Research paper Immobilization of nitrifying bacterial consortia on wood particles for bioaugmenting nitrification in shrimp culture systems N.J. Manju a , V. Deepesh a , Cini Achuthan a , Philip Rosamma b , I.S. Bright Singh a, ⁎ a National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Lakeside Campus, Cochin, Kerala, India, 682016 b Department of Marine Biology, Microbiology and Biochemistry, School of Ocean Science and Technology, Cochin University of Science and Technology, Cochin, Kerala, India, 682016 abstract article info Article history: Received 4 January 2009 Received in revised form 9 May 2009 Accepted 11 May 2009 Keywords: Nitrifying bacterial consortia TAN Bioremediation Bioaugmentation Immobilization Wood particles Shrimp grow out systems under zero water exchange mode demand constant remediation of total ammonia nitrogen (TAN) and NO 2 - – N to protect the crop. To address this issue, an inexpensive and user-friendly technology using immobilized nitrifying bacterial consortia (NBC) as bioaugmentors has been developed and proposed for adoption in shrimp culture systems. Indigenous NBC stored at 4 °C were activated at room temperature (28 °C) and cultured in a 2 L bench top fermentor. The consortia, after enumeration by epifluorescence microscopy, were immobilized on delignified wood particles of a soft wood tree Ailantus altissima (300–1500 μm) having a surface area of 1.87 m 2 g -1 . Selection of wood particle as substratum was based on adsorption of NBC on to the particles, biofilm formation, and their subsequent nitrification potential. The immobilization could be achieved within 72 h with an initial cell density of 1 × 10 5 cells mL -1 . On experimenting with the lowest dosage of 0.2 g (wet weight) immobilized NBC in 20 L seawater, a TAN removal rate of 2.4 mg L -1 within three days was observed. An NBC immobilization device could be developed for on site generation of the bioaugmentor preparation as per requirement. The product of immobilization never exhibited lag phase when transferred to fresh medium. The extent of nitrification in a simulated system was two times the rate observed in the control systems suggesting the efficacy in real life situations. The products of nitrification in all experiments were undetectable due to denitrifying potency, which made the NBC an ideal option for biological nitrogen removal. The immobilized NBC thus generated has been named TANOX (Total Ammonia Nitrogen Oxidizer). © 2009 Elsevier B.V. All rights reserved. 1. Introduction Expansion and intensification of aquaculture have been taken up as means for attaining food security in several tropical countries triggered by the rapid decline in ocean fisheries. However, this intensification has led to increased organic loading culminating in deterioration of water and sediment quality especially with high total ammonia nitrogen (TAN) (Shan and Obbard, 2001). As ammonia build up in aquaculture systems is deleterious to the rearing stock, their mitigation is of primary concern (Paungfoo et al., 2007) for sustainability. Optimum shrimp growth demands less than 0.1 ppm unionized ammonia (1.33 to 1.53 mg L -1 TAN at pH 8.0 and 28–30 °C) (Shan and Obbard, 2001), which otherwise leads to poor feed intake, retarded growth, poor survival and high susceptibility to diseases. Such situations are negotiated by water exchange up to 40% intermittently to facilitate removal of the toxic waste metabolites (Deb, 1998). While doing so a higher concentration of ammonia is discharged into the receiving waters (Jones et al., 2001), a situation often correlated with eutrophication (Shan and Obbard, 2001). Therefore, management of water quality in shrimp culture systems is an essential prerequisite for maximizing productivity and minimizing the impacts of shrimp culture effluent discharged into the surrounding environment. Sequestering ammonia from culture systems has been achieved by chemical (Gräslund and Bengtsson, 2001) and biological filters (Malone and Pfeiffer, 2006) and through in situ application of microbial amend- ments (Rombaut et al., 2003). In small scale shrimp grow out systems, zero or limited water exchange system based on chemical and biological filtrations and recirculating aquaculture systems is not practical, and economically not viable (Schryver et al., 2008). Main constraints are high capital cost, and technical problems related to their establishment and operation (Shan and Obbard, 2001). An alternative for this is the bio-flocs technology (BFT) (Avnimelech, 2006; Crab et al., 2007), where hetero- trophic bacteria and algae are grown into flocs under controlled conditions within the culture ponds. The intensive growth of heterotrophic bacteria immobilizes inorganic nitrogen depending on the C/N ratio. However, factors responsible for their dynamics and their effects on growth and survival of cultured species warrant further investigation to exploit the merits of BFT (Crab et al., 2007). Under such situations use of nitrifying bacteria as bioaugmentors has been found to be a better option, and biological nitrification can be sustained by maintaining optimal conditions for their proliferation. Both the groups of nitrifiers, ammonia and nitrite oxidizers, involved in nitrification are obligate autotrophs, and slow growers, and have different levels of sensitivities to environmental factors. Aquaculture 294 (2009) 65–75 ⁎ Corresponding author. Tel./fax: +91 484 2381120. E-mail address: bsingh@md3.vsnl.net.in (I.S.B. Singh). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.05.008 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online