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Bioremediation of aquaculture wastes

Pamela Chavez-Crooker1,2 and Johanna Obreque-Contreras1
Environmental impacts of wastes from large-scale, intensive aquaculture are substantial and can lead to complex ecosystem changes. The application of known and new technologies can capture inorganic nitrogen from water and reduce organic enrichment of sediments. Biological methods, including Integrated Multi-trophic Aquaculture are now gaining interest for increasing in situ removal of nitrogen and other nutrients at sea cage sites. Several studies on biological nitrogen removal through nitrication, denitrication and anaerobic ammonium oxidation (anammox) have been reported and a number of bacterial groups active in this regard have been described. Nevertheless, additional efforts need to be focused on remediation of aquaculture wastewater and marine sediments. Conventional treatment systems have several disadvantages. Development of more efcient reactor systems and a holistic, integrated approach to waste treatment would allow more environmentally balanced aquaculture practices.
Addresses 1 Biotecnologas Aguamarina S.A., Esmeralda 1807 of. 101, Antofagasta, Chile 2 Instituto de Biotecnologa de Tarapaca (IBT), Universidad Arturo Prat, Cordunap, Avenida Playa Brava 3256, Iquique, Chile Corresponding author: Chavez-Crooker, Pamela (pchavez@aguamarina.cl)

proceeds by anaerobic sulfate reduction [3]; simultaneously, the lack of oxygen inhibits aerobic nitrication and denitrication processes [4]. Lack of sufcient oxygen leads to the death or migration of the sediment macrofauna responsible for bioirrigation, and thus to a decline in aerated water within sediments and a further spread of anoxia. Upon the loss of bioirrigation, pelagic benthic coupling becomes reduced for these anoxic, azoic sediments. The net effect of organic enrichment in sediments is to move the ecosystem to the one dominated by bacteria, ciliates and meiofauna, where the trophic links to the next level of the food web are broken [57]. Under these conditions, the predominant bacteria are anaerobes, mainly sulfate reducers and methanogens [5]. Pohle et al. [8] reported one study of benthicpelagic coupling for salmon aquaculture. Although cause and effect are not yet well established, it seems probable that organic enrichment impacted this coupling in a way that excluded some species and encouraged others. Organic enrichment also can lead to an increased presence of pathogenic bacteria. Studies on surface sediments of a well-established sh farm showed that benthic bacteria levels were closely related to organic enrichment and their concentration was three times higher in stations beneath the cages. Counts (colony forming units (CFU)) of heterotrophic bacteria indicated a shift toward Gramnegative bacteria, with a predominance of Cytophaga/ Flexibacter-like bacteria (CBF), and the occurrence of pathogenic bacteria (such as Vibrio) in sediments beneath the cages. In contrast, Gram-positive bacteria were more prevalent in the control site, where they represented up to 90% of total isolates [6]. Sediments close to aquaculture facilities can become enriched reservoirs of viruses associated with organic detritus [9]. Labrana et al. [10] showed that IPNV in Chilean salmon farms can remain active in fresh water sediments for weeks and that the virus can be detected in areas with a previous history of IPNV outbreaks. Also, examples are known from shrimp aquaculture where water and soil from the cultivation ponds contributed importantly to the spread of viruses to neighboring ponds or farms [11]. In view of the potential environmental impacts as well as economic impacts through disease transmission, it is relevant to suggest that improvement in aquaculture waste management is a highly desirable objective. Removal of nitrogen and phosphorus from the water column to mitigate eutrophication along with improved wastewater and sediment treatments that reduce the level
Current Opinion in Biotechnology 2010, 21:313317

Current Opinion in Biotechnology 2010, 21:313317 This review comes from a themed issue on Evironmental biotechnology Edited by Sharon Borglin and John van der Meer Available online 27th April 2010 0958-1669/$ see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.04.001

Introduction
The global growth of aquaculture industries has brought an increase in negative environmental impacts through the discharge of substantial amounts of polluting efuents containing uneaten feed and feces. This organic enrichment causes environmental deterioration of both the receiving water bodies and sediments [1]. The initial effect of adding large amounts of decomposable organic waste to marine sediments is increased metabolic activity by aerobic bacteria. Their demand for oxygen results in localized hypoxia or anoxia, killing the most susceptible aerobic life forms [2]. In the case of sediment in sh-cage footprints, much of the continuing metabolism then
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314 Evironmental biotechnology

of organic matter thereby, biological risk, will do much to make aquaculture a sustainable farming practice for the long term.

ammonia serve as principal nitrogen sources for the growth of some microorganisms. Biological denitrication: Denitrication is the main mechanism for converting xed nitrogen to N2 gas, which returns to the atmosphere. It occurs under low oxygen conditions, in energy-generating reactions where oxides of nitrogen, including nitrate, nitrite, and nitric and nitrous oxides, are used as electron acceptors in place of oxygen, nally becoming reduced to N2 gas as the end product [25]. A broad spectrum of microorganisms is capable of denitrication reactions, including various bacteria, Archaea and Eukarya [26,27,28]. Known denitrifying bacteria and archaea possess several clusters of genes involved in denitrication [29]. These genes encode four metalloenzymes: nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, which sequentially reduce nitrate to N2 (NO3, NO2, NO, N2O, N2) [29]. Anammox: Anaerobic ammonium oxidation is another route to denitrication. It was rst discovered in anoxic (denitrifying) bioreactors of wastewater treatment plants [30], where novel organisms related to Planctomycetales were found to be capable of oxidizing ammonium using nitrite as electron acceptor [26]. First thought an oddity, it was later found that anammox was responsible for 24 67% of nitrogen loss in marine sediments [31]. Anammox activity now appears to account for 2040% of all nitrogen loss in the ocean [3236]. It is also present in coastal and estuarine sediments [26,31,3740], anoxic basins [33,34], oxygen minimum zones [35,36,41,42], mangroves [43], sea-ice [44] and freshwater lakes [45]. It seems likely that anammox activity would be a major contributor to denitrication of anoxic aquaculture-derived sediments.

Treatment of wastes
Bioremediation of water and sediments contaminated by sea cage aquaculture, and of efuents discharged by landbased aquaculture activities, brings into play many organisms, including bacteria, microalgae and macroalgae. For sea cage operations, bioremediation techniques conducted on-site are likely more feasible than land-based treatments. However, for some aquaculture operations a combination of standard waste treatment methods and other bioremediation techniques may be needed [12,13].
Integrated Multi-trophic Aquaculture

Integrated Multi-trophic Aquaculture (IMTA) strategies [14] have been described as a key development for aquaculture sustainability [1517]. IMTA integrates a number of complementary organisms at a farm site in order to optimize nutrient utilization and reduce solid waste that goes to sediments. The waste from one organism becomes a source of energy for others, thereby moving toward a better ecosystem balance [17]. Valuable seaweeds strip ammonium, nitrate and phosphorous excreted by sh from the water column, simultaneously gaining nutrients for growth and removing aquaculture pollutants. Shellsh and other lter feeders convert signicant amounts of particulates from uneaten sh feed and feces into harvestable body biomass. Bottom feeders can also be integrated into the system to help work sediments, removing organics and enhancing bioirrigation. Although IMTA may not remove all inorganic and organic waste from aquaculture farms, combining this approach with biotechnological applications such as integrated anaerobicaerobic reactors for waste treatment need to be investigated at industrial pilot scale [18,19].
Microbial nitrication and denitrication in sediments

Technological applications
Land-based aquaculture and biological lters

Biological nitrication: Under aerobic conditions, two groups of bacteria convert ammonium to nitrite and then to nitrate (NH3NO2NO3). This is a process that consumes a great deal of oxygen and can lower dissolved oxygen in the area. The aerobic ammonia-oxidizing bacteria (AOB) oxidize ammonia to nitrite via hydroxylamine and then the nitrite-oxidizing bacteria (NOB) oxidize nitrite to nitrate. The AOB are frequently combined with another group, the ammonia-oxidizing archaea (AOA), known together as the ammonia-oxidizing microorganisms (AOM) [2022]. Autotrophic bacteria in these groups use the reducing power of the nitrogenous substrates to x CO2 via the CalvinBenson cycle as their source of carbon. The ammonia oxidizers belong predominantly to the b-subclasses and g-subclasses of the Proteobacteria [23], whereas the nitrite oxidizers belong to the a-subclasses and g-subclasses of the Proteobacteria and the phylum Nitrospirae [24]. Not surprisingly, the nitrate and
Current Opinion in Biotechnology 2010, 21:313317

Environmental impacts are minimized by taking aquaculture operations onto land where waste products can be accessed more readily. For ow-through systems the gain is marginal as large volumes of wastewater are still discharged into a receiving water body. However, for recirculating systems, the volume of the waste streams becomes more manageable and various treatment options can be considered. Protein is the major component of sh feed, and is used by sh for both growth and energy production. During protein metabolism, ammonium, which is toxic to sh, is generated as an end product [1]. To maintain good water quality, recirculating systems use biological lters in which heterotrophic bacteria break down organic wastes and aerobic nitrifying bacteria convert ammonium to nitrate which is eventually discharged in the waste stream. Maintaining biological lters requires care as the bacteria responsible for ammonium oxidation are very sensitive to operating conditions, particularly pH. For
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 Bioremediation of aquaculture wastes Chavez-Crooker and Obreque-Contreras 315

larger aquaculture operations, it becomes feasible to construct an on-site, dedicated wastewater treatment facility to remove the nitrate and treat solid wastes before release to the environment. Treatment of aquaculture wastewater: A wide range of physical, chemical, and biological processes can contribute to the removal of nitrogen from wastewater in biological treatment systems. Treatment facilities and reactors take on many designs. All use processes that are derived from an integration of various components of the natural biogeochemical cycling of nitrogen in the environment; however, they are operated so as to maximize the kinetics of the activities during remediation treatments. A treatment system that combines total microbial nitrication with anammox (to reduce nitrite) seems to be more cost effective than most others [46]. The desired reactor conguration depends on the waste characteristics such as the concentration of organics and ammonium. In all cases, the key step appears to be partial nitrication [47]. Processes that use partial nitrication or combine partial nitrication with anammox in suspended biomass or biolm reactors have been developed, including a new process that is based on anaerobic ammonia oxidation coupled to conversion of nitrate into nitrite, driven by sulphide [48]. More studies are needed on microbial processes controlling nitrogen removal in immobilized systems such as biolters or biolms and in wetlands used for the bioremediation of wastes [49]. Wetlands are sometimes linked to recirculating aquaculture systems as a nal treatment phase before complete release of efuents to the environment. In one test of such a wetland system, small-scale treatments of efuents from a trout farm were conducted using a constructed wetland with subsurface ow. With loading rates involving large volumes of water, and consequently, short retention times, wetland treatment of dissolved nutrients was effective only for the ammonium and nitrites while nitrate and phosphate showed no, or even negative, treatment effects through the wetland passage [50]. More effective removal of the nitrogen could be expected with longer retention times. Some examples for nitrogen removal in aquaculture systems using simple in situ biological reactors have been reported. Kumar et al. [51] described a rapid setting up of nitrication using ammonia oxidizers (native and exogenous strains) in stringed bed suspended bioreactors (SBSBR) in a shrimp hatchery. De Scheryver and Verstraete [52] reported the potential of batch reactors (SBRs) as an alternative bio-ocs technology approach in aquaculture. Manju et al. [53] reported immobilized nitrifying bacterial consortia as bioaugmentators for in situ applications in shrimp aquaculture. Lyles et al. [54] reported a biological treatment (SBRs) for shrimp aquaculture wastewater.
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Conclusions
The aquaculture industry, especially intensive salmon aquaculture in near-shore waters, is facing an important challenge in maintaining social, economic and environmental sustainability. Recently, in Chile, a single disease caused the loss of millions of dollars and thousands of jobs through the closure of hundreds of production centers. The role of waste management, or the lack thereof, is not known in this instance; however, it demonstrates the scale of costs associated with outbreaks that could be derived from pathogens harbored in waste sediments. Several strategies and treatment technologies are being available for enhanced aquaculture waste treatments and for in situ remediation strategies. The aquaculture industry needs to consider these carefully and integrate better waste management into their operations as an investment that will help them sustain and augment their production while still protecting the environment.

Acknowledgements
We would like to thank the Chilean Government for support through CONICYT and FONDEF Grant DO8I1027. A special thanks goes to Dr Thierry Chopin for providing copies of reprints relating to IMTA used for this manuscript.

References and recommended reading

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