Introduction

Aquaculture globally has undergone tremendous growth during the last fifty years from a production of less than a million tonnes in the early 1950s to over 50 million tonnes in the year 2008 (FAO, 2009). Modern intensive and semi-intensive aquaculture practices involve use of supplementary feeds rich in protein (as much as 25-40 percent). In high intensity aquaculture, water quality becomes a limiting factor. Fish and shrimp accumulate about 20-25% of protein and the rest is released to the pond as ammonium and organic nitrogen (Boyd and Tucker, 1998). These proteinaceous wastes result in total ammonia nitrogen (TAN) and biochemical oxygen demand (BOD). It is estimated that in aquaculture ponds, for every kilogram of feed containing 35 percent protein, about 50.4g of ammonia nitrogen is generated (Ebeling et al., 2006). Ammonia is also a major end product of protein catabolism, excreted by fish, crustaceans, and molluscs into the culture system. These nitrogenous organic wastes stimulate proliferation of heterotrophic microbes. Total ammonia-nitrogen (TAN) is composed of unionised (NH₃-N) and ionised forms (NH₄⁺). The unionised ammonia is most toxic to aquatic organisms as it can readily diffuse through cell membranes and is highly lipid-soluble. Nitrite (NO₂) an intermediate product of nitrification is also one of the toxic forms of nitrogen that can be found in aquaculture ecosystems. These substances, despite being toxic to the cultured animals per se, increase their susceptibility to diseases, particularly shrimp, which are bottom dwelling organisms. Hence, it is extremely important that these organic pollutants generated during aquaculture have to be treated using conventional better management practices(BMPs) and also by in situ or ex situ bioremediation approaches in order to achieve optimal aquaculture productivity. This article provides a brief overview of strategies of mitigating nitrogenous wastes in aquaculture.

Strategies for mitigating NH₃ and H₂S in Aquaculture

Remediation of aquaculture wastes needs to be addressed by developing appropriate strategies, keeping in view the specific requirements in the hatcheries and grow-out ponds. A number of approaches have been adopted for the removal of nitrogenous organic wastes in aquaculture ponds and hatcheries with varying degrees of success. In grow-out ponds, simple physical measures such as aeration, ozonation and replacement with freshwater on a regular basis have been practiced to provide good water quality to the cultured animal. The paddle wheel aerators, despite providing oxygenation, create a circular motion to water, facilitating concentration of wastes at the centre of the aquaculture ponds (Fig.1). Waste mitigation strategies in aquaculture include use of bioaugmentation probiotics (an example of in situ bioremediation) and use of biofilters or bioreactors (Ex situ bioremediation) for the management of water quality. The latter are used mainly in maturation systems and hatcheries. Certain innovative aquaculture practices utilizing microbial and algal biomass such as ‘active suspension ponds’ and ‘partitioned aquaculture’ for management of nitrogenous wastes for maintaining water quality in aquaculture ponds have been proposed.

Fig. 1. Shrimp farm with aerators being operated to provide dissolved oxygen in
water and to concentrate wastes at the centre of the ponds

Bioaugmentation

The use of different metabolic pathways of microbes to stimulate autochthonous degradation processes to mitigate undesirable hazardous substances is the strategy of in situ bioremediation adopted in aquaculture. Bioaugmentation has been applied in aquaculture with exogenous microbes as “probiotics”, the microbes with nitrification, denitrification and sulfur oxidation potential and ability to digest detritus for management of organic matter in aquatic ecosystems. The use of ‘probiotics’ in aquaculture has been extensively reviewed by Vershuere et al. (2000) and is defined 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 or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment”.

A wide range of microbes including nitrifiers, sulphur bacteria, Bacillus spp. and Pseudomonas spp. are commercially sold for in situ bioremediation in aquaculture for improving water quality by mineralization of organic matter to carbon dioxide and remove nitrogen from the pond ecosystem by nitrification and denitrification (Antony and Philip, 2006). The most common probiotic bacteria used in ponds or hatchery tanks are Bacillus spp. These bacteria being spore formers and resistant to relative extreme physicochemical conditions have the ability to occupy a wide range of niches and degrade a wide variety of substrates. The rationale is that the Bacillus spp. are generally more efficient in converting organic matter into CO₂ than the gram-negative bacteria, and convert a greater percentage of organic carbon to bacterial biomass or slime (Stanier et al., 1963). By maintaining higher levels of Bacillus spp in the pond, it is believed that the buildup of dissolved and particulate organic carbon can be minimized during the culture cycle.

It appears that there is potential to employ beneficial microbes in aquaculture to improve water quality. But a number of issues such as the stochaic and deterministic factors that govern the microbial diversity and their population dynamics, the fate of exogenous microbe introduced into a new dynamic aquatic ecosystem and sustaining its activity, implications on the natural microbial ecology and impact on the environmental parameters have to be still understood. Boyd (1995) suggested that the bioaugmentation probiotic application must improve water quality, including increase in DO, prevention of off flavour, reduction of nitrogen and phosphorus levels, promoting organic mater decomposition, optimal algal boom, exclusion of pathogens from the production system, enhancement of animal’s physical condition, indicators to improve the animals’ overall health, appearance, better average size and weight of the animal, and restoration of normal appetite and feed consumption.

Mitigating NH₃ and nitrite using recirculating aquaculture systems (RAS)

The primary objective of the recirculating aquaculture system is to save water, reduce health risks to aquatic animals caused by exchange of water that may contain contaminants and pathogens, and control pollution (Eding et al., 2006). In RAS, removal of total ammonia nitrogen (TAN), particularly the unionized form of ammonia is the main objective and the process relies mainly on microbial metabolism (See box). RAS have been identified as one of the main research areas by NOAA’s Aquaculture Policy (http://swr.ucsd.edu/find/bill/aquapol.htm). RAS have been used successfully in aquaculture for the past 20 years, and are now increasingly used in shrimp maturation, hatcheries, nurseries, and ornamental fish breeding which require oligotrophic-grade water quality. Water is recycled through an external biofilter, where it gets purified (Gutierrez-Wing and Malone, 2006). These systems are well tested, proven efficient and are now commercially available. Basic elements of RAS design consider i) maximum biomass of fish / shrimp in the culture system, ii) maximum load of feed used and iii) waste production in the culture system on a diurnal basis. In addition, water quality characteristics required in the culture system are also taken into account in order to regulate flow rates. Components in RAS that perform four critical processes are: i) mechanical filtration to remove suspended solids, ii) foam fractionation for removal of small suspended particles and surfactant molecules, iii) degasification to remove excess carbon dioxide, iv) biofiltration for nitrification of ammonia, and v) aeration to replenish oxygen (Fig. 2).

Fig. 2. Components of Recirculating Aquaculture Systems

Recent approaches for the mitigation of NH₃

With the discovery of anaerobic ammonia oxidizing (anammox) bacteria, new approaches of ammonia bioremediation have evolved during the past decade. Anammox are obligate anaerobic chemolithoautotrophs, belonging to planktomycetes group and are extremely slow growing in nature with 11 days of doubling time. The new approaches include processes such as combined SHARON-Anammox process (Single reactor system for High Ammonium oxidation Over Nitrite- anaerobic ammonia oxidation) and CANON (completely autotrophic nitrogen removal over nitrite) (Paredes et al., 2007).

The combined SHARON-Anammox process is based on partial nitrification, followed by denitrification by anaerobic ammonia oxidizing (anammox) bacteria. In the SHARON process, oxidation of ammonium is regulated to proceed only up to nitrite production. The process is combined with anammox process wherein, the subsequent oxidation of nitrite takes place. The anammox process is the denitrification of nitrite with ammonium as electron donor. Hence the anammox process requires nitrite (produced through partial nitrification by nitrifying bacteria) as an electron acceptor. One of the problems with anammox process is the long start-up time because of the extremely slow growing nature of these bacteria. The SHARON-Anammox process has been patented and implemented for wastewater treatment in Rotterdam in The Netherlands.

The CANON process is also based on partial nitrification, wherein, oxidation of ammonium rich wastes is carried out sequentially by aerobic chemolithotrophs such as Nitrosomonas spp. and anaerobic anammox bacteria to dinitrogen gas (Paredes et al., 2007). Nitrosomonas spp. are basically aerobic organisms, but can also survive under anaerobic conditions, whereas, the anammox bacteria are obligate anaerobes. Hence, these two groups of bacteria could be co-cultured under oxygen limiting conditions. The aerobic chemoautotrophs (Nitrosomonas spp., Nitrosobacter spp., and Nitrosospira spp.) oxidize ammonium to nitrite and consume oxygen. The anammox bacteria subsequently oxidize ammonium along with the nitrite produced by the aerobic chemoautotrophs to dinitrogen and nitrate.

Conclusion

Predominant wastes generated during aquaculture include rapidly degradable organic nitrogenous wastes and to some extent reduced sulfur compounds, and these affect the shrimp and fish being cultured unless measures are employed to maintain their levels tolerable by the culture species. The perspective is essentially to provide a clean environment for the aquatic animal being cultured, in order to achieve optimal production from aquaculture. Simple management techniques such as aeration and ozonation have been practiced in semi-intensive and intensive aquaculture for providing adequate oxygen to the cultured animal, while oxidizing organic wastes generated during the culture process. However, these measures need to be supplemented through eco-friendly bioremediation tools in order to achieve freedom  from  organic  loading. In situ bioremediation  has  been widely applied in aquaculture through bioaugmentation, using indigenous or exogenous microbes called ‘probiotics’ which ameliorate water quality (Wang et al., 2005). However, their efficacies are uncertain. Policy guidelines on the use of bioaugmentation probiotics in aquaculture do not exist. Application of biofilms and microbial mats independently for bioremediation of aquaculture wastes is still under research and development. However, these are the major microbial ecosystems in recirculating aquaculture systems that remove nitrogenous wastes. In hatcheries, in addition to bioaugmentation probiotics, importance of RAS has been recognized for management of nitrogenous wastes especially in maturation and larval rearing facilities. RAS rely on use of biological filters / bioreactors for the removal of toxic wastes such as ammonia and nitrite. New technologies such as SHARON-ANAMMOX and CANON are being recently explored for application in aquaculture for the mitigation of nitrogenous wastes (Tal and Schrier, 2008). However, because of the initial high installation costs with RAS and the newer technologies and falling prices of shrimp and fish, their use in grow-out systems is becoming prohibitive.

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