BIOREMEDIATION AND BENEFICIAL MICROBES IN AQUACULTURE | Prasenjit Barman and Partha Bandyopadhyay #

1Research & Development Division, FINRAY BIOTECH INC., Mahavir Estate, Opp.

RDC Plant, Santej- 380060, Gandhinagar, Gujarat.

#FINRAY BIOTECH INC., Kolkata, West Bengal – 700078, INDIA.

#Corresponding Author:Partha Bandyopadhyay, Email: finraybiotech@gmail.com

Introduction

Aquaculture not only contributes to the world’s seafood demand but also plays a pivotal role in the national economy and poverty reduction plans of many countries around the world. In aquaculture, Shrimp (or prawn) farming is widespread throughout the tropical world. Presently, White leg shrimp, Penaeus vannamei, is the most widely cultured species across the world. However, this industry is beset by disease, mostly due to bacteria (especially the luminous Vibrio harveyi) and viruses. The high density of animals in hatchery tanks and ponds, with high inputs of protein-rich feed especially in intensive culture systems is conducive for bacterial growth and for the spread of pathogens. At a time when capture fisheries is levelling off, aquaculture production continues to increase. 

The increase in production is greatest in developing countries where, about 93 percent of aquaculture production originates. Aquaculture was once considered an environmentally sound practice because of polyculture practices and integrated systems of farming, which ensured optimum utilization of farm resources, including farm wastes. Presently, increased fish production is being achieved by the expansion of land and water under culture and the use of more intensive and modern farming technologies that involve higher usage of inputs such as water, feed, fertilizer and chemicals. As a result, aquaculture is now considered as a potential polluter of the aquatic environment and a cause of degradation of wetland areas. Aquaculture generates considerable amount of wastes, consisting of metabolic by-products, residual food, fecal matter and residues of prophylactic and therapeutic inputs, leading to the deterioration of water quality and disease outbreaks. Bioremediation - the application of microbes/enzymes to the ponds, is the method currently in use for improving water quality and maintaining the health and stability of aquaculture systems. Bioremediation involves mineralization of organic matter to carbon dioxide, maximizing primary productivity that stimulate shrimp production; nitrification and denitrification to eliminate excess nitrogen from ponds and maintaining diverse and stable pond community, where the pathogens are excluded from the system and desirable species get established. Apart from organic matter degrading (detritivorous)heterotrophic bacteria, nitrifying, denitrifying and photosynthetic bacteria are also generally employed in bioremediation. The major impact on the receiving water bodies are eutrophication, silting, oxygen depletion and toxicity of ammonia and sulfide. High organic load increases the oxygen demand in water bodies. This eventually reduces dissolved oxygen levels in aquaculture systems. The urine and faeces from the aquatic animals can cause high content of ammonia nitrogen and an increase of BOD. Ammonia is the main nitrogenous waste that is produced by fish via metabolism and is excreted through the gills.

Waste production in aquaculture

The physical, chemical and biological conditions of the culture environment have an influence on the health and productivity of shrimp. Exposure of shrimps to toxins like hydrogen sulphide, ammonia and carbon dioxide lead to stress and ultimately to disease. The types of wastes produced in aquaculture farms are basically similar. However, there are differences in quality and quantity of components depending on the species cultured and the culture practices adopted. The wastes in hatcheries or aquaculture farms can be categorized as: residual food and fecal matter, metabolic by-products,residues of biocides and biostats, fertilizer derived wastes, wastes produced during molting and collapsing algal blooms. 

Organic detritus & bioremediation 

The dissolved and suspended organic matter contains mainly carbon chains and is abundantly available to microbes and algae. A good bioremediation must contain microbes that are capable of effectively clearing carbonaceous wastes from water. Further it would be very supportive, when these microbes multiply rapidly and have good enzymatic capability. Members of the genus Bacillus like Bacillus subtilis, B. licheniformis, B. cereus, B. coagulans and species Phenibacillus polymyxa are good examples of bacteria suitable for bioremediation of organic detritus. However, they are not normally present in required quantities in the water column, their natural habitat being the sediment. When certain Bacillus strains are added to the water in sufficient quantities, they can make an impact. They compete with the bacterial flora naturally present for the available organic matter, like leached or excess feed and shrimp faeces. As a part of bio-augmentation, the Bacillus can be produced, mixed with sand or clay and broadcasted to be deposited in the pond bottom. Lactobacillus is also used along with Bacillus to break down the organic detritus. These bacteria produce a variety of enzymes that break down proteins and starch to small molecules, which are then taken up as energy sources by other organisms. The removal of large organic compounds reduces water turbidity.

Nitrogenous compounds & bioremediation

Nitrogen applications in excess of pond assimilatory capacity can lead to deterioration of water quality through the accumulation of nitrogenous compounds (ammonia and nitrite), causing toxicity to fish and shrimp. The principal sources of ammonia are excretion and sediment flux derived from the mineralization of organic matter and molecular diffusion from reduced sediment. Bacteriological nitrification is the most practical method for the removal of ammonia from closed aquaculture systems and it is commonly achieved by the setting of sand and gravel bio-filters through which water is allowed to circulate. The ammonia oxidizers are placed under five genera Nitrosomonas, Nitrosovibrio, Nitrosococcus, Nitrolobus and Nitrospira. Nitrification not only produces nitrate but also alters pH towards the acidic range, facilitating the availability of soluble materials. The vast majority of aquaculture ponds accumulate nitrate, as they do not contain a denitrifying filter. Denitrifying filters help to convert nitrate to nitrogen. It creates an anaerobic region where anaerobic bacteria can grow and reduce nitrate to nitrogen gas. Nitrate may follow several biochemical pathways, following production by nitrification.

Phosphorous & bioremediation

Phosphorus normally has limitations in a freshwater environment. Any deviation from the normal NO3/PO4 ratio is believed to be dependent on, which influences the rate of nitrification or bacterial regeneration of phosphorous, available in organisms mainly as phospholipids and nucleoproteins. Phosphorous is generated from organic compound as PO4 by certain bacteria that produce enzymes such as phosphatases and phytases. The solubility of inorganic phosphatases is primarily a function of pH. Bacteria are capable of liberating PO4 from these compounds through the production of organic and mineral acids.

Hydrogen sulphide (H2S) & bioremediation

Sulphur is of some interest in aquaculture because of its importance in anoxic sediments. In aerobic conditions, organic sulphur decomposes to sulphide, which in turn gets oxidized to sulfate. Sulfate is highly soluble in water and so gradually disperses from sediments. Sulphide oxidation is mediated by micro organisms in the sediment, though it can occur by purely chemical processes. Organic loading canstimulate H2S production and reduction in the diversity of benthic fauna. Hydrogen Sulphide is soluble in water and has been suggested as the cause of gill damage and other ailments in fish. Unionized H2S is extremely toxic to fish that may occur in natural waters as well as in aquaculture farms. Bioassays of several species of fish suggest that any detectable concentration of H2S should be considered detrimental to fish production. Photosynthetic benthic bacteria that break H2S at pond bottom have been widely used in aquaculture to maintain a favorable environment. These bacteria contain bacterio-chlorophyll, that absorb light and perform photosynthesis under anaerobic conditions. They are purple and green sulphur bacteria that grow at the anaerobic portion of the sediment - water interface. Photosynthetic purple non-sulphur bacteria can decompose organic matter, H2S, NO2 and harmful wastes of ponds. The green and purple sulphur bacteria split H2S to utilize the wavelength of light not absorbed by the overlying phytoplankton. The purple and green sulphur bacteria obtain reducing electrons from H2S at a lower energy cost than H2O splitting photoautotrophs and thus require lower light intensities for carrying out photosynthesis. Chromatiaceae and Chlorobiaceae are the two families of photosynthetic sulphur bacteria that favour anaerobic conditions for growth while utilizing solar energy and sulphide. Chromatiaceae contain sulphur particles in cells but Chlorobiaceae precipitate them out. The family Rhodospirillaceae is not of any use for H2S removal, but can be used as efficient mineralizers at pond bottom, as they grow in both aerobic and anaerobic conditions as heterotrophic bacteria, even in the dark without utilizing solar energy. The common examples of photosynthetic bacteria of importance in aquaculture are Rhodospirillum, Rhodopseudomonas, Chromatium, Thiocystis, Thiospirillum, Thiocapsa, Lamprocystis, Thiodictyon, Thiopedia, Amoebobacter, Chlorobium, Prosthecochloris,Pelodictyon and Clathrochloris. For bioremediation of H2S toxicity, the bacterium that belongs to Chromatiaceae and Chlorobiaceae can be mass cultured and can be applied as pond probiotic. Being autotrophic and photosynthetic, mass culture is less expensive and the cultured organisms can be adsorbed on to the sand grains and applied, so that they may reach the pond bottom to enrich the hypolimnion and ameliorate H2S toxicity.

Conventional approaches for addressing challenges in aquaculture

The rearing of fish in reticulated systems results in a highly artificial environment which has a propensityfor the accumulation of waste metabolites andwhich promotes the growth of pathogenic bacteria. Management considerations for aquaculture operations include nutrition, water quality, physical parameters as well as pathogen and disease control. A wide range of chemicals such as topical disinfectants, organophosphates, antimicrobials and parasiticides are often used to deal with disease and water quality. Water quality is traditionally managed through conventional filtration systems, which are sensitive to process fluctuations and can result in mass mortality when the systems crash.

Conventional biofiltration

Normally the oxidation of ammonia to the morebenign nitrate ion occurs through ammonia and nitrite oxidizing obligate chemoautotroph such as Nitrosomonas and Nitrobacter spp. These are slow growing and sensitive to fluctuations in environmental conditions. Removal of nitrate and nitrite is a challenge in intensive aquaculture operations. System fluctuations, resulting from the sensitivity of natural filter bacteria, often lead to accumulation of ammonia, nitrite, nitrate and phosphate. Although the concentration of these residues can be reduced by the addition of fresh water, purges of effluent containing high concentrations of these compounds into natural river and seawaters, results in a deterioration of the environment and can lead to algal blooms, which may be detrimental to natural ecosystems. High capital investment is thus required for installation of larger scale filtration systems to compensate for the seinefficiencies of conventional filtration. At present heterotrophic nitrifying and aerobic denitrifying bacteria are frequently used in aquaculture for the removal of toxic nitrogenous pollutant which removes ammonia, nitrate and nitrite faster than chemoautotrophs. Heterotrophic organisms work properly in aerobic condition, which is a very essential parameter for the sustainable shrimp culture.

Biological solutions as alternatives for addressing challenges in aquaculture

Given the challenges in conventional aquaculture practices, alternative methods for disease control and enhancement of water quality are desperately required. Micro-organisms play important roles in aquaculture, particularly with respect to nutrient cycling and the nutrition of the cultured animals, water quality, disease control and the environmental impact of effluent. Beneficial microbes can be used to alter or regulate the composition of bacterial flora in a water system to optimize fish production by reducing pathogen concentration, by improving water quality through reduction of waste ions and through accelerated mineralization and nitrification, by reducing algal growth and by accelerating sediment decomposition. These biological agents also confer the added advantage of natural integration into existing ecosystems and present opportunities for development of multi-effect products which are attractive to endusers. The marketing of biological and “organic certified” solutions for enhancement of fish health has also gained consumer acceptance. The use of beneficial microbes is a more appropriate remedy than the use of chemicals, but successful application requires an understanding of the ecological processes occurring in aquaculture systems, of the agents responsible for disease and knowledge of the beneficial characteristics of bacteria to be used as biological agents.

Biological agents

Microbial webs are an integral part of all aquaculture systems and have a direct impact on productivity, especially in intensive culture operations. The quality of water and health of the cultured species is governed by the activities of a diversity of microbes with different roles and interactions in the ecosystem. There are distinct uses of bacterial supplements in

 

aquaculture for bio-augmentation as probiotics as well as biocontrol and bioremediation agents. Bio-augmentation refers to the augmentation of the environment with microbes to result in enhanced fish health, while probiotics are normally associated with feed and digestion. A strict definition of biocontrol agents is that they are microorganisms that are antagonistic to pathogens. In some instances,however the description of biocontrol agents transcends the boundary between bio-augmentation, and the exclusion of pathogens. Bioremediation refers to the breakdown of pollutants or waste by microbes.Probiotics can be defined as a cultured product or live microbial feed supplement, which beneficially affects the host by improving its intestinal balance (Fig. 1). The important components of this definition reflect the need for a living microorganism and application to the host as a feed supplement. A broader definition is that of a live microbial supplement, which beneficially affects the host animal by improving its microbial balance. In a third proposed definition, a probiotic is any microbial preparation, or the components of microbial cells, with a beneficial effect on the health of the host. It is thus apparent that there are variations in the actual application of the terminology associated with biological agents. Based on the observation that organisms are capable of temporarily modifying the bacterial composition of water and sediment, it was suggested that the definition should include the addition of live naturally occurring bacteria to tanks and ponds. Given the broad-spectrum effects of microbial consortia used in aquaculture, some scientists described a biological agent as a live microbial adjunct, which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed and enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment. The range of biological treatments examined for use in aquaculture has encompassed both Gram-negative and Gram-positive bacteria, bacteriophages, yeasts, unicellular algae, enzyme preparations and plant extracts. Microbes have been successfully applied to aquaculture systems via inclusion in artificial or live feed, by addition to bio-filtration systems and by direct addition to water. Most biological treatments used in aquaculture belong to the genera Lactobacillus, Vibrio, Bacillus, or Pseudomonas, although other genera have been applied to a lesser extent.

Probiotics: An essential tool in intensive shrimp aquaculture

As aquaculture develops, the industry faces several  challenges related to its environmental impact. The

application of beneficial bacteria (probiotics) is not only associated with gut health (feed probiotics), but also with bioremediation that improves the environment (water and soil) in which the animals are reared (Table. 1). A key factor for successful aquaculture is to understand the interactions between the microbial environment, gut flora and immune system of the shrimp, as well as factors that determine the persistence of microbial species in the internal and external microbial ecosystems. While natural ecosystems are balanced, the farming environment favours the growth of microorganisms as it is rich in nutrients and feed waste.

Conclusion

The traditional practice of extensive land-based aquaculture is under pressure, due to limitations in available space and environmental considerations. This has led to the increased use of more intensive reticulated systems which also offer the benefit of greater control of physiological culture conditions. While intensive systems offer the advantages of increased stocking densities and higher production throughput, challenges include water quality and increased disease prevalence among others. These are driving the adoption of environment friendly solutions, that meet consumer expectations and comply with regulatory requirements. Beneficial microbes provide an attractive option. Issues that require attention to accelerate the adoption of biological solutions include elucidation of the mode of action of commercially beneficial microbes and demonstration of clear costbenefit advantages for commercial products. Readers may kindly contact the authors for references