculum preparation.
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Figure 4: Observation of biofloc volume in imhoff cone.
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Applications in Aquaculture

Various studies have analyzed the use of probiotic bacteria to promote the health of the organisms.^{4,5,15,38,56,60,91,138} Conducted research on probiotics has shown many beneficial impacts to the health of cultured animals, including growth and immunity.^39,134,138 Probiotics have many mechanisms of action: the competitive exclusion of pathogenic bacteria, serving as a nutrient source and contributing to enzymatic digestion of animals, beneficial effects on water quality, and improvement of the animal’s immune response.^15,34,98,138 Many bacteria are being explored to be used as a probiotic strain as they contain the growth, immune stimulatory effects and resistance against pathogenic microbes.⁷³ Previous studies indicated that the addition of probiotics in the water or feed increases growth, immunity, reduces animals to expose pathogenic bacteria and stops the growth of harmful pathogens.^{94,108,112,140} There is rapidly growing literature on the application of probiotics which indicates that it is one of the important methods developed to control disease at the farm; therefore, the addition of probiotics is common practice in fish farming.¹⁸ Aquatic farming is surrounded by environment that facilitates the natural uptake of potentially harmful opportunistic pathogens by animals through the water.²⁴ Surrounding bacteria are continuously ingested when the fish is drinking; thus, harmful pathogenic microorganisms will reach high densities in the animal tissues. Especially this is the case with filter feeders which ingest bacteria at a high rate from the culture water, causing a bacterial infection to the animals.¹³⁵ Previous reports in fish suggested that probiotics reduce the loads of harmful bacterial in the fish tissues.^{15,34,73,74,94,140} It has been reported that lactic acid bacteria such as lactobacilli and bifidobacteria are helping to reduce the gastrointestinal tract (GIT) pH by converting lactose into lactic acid.¹²² In this manner, colonization of many bacteria in the GIT is barred. Previous studies have also proven that spore-forming Bacillus sp generates antimicrobial peptides which offer immuno-stimulatory effect to the animals.¹⁶ Probiotic strains are either used as a single bacterial strain or multi strains which contain more than one strain. The available evidence seems to suggest that multi strain probiotics deliver synergistic effect which leads to an extra protection to the animal health.¹³² Dalmin and co-authors indicated that Bacillus sp. enhanced the growth, immunity and water quality in Penaeus monodon culture system.³³ The findings from previous results confirmed that rainbow trout improved the activity of leukocytes, phagocytes and the resistance against Vibrio sp. when Clostridium butyricum bacteria were orally given.¹¹⁵ Besides, it was also reported that lactic acid bacterium Lactobacillus rhamnosus in the feed administration motivated the respiratory burst activity in rainbow trout.⁹⁶ Some available reports in the literature appears to support that some probiotic bacteria are effective against some viruses such as Infectious hematopoietic necrosis virus (IHNV), Oncorhynchus masou virus (OMV) and Poliovirus.^41,57,72

Overview of some of the study conducted in fish with reference to probiotics supplementation is listed in the table 2.

Advantages of Probiotics Application in Fish Culture Systems

Probiotics are single bacteria act as a growth promoter via improving the digestibility of nutrients through the colonization of beneficial bacteria in the gastrointestinal tract of the animals.¹⁵
Probiotics improve the water quality.^34,98 Most of the gram-positive probiotic bacteria are are very efficient in converting the organic matter to CO_2.¹³⁸ Also, most of the probiotic bacteria which are heterotrophic in nature are efficiently utilize the toxic nitrogenous matters available in the pond water for their growth.¹⁰⁵
Probiotic bacteria also have synergetic effect.¹²⁷ It communicates each other’s and does not allow harmful pathogens to attach for the binding sites for nutrients.¹⁴³
Probiotic bacteria have capacity to increase the rate of expression of immune related genes that improve the immune status of the animals.^99,100 Probiotics also have phagocytic, antibacterial and antiviral activity against pathogenic bacteria.^96,115
Probiotic microorganisms show bactericidal and bacteriostatic effect against pathogenic bacteria due to the inhibitive influence of probiotic bacteria against harmful bacteria, production of enzymes that kills the harmful bacteria and creating the acid pH in the intestine of animals to kill the harmful bacteria lives in the low pH.⁸⁷
Probiotic bacteria produce the digestive enzymes such as proteases, amylases and lipases to improve the nutrient digestion.⁸⁷ It also promote the growth factors such as vitamins, fatty acids, and amino acids to metabolise the digested nutrients to the cells for absorption.¹⁵
Probiotics also offers stress tolerance to the animals by reducing the metabolic¹³⁶ and oxidative stress factors.²⁵
Certain probiotics include B. subtilis produce essential vitamins such as vitamin B1 and vitamin B12 which help in the animal’s growth, metabolism and reproduction.^55,72

Present status on influence of exogenous probiotic bacteria on biofloc based fish culture systems

Exogenous supplementation of probiotics to the biofloc is very recent area, still in the experimental level studies. So far, only few attempts have been successfully performed globally to identify the beneficial effects of exogenous probiotic strains on biofloc based aquaculture.^{1,31,62,77,146} An attempted works documented the positive results on growth and survival of the aquatic animals.^{1,31,62,77,146} In-vitro study by Hutabarat and co-authors (2013) reported that when biofloc inoculated with probiotic bacterium, Bacillus cereus produced higher amounts of polyhydroxybutyrate (PHB) which is one of the main component believed to have role in the energy reserve and growth acceleration in fish.⁶⁸ Crab and co-authors reported that biofloc based brine shrimp tanks immunized with Bacillus sp. were reduced almost five times of pathogenic vibrio load than the control tanks.³¹ Krummenauer and co-authors analyzed the effect of commercial bacterial probiotics on a Litopenaeus vannamei based biofloc culture system.⁷⁷ In this study, Vibrio parahaemolyticus infected to animals, and results show that experimental fishes received probiotic strains on biofloc system had significantly higher growth rate and survival than that of control. Aguilera-Rivera and co-authors observed the probiotic effect of biofloc in Pacific white shrimp, Litopenaeus Vannamei. This study reported that Vibrio load was reduced in the biofloc tanks treated with the probiotics.¹ The study conducted by Yusuf and co-authors (2015) reported that biofloc added with Bacillus sp., showed highest growth and feed conversion ratios in African catfish.¹⁴⁶ The findings of Hapsari, 2016 also supported that biofloc inoculated with Bacillus cereus improved the growth performances and reduced the FCR in African catfish.⁶² It can also be noted that authors of the paper are presently working in the area of current topic, results yet to be published. Nevertheless, schematic representation of the technology, experimental set up, components required for biofloc preparation, besides with the observation during the work was displayed in figure 1-4.

Pros/cons and future prospects of Biofloc technology together with supplementation of exogenous probiotic bacteria

Results of recent studies supported that supplementing the probiotics to biofloc helps in the growth, digestion, metabolism and disease resistance to the animals together with improving the water quality in the culture systems.^{1,31,62,77,146} This is probably a result of the ability of supplemented probiotic bacterial groups that dominate the other bacteria to minimize the pathogenic loads in the fish tissues. Despite the presence of bacteria in the biofloc or supplemented probiotics bacteria exhibits the mitigating effects on pathogenic microbes in the fish that ensures the non-specific immunity to the cultivable organisms, there is still dearth of knowledge in understanding the exact mechanisms behind how bacteria communicate each other (quorum sensing) to perform these effects. Therefore, future studies must be required in these areas to disclose the exact scientific reasons for these, so that this technology will be more scientific oriented. Bioflocs together with probiotics is reliable technique to aquaculture industry, but farmers needed to be satisfied with economic benefit; thus, require economic study. As the immunological effect differs among the strains and species; hence the specific actions of different strains must be explored among the different cultivable animals.¹³⁸ The utilization of probiotic strains is species-specific,¹⁴⁵ therefore, this technique should be scrutinized with various commercially cultivable fishes. An animal’s capacity to utilize various components varies among species^{10,13,21,30,64,123,131} therefore, animals preference utilize biofloc and probiotics must be well studied. The feed utilization test must be done to identify potential candidate species would be best for the culture; and also at what extend these techniques can reduce the feed cost must be available to the farmers for the implementation of this technology. Research must also explore how biofloc contributes to the improvement of growth and animal health performances. Bioflocs have an adequate amount of protein, lipid, carbohydrate and ash content for use as an aquaculture feed.²⁹ However, the composition of amino acid and fatty acid is less studied; thus, careful investigation on nutritional composition must be completed to find out whether % of any nutrients in excess which responsible for the improvement of growth in the animal.

Conclusion

In conclusion, the paper demonstrated the new technique i.e. “Supplementation of probiotics to the biofloc”. Nevertheless, according to the best of author’s knowledge, at present no farmers in the country are following this technique. Therefore, keeping the practical advantages discussed in the technique, we hope that this technology will be shortly disseminated to be implemented by the fish and shrimp farmers of India and other countries for the sustainable production of aquatic animals in the cost effective and environmental friendly approach.

Acknowledgements

Authors are thankful to the Director of ICAR- Central Institute of Fisheries Education (CIFE), Mumbai, India for the provision given to carry out this study. Authors would like to thank scientists, staffs and students of Aquaculture Division (CIFE) for the contribution and support in the present study.

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