Aquaculture 283 (2008) 2935

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

The biooc technology (BFT) in indoor tanks: Water quality, biooc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus)
M.E. Azim , D.C. Little
Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, United Kingdom

A R T I C L E

I N F O

A B S T R A C T
The present study evaluates the biooc technology (BFT) in light-limited tank culture of Nile tilapia (Oreochromis niloticus). Two biooc treatments and one control were managed in 250 l indoor tanks: BFT fed a diet of 35% crude protein (CP), BFT fed a diet of 24% CP, and clean water control without biooc with 35% CP. BFT tanks were aerated and agitated using a dome diffuser. Three kg of Nile tilapia were stocked in each tank. Feed was applied at 1.5% of the total sh biomass daily in each tank. Wheat our was added in BFT tanks to maintain an optimum C:N ratio for heterotrophic production. The total suspended solid (TSS) level was maintained at around 500 mg l 1 in BFT tanks. The nutritional quality of biooc was appropriate for tilapias. Fish survival was 100%. Net sh production was 45% higher in the BFT tanks than in the control tanks conrming the utilization of biooc by sh as food. There was no difference in sh growth/production between 35% and 24% CP fed tanks under BFT. Welfare indicators in terms of n condition, gill histology, proximate composition, blood haematocrit and plasma cortisol levels were compared and no signicant differences between BFT and control tanks were recorded indicating no increased sh stress due to the presence of biooc. However, overall sh growth and production was poor in terms of commercial feasibility. A modied system design that would allow enhanced feed and biooc utilization is proposed. 2008 Elsevier B.V. All rights reserved.

Article history: Received 13 April 2008 Received in revised form 24 June 2008 Accepted 24 June 2008 Keywords: Biooc Activated suspension technique Fish welfare Tank culture Tilapia

1. Introduction The basic principle of the activated suspension technique (AST), recently referred to as biooc technology (BFT) is the retention of waste and its conversion to biooc as a natural food within the culture system. This is done by constant aeration and agitation of the water column and addition of carbon sources as organic matter substrate to allow aerobic decomposition and maintain high levels of microbial oc in suspension in fed and/or fertilized ponds (Avnimelech et al., 1986; Hargreaves, 2006). Theoretically, increased C:N ratio through carbon addition enhances conversion of toxic inorganic nitrogen species to microbial biomass available as food for culture animals. The optimum C:N ratio in an aquaculture system can be maintained by adding different locally available cheap carbon sources and/or reduction of protein content in feed (Avnimelech, 1999; Hargreaves, 2006).Goldman et al. (1987) found C:N ratios N10:1 were optimal for optimizing biooc production while minimizing ammonia regeneration. Typically only 2025% of fed protein is retained in the sh raised in intensive systems (Avnimelech, 2006), the remainder being lost to the

Corresponding author. Department of Physical and Environmental Sciences, University of Toronto, 1265 Military Trail, Toronto M1C 1A4, Ontario, Canada. Tel.: +1 416 287 7690; fax: +1 416 287 7279. E-mail address: eazim@utsc.utoronto.ca (M.E. Azim). 0044-8486/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.06.036

system as ammonia and organic N in feces and feed residue. Microbial breakdown of organic matter leads to the production of new bacteria, amounting to 4060% of the metabolized organic matter (Avnimelech, 1999). Under optimum C:N ratio, inorganic nitrogen is immobilized into bacterial cell while organic substrates are metabolized. The conversion of ammonium to microbial protein needs less dissolved oxygen compared to oxygen requirement for nitrication (Avnimelech, 2006; Ebeling et al., 2006) suggesting the preference of heterotrophic community rather than nitrifying bacteria in the BFT system. In addition, the growth rate and microbial biomass yield per unit substrate of heterotrophs are a factor 10 higher than that of nitrifying bacteria (Hargreaves, 2006). Although some commercial production based around the concept of BFT has been used since the early eighties (Sering, 2006), the knowledge base concerning the technique is still undeveloped. The benets of this technology are documented mainly for shrimp culture (e.g.Burford et al., 2004; Wasielsky et al., 2006) and to a lesser extent for nsh culture (Milstein et al., 2001; Sering, 2006). Preliminary studies on tilapia culture in activated suspension ponds indicated that the sh grew well on low protein feed and fed on suspended particles leading to additional savings in feed costs; also the technology greatly increased the water use efciency (Avnimelech, 1999; Milstein et al., 2001; Sering, 2006). The technology was tested in aquaculture ponds where both autotrophic and heterotrophic microorganisms interact. Algae and bacteria have a range of stimulatory or inhibitory effects on

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M.E. Azim, D.C. Little / Aquaculture 283 (2008) 2935

each other (Cole, 1982) resulting in complexity of water quality dynamics. Senescent algae or algal detritus are a major source of organic substrate for bacterial growth whereas living algae provide oxygen for decomposition. In return, bacteria regenerate inorganic nutrients and vitamins that stimulate algal productivity. On the other hand, both groups produce substances that are antagonistic to growth of each other (Cole, 1982). Light limitation of autotrophic production also likely restricts its economic viability to applications where outdoor culture is possible whereas a heterotrophic production could be applied in light-limited indoor systems. However, the contribution of biooc to sh production was not quantied so far and the relative importance of bacteria within the system for both maintaining water quality and as food was not distinguished. Our preliminary study conducted in light-limited indoor tanks without sh indicates that the biooc quality and quantity were appropriate for sh culture and water quality parameters can be managed properly using the BFT (Azim et al., 2008). The nutritional value of biooc to aquatic animals is dependant on several factors: food preference, not only the ability to both ingest and digest it but also the density of suspended particles (Hargreaves, 2006). Tilapias being capable of both lter feeding and detritivory are ideal candidates for such system (Dempster et al., 1995; Azim et al., 2003). However, if successful, BFT might be a promising alternative technology to conventional recirculation aquaculture system (RAS) in which formulated diets including high levels of protein and complex external ltration are required. The main objective of the present experiment was to test the suitability of biooc technology in light-limited indoor tanks for rearing Nile tilapia Oreochromis niloticus. The specic objectives were as follows: (1) to quantify the contribution of biooc to sh growth and production, (2) to evaluate sh welfare in BFT managed tanks, (3) to test the effects of crude protein level in supplemental feed on sh growth and productivity in BFT tanks (4) to investigate the effects of protein level/C:N ratio of feed on proximate and taxonomic composition of biooc and (5) to compare dissolved inorganic nitrogen dynamics. System management aspects have also been discussed. 2. Materials and methods 2.1. Tank facilities and experimental design The experiment was carried out in ber glass indoor tanks (250 l each) situated in the Tropical Aquarium Laboratory of the Institute of Aquaculture, University of Stirling, UK. Two treatments (with four replications) and one control (with two replications) were compared: Biooc tank fed with 35% crude protein (treatment BFT35), BFT fed with 24% crude protein (treatment BFT24). As control, similar size duplicate tanks were placed within a conventional recirculating aquaculture system (RAS) and stocked with sh fed with similar amount of a 35% crude protein diet (RAS35). Biooc was already developed in the treatment tanks during the previous experiment (350 mg l 1 TSS). The levels were initially equilibrated through exchanging water between the tanks and adding water for evaporation loss was done. Tanks were aerated and agitated continuously using dome diffusers (AS9, 17.5 cm 3.75 cm, Aquatic Services, UK) connected to an air pump. Tanks were always covered with lids. 2.2. Fish stocking and tank management Mixed sex Nile tilapia O. niloticus, of individual weight ranging from 80 to 120 g were stocked at 12 kg m 3 in each tank. Pelleted feeds (3 mm) were formulated using plant ingredients and made locally using the technique developed in the Institute (Kim Jauncey, personal communication). Percentage of raw ingredients and proximate

composition, energy content and C:N ratios of the experimental diets are given in Table 1. Feeding rates were based on observation of feeding behaviour of sh in the BFT treatments during the rst several days and xed at 1.5% of the total stocked biomass daily, and adjusted fortnightly after weighing a sh sample. The same amount of feed was applied to all treatment and control tanks. Daily feed rations were split into two equal amounts given at 0900 and 1800 h to all tanks. Wheat our was also added at a rate of 60% feed applied to BFT tanks to maintain an optimum C:N ratio of bacteria (Avnimelech, 1999). Flour was completely mixed with tank water and spread to the tank surfaces in the afternoon. The sh were cultured for a period of 12 weeks between June and September 2006. Whenever oc levels exceeded 500 mg l 1in any tank, a oc separator was run for 812 h to settle and remove the oc from the tanks. When the pH of water dropped below 6.5, NaHCO3 was added to raise the pH to 7.5. Addition of water for evaporation loss and oc removal was done on a weekly basis. 2.3. Assessment of water quality parameters Water temperature, dissolved oxygen (portable DO meter WTW Oxi 340i) and pH (portable HANNA pH/ORP/Temperature meter HI 991002) were determined twice weekly. Total alkalinity was measured by acid titration on a weekly basis followingStirling (1985). Total ammonia nitrogen (TAN), nitrite (NO2N) and nitrate (NO3N) were analyzed spectrophotometrically with an autoanalyzer (BRAN LUBBE AutoAnalyzer 3) according to standard methods (APHA, 1998). Water samples (50 ml) were collected weekly at around 12:00 h from each tank and ltered under vacuum pressure through pre-dried and preweighed GF/C lter paper. The ltered water was used for nutrient analysis and the lter paper for total suspended solid (TSS) determination. Total suspended solid (TSS) and biochemical oxygen demand (BOD5) of water were measured on a weekly basis followingStirling (1985). After ltering water for nutrient analysis, the pre-dried and weighed lter paper containing suspended materials was dried in an oven until constant weight. Dried samples were weighed to 0.01 mg using a Mettler AC 100 balance. The TSS was calculated from the weight differences. For BOD determination, water samples were collected in plastic bottles from each tank. Two diluted samples in each tank and two controls (with only distilled water) were used in 170 ml BOD bottles. Samples were diluted 10 times initially and 20 times at the end of the experiment with distilled water. The initial oxygen concentrations in the samples were determined using a BOD YSI probe and DO meter (YSI model 57). The necessary chemicals were added to each bottle in case of diluted samples according toStirling (1985). The samples were rmly closed with stopper and placed in an incubator (Gallenkamp Cooled Incubator) in the dark at 22 C for 5 days. At the end of this time, the dissolved oxygen level was determined again and BOD5 was calculated in mg l 1 (Stirling, 1985).
Table 1 Diet formula and biochemical composition of the experimental diet High protein diet Feed ingredients (%) Soy meal Wheat meal Vegetable oil Molasses Biochemical composition (%DM) Moisture Crude protein Crude lipid Ash Crude bre Energy (kJ g 1) C:N ratio 60 30 5 5 Low protein diet 30 60 5 5

8.51 35.13 6.23 5.23 2.63 19.6 8.4

4.51 23.88 6.64 3.83 2.13 19.1 11.2

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2.4. Imaging and identication of microorganisms in biooc The 3-D images of bacterial oc were taken using a confocal microscope (Leica TCS 4PI) on a biweekly basis. Water samples were collected from one tank in each treatment and processed immediately for microscopy. Each sample was diluted with pre-ltered water of the same tank, stained with uorescence 4-6-Diamidino-2-phenylindole (DAPI), and observed and photographed at magnication 10. Taxonomic composition of other organisms in the water was studied in three consecutive weeks in the middle of experimental period. Well-mixed water samples were collected in 30 ml plastic bottles and the organisms were killed by adding several drops of Lugol's iodine. One ml sample was placed on the counting chamber of the Sedgewick Rafter counting cell (S-R cell) and left for several minutes to allow organisms to settle. Then the organisms on 10 randomly selected elds of the chamber were identied and counted using magnication of 2040 of a binocular microscope (Olympus LH50A). The number was multiplied by 100 to give the results in total number of organisms per ml water. This counting was repeated three times in each sample in each tank. The keys used for taxonomic identication of the microorganisms were Donner (1966), Patterson and Hedley (1992) and Nufeld Advanced Science (1970). 2.5. Determination of biochemical composition of biooc Biooc samples for biochemical analysis were collected on a fortnightly basis. Concentrated oc samples collected from each tank using a oc separator were dried in an oven at 102C until constant weight and then preserved in a refrigerator. At the end of the experiment, tank-wise pooled samples were ground and processed for proximate analysis followingAOAC (1990). For ash contents, a known amount of dry sample was burnt in a mufe furnace at 550 C for 4 h and the ash cooled and weighed. The crude protein content was determined by the Kjeldahl method. Lipid content was determined with Soxhlet apparatus. The energy content was determined by a bomb calorimeter (Gallenkamp Autobomb). The C:N ratios were determined using a CHN analyzer (Perkin Elmer 2400 Series II). Biooc samples for fatty acid analysis were collected at the end of the experiment and freeze-dried. Fatty acid analysis was undertaken by a modied direct methylation method (Christie, 2003) using an N-Evap 112 Liquid Gas Chromatograph (Organomation Associates Inc.). 2.6. Determination of sh welfare indicators More than 50% of sh in each tank were sampled fortnightly, anaesthetized and length and weight measured individually. These sh were also checked for any skin or n damage. At the end of the experiment, one sh in each BFT treatment tank and two sh in each RAS35 tanks were sacriced, the 1st and second gills from one side were removed and preserved in 10% buffered formalin. The samples were then processed for histology. Unpreserved gills were examined for parasites using a binocular microscope. Samples of skin mucus were also checked under microscope. At the end of the experiment, 10 sh were netted randomly from each BFT35 and RAS35 tank. They were mildly anaesthetized with benzocaine in a bucket, weighed and measured. Blood samples were collected from the dorsal caudal aorta using heparinised syringes and placed in microcentrifuge tubes. After blood sampling, sh were individually tagged (internally) and recovered in another bucket with clean water and air supply. Then they were transferred to another sh tank within a RAS until they were blood sampled for second time after 1 h following the same procedure mentioned above. Haematocrit (% packed red cell volume) was measured after centrifuging sub-samples of whole blood collected the rst time using heparinised micro-haematocrit tubes for 5 min at 5000 g. The remaining blood was centrifuged at 1200 g for 15 min and the plasma

was separated and stored at 70 C. Concentrations of plasma cortisol were determined using the radioimmunoassay described by Ellis et al. (2004) with the modications to the steroid extraction. Aliquots (200 l) of plasma were extracted with 1 ml ethyl acetate and after thorough mixing and centrifugation (430 g for 10 min), 200 l of chilled (4 C) supernatant were pipetted into duplicate polypropylene tubes, which were evaporated to dryness under vacuum at 35 C and before cooling to 4 C. Proximate composition and energy content of sh were also compared. Ten randomly selected sh at stocking and three sh in each tank at harvest were analyzed using the similar methods described above for biooc. 2.7. Statistical analysis Water quality parameters were compared by two-way repeated measures ANOVA with treatment (system type) as main factor and sampling date as repeated measures factor (Gomez and Gomez, 1984). The sh yield and blood parameters and biochemical composition of oc and sh were compared using one-way ANOVA. If main effects were signicant, differences among the treatments were tested with Tukey's multi-comparison test of means. The analyses were run at 5% signicance level using Statistica package. 3. Results and discussion 3.1. Water quality Average water temperature (28 C, range 2630 C), DO concentrations (6 mg l 1, range 3.07.5 mg l 1) and pH (6.7, range 5.08.5) were within the range for tropical sh culture except for low pH levels observed, and then corrected for, on several occasions. While total alkalinity was very stable in the control tanks (1827 mg l 1) it uctuated very much in the tanks with biooc (8250 mg l 1). This indicated that the biooc systems lose buffering capacity and therefore required frequent additions of NaHCO3. High rates of nitrication, indicated by constant nitrate accumulation (discussed later) coupled with ammonia immobilization into bacteria (Azim et al., 2008) were observed in BFT treatments. It is reported that nitrication requires approximately 4 mg O2 and 8 mg HCO for oxidizing 1 mg 3 TAN (Gujer and Jenkins, 1974; Sharma and Ahlert, 1977). However, continuous vigorous aeration ensured that dissolved oxygen was not limiting. The average TSS levels were 16, 597 and 560 mg l 1 in the control, BFT35 and BFT24, respectively. Efforts were made to keep the TSS level at 500 mg l 1 using a oc separator but the levels became uncontrollable using this regime at the last two sampling dates reaching up to 1000 mg l 1. Sometimes oc settled to the bottom of the separator and sometimes oated on the surface, suggesting qualitative changes in ocs. This characteristic as also observed by Murray and Little (submitted for publication) in light-limited BFT systems. Therefore, characterization of oc and improved approaches to its removal are a pre-requisite for effective management of this system. Optimum oc levels in terms of TSS level in relation to the sh stocking density needs further investigation. The BOD level corresponded to the TSS level with mean values of 7, 87 and 88 in the control, BFT35 and BFT24, respectively with a peak of 290 mg l 1 at the last sampling date in the biooc treatments. Dissolved inorganic nitrogen (TAN, NO2N and NO3N) concentrations throughout the experimental period are shown in Fig. 1. A high degree of uctuation in TAN and NO2N levels was observed in biooc treatments (high standard deviation) during the entire experimental period. TAN concentrations varied signicantly between treatments and sampling dates with higher mean values in the tanks fed 35% CP with biooc as compared to the control tanks without biooc (Fig. 1A). The NO2N concentrations in both biooc treatments were signicantly higher than in the control (Fig. 1B). There were also signicant

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M.E. Azim, D.C. Little / Aquaculture 283 (2008) 2935 Table 2 Mean ( SD) proximate composition, energy content and C:N ratio of biooc in high and low protein fed tanks Composition (%DM) Crude protein Crude lipid Ash Crude bre Energy ((kJ g 1) C:N ratio 35% CP Feed (C:N = 8) 37.93 2.38 3.16 0.31 13.38 1.35 6.27 0.44 18.62 0.43 7.2 0.43 24% CP Feed (C:N = 11) 38.41 3.62 3.23 0.21 11.83 0.80 5.71 1.86 19.04 0.22 7.3 0.22

(Ebeling et al., 2006). The relative importance of each varies with system type and production intensity. In the present experiment, although immobilization of heterotrophic bacteria was encouraged, the system was dominated by nitrifying bacteria. Further research on how to minimize nitrication in BFT systems is needed. Despite the fact that carbohydrate was added based on TAN: CHO of 1: 20 (Avnimelech, 1999), TAN and NO2N concentrations were elevated, sometimes reaching critical levels in BFT tanks during the experiment. The CHO requirement to minimize TAN needs further evaluation. The calculation might include NO2N concentration along with TAN concentration as also suggested byMurray and Little (submitted for publication). 3.2. Nutritional quality of biooc as sh food The biochemical compositions and energy contents of biooc are given in Table 2. There were no signicant differences in any nutritional parameters between the 35% and 24% CP fed treatments, indicating that the biooc quality was independent of feed quality

Table 3 Fatty acid composition (% lipid) of biooc in high and low protein fed tanks Fatty acid C14:0 C15:0 C16:0 C18:0 C20:0 C22:0 C24:0 Total saturated 16:1n-9/n-7 18:1n-9 18:1n-7 20:1n-11/n-9 20:1n-7 Total monounsaturated 18:2n-6 20:2n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6 Total n-6 PUFA 18:3n-3 18:4n-3 20:3n-3 20:4n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 PUFA 16:2 16:3 16:4 Total PUFA Unknown Total Total lipid % 35% CP Feed 2.48 0.77 19.10 6.24 1.44 1.31 0.33 34.92 7.74 8.51 11.05 0.80 0.00 28.10 15.38 0.55 0.40 3.55 0.34 3.28 23.50 0.65 0.06 0.00 0.00 0.46 0.00 0.74 1.91 0.00 1.32 0.03 26.76 10.22 100.00 3.16 24% CP Feed 2.02 0.70 17.88 7.27 0.87 1.06 0.40 30.20 7.15 10.08 11.28 0.74 0.13 29.38 16.68 0.50 0.46 3.11 0.59 4.47 25.81 0.73 0.05 0.02 0.06 0.39 0.09 0.77 1.38 0.02 0.80 0.08 28.09 12.33 100.00 3.23

Fig. 1. Dissolved inorganic nitrogen in different treatments throughout the experimental period. (A) TAN, (B) NO2N and (C) NO3N. Values are means ( standard deviation) of four and two replications in each sampling date respectively for treatment and control.

time effects and treatmenttime interactions indicating that the concentrations in different treatments behaved differently over time. The NO3N accumulation in the systems started on the 3rd sampling date (Fig. 1C). Similar to TAN, the NO3N concentrations in the treatment BFT35 were unstable throughout the experimental period. In contrast to TAN levels, NO3N concentrations increased gradually in BFT24 tanks throughout the experiment indicating better nitrication in low protein fed tanks. While TAN and NO2N concentrations were very unstable, NO3N concentrations were more stable and tended to increase in the biooc treatments throughout the experiment. The concentrations were signicantly higher in the high protein fed tanks followed by low protein fed tanks and control tanks. There were signicant time effects and treatment time interactions as well indicating the different trends of the concentrations irrespective of the treatments over time. There are three principal pathways to remove hazardous N species in aquaculture: (1) photoautotrophic removal by algae, (2) immobilization by heterotrophic bacteria as proteinacious microbial biomass and (3) chemo-autotrophic oxidation to nitrate by nitrifying bacteria

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Table 4 Growth performance of Nile tilapia in activated suspension and clean water tanks fed different levels of protein feed Parameters Initial individual weight (g) Final individual weight (g) Survival (%) Individual weight gain (g) Net yield (kg m 3) FCR 35% CP without biooc 99.61 13.74 127.51 28.17b 100 27.9 0.69b 3.35 0.08b 4.97 0.12a 35% CP with biooc 100.69 13.61 140.72 27.26a 100 40.04 3.04a 4.80 0.60a 3.51 0.44b 24% CP with biooc 98.45 12.71 138.58 24.99a 100 40.08 4.34a 4.90 0.59a 3.44 0.45b

Data shown are Means ( SD) for m3 water and 12 weeks culture period. In each row, different superscript letters indicate signicant difference at 0.05 level (ANOVA and Tukey test).

fatty acid (15%) than in the present study.Jauncey (2000) reviewed that about 0.51% n-6 fatty acid of dietary lipid required for Nile tilapia. However, a better quality biooc was reported in the similar system without sh (Azim et al., 2008) indicating that in situ oc utilization by sh could have an effect on the biochemical composition of oc.
Fig. 2. A 3-D image of six weeks old bacterial ocs stained with 46-diamidino-2phenylindole (DAPI) using a confocal microscope.

3.3. Taxonomic composition of biooc A 3-D picture of two individual bacterial ocs using a confocal microscope is shown in Fig. 2. The blue channel dominating the picture indicates the clusters of bacterial nuclei. There are trace amounts of mucopolysaccharides, typically secreted by bacteria (green channel). The ocs were more or less similar in shape but varied in size ranging from 50 to 200 m during the experiment. The abundance and taxonomic composition of different ocassociated organisms are given by treatment in Fig. 3. Three groups of organisms were identied: Protozoa, Rotifera and Oligochaeta. Among protozoans, three genera, namely, Paramecium, Tetrahymena and Petalomonas dominated. Four genera of rotifers were identied, namely, Lecane, Trichocerca, Polyarthra and Asplanchna. Only Tubifex was found in the group Oligochaeta. The total numbers of organisms were signicantly higher in the low protein fed tanks followed by the high protein fed tanks and control (Fig. 3).These microorganisms were observed grazing on the ocs using fresh samples under microscope. In natural system, coupling between bacteria and heterotrophic nanoagellates was such that about 1070% of the water column was daily cleared of bacteria (Bloem et al., 1989). 3.4. Fish yield parameters The sh yield parameters are given in Table 4. Tilapia survival was 100% in all treatment and control tanks. Individual sh weight at harvest was 910% higher in the BFT treatments than in the control. The BFT treatments contributed 4446% greater individual weight gain and net sh production than those in the control conrming the

applied in this experiment. A similar observation was made byAzim et al. (2008) using the similar feeds and system but without any sh. Goldman et al. (1987) also reported that the C:N ratio of bacteria varied little (56:1) despite a wide variation in substrate C:N ratio (1.510:1). Jauncey (2000) suggested 2530% crude protein and 68% crude lipid in tilapia diets for larger sh.Chou and Shiau (1996) reported that 5% dietary lipid appeared to be sufcient to meet the minimal requirement of juvenile hybrid tilapia (O. niloticus O. aureus), but a level of 12% was needed for maximal growth. It is also not desirable to have a bre content exceeding 812%, and ash content 12% in diets for sh, as the increase in bre content would consequently result in the decrease of the quantity of a usable nutrient in the diet (De Silva and Anderson, 1995). The biooc quality in terms of sh nutrition containing 38% protein, 3% lipid, 6% ber, 12% ash and 19 kJ g 1 energy (on dry matter basis) in the present study was appropriate for tilapias except for the very low crude lipid levels. The biooc was further analysed to see whether it contained essential fatty acids for the sh (Table 3). There were 2728% polyunsaturated, 2829% monounsaturated, 3035% saturated fatty acids and 1012% unknown peaks. Tacon et al. (200) estimated 3538% crude protein, 59% crude lipid, 710% ash, and 18 19 kJ g 1 energy in the biooc collected from zero-exchange outdoor system cultured with shrimp and fed different formulated and commercial diets. They also reported a lower level of polyunsaturated

Fig. 3. Abundance of biooc associated organisms in different treatments. Values are means ( standard deviation) of four replications and three sampling dates for treatment (N = 12) and two replications and three sampling dates (N = 6) for control.

Fig. 4. Blood cortisol level in sh cultured in tanks with and without biooc and under pre-stressed and post-stressed conditions. Values are means ( standard deviation) of 10 sh for both treatment and control.

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Table 5 Mean (SD) proximate composition and energy content of sh at stocking and harvest in BFT and RAS tanks fed different levels of protein feed Composition (%DM) Stock Harvest 35% CP without biooc Moisture Crude protein Crude lipid Ash Energy ((kJ g 1) 77.50 55.13 19.15 20.37 21.23 71.25 1.06 55.28 2.12 25.88 2.84 16.61 0.54 23.06 0.69 35% CP with biooc 69.03 1.34 53.41 2.74 27.83 2.84 17.05 0.78 23.34 0.55 24% CP with biooc 67.83 0.76 49.55 1.75 32.85 2.00 15.92 0.39 24.20 0.40

utilization of biooc by sh as food. Most of the tilapias are known to utilize in situ produced food particles including suspended bacteria (Beveridge et al., 1989; Beveridge and Baird, 2000).Avnimelech (2007) also conrmed the biooc uptake by Mozambique tilapia using stable nitrogen isotope labelling technique. However, there was no signicant difference in sh growth/production between 35% and 24% CP fed tanks under the BFT. The FCR value was signicantly higher in the control than in the BFT treatments. Although there was clear evidence that the biooc signicantly contributed to the growth and production of sh, the poor FCR and level of production was well below commercially viable levels.Little et al. (2008) reviewed nal biomass levels of 1028 kg sh m 3 achieved in indoor and outdoor BFT systems. These productions are even far below its counterpart traditional recirculating aquaculture systems in which standing biomass exceeding 100 kg sh m 3 with oxygenation and 7080 kg sh m 3 with aeration (Timmons et al., 2002). However, there might be several reasons attributed to the poor sh growth and production. Increased turbidity due to biooc reduces the visibility and hence articial feed intake. Although oc separator was used, it was not easy to maintain 500 mg l 1 TSS level, and very often the level reached to 1000 mg l 1 TSS especially during the second half of the experiment. Maintaining optimum oc levels was also identied as a critical issue in managing BFT systems byLittle et al. (2008). Secondly, water quality parameters were not stable: high uctuation of pH and alkalinity, high concentrations of inorganic nitrogen species might have chronic effects on sh health. Thirdly, some levels of sh reproduction occurred in the tanks inevitably reducing their growth. However, most of the sh seeds did not survive until the end of the experiment, might be due to adverse water quality on some occasions, rapid agitation of water and also cannibalism. 3.5. Fish welfare indicators Visual and microscopic observations of skin, n and gill indicated no physical damage in any sh. Out of eight gills examined by

histology, four were perfectly normal in the treatment BFT24. Some primary laments of the remaining four gills had minor distal extremity thickenings. Also one gill had couple of very minor nodular focal thickenings lower down on two laments. In treatment BFT35, six gills looked perfectly normal, one had thickening of distal extremities of some primary laments and the remaining gill displayed telangiectasis in ve adjacent laments near distal extremities. In the control tanks, out of eight gills, four were perfectly normal, the remaining had minor distal extremity thickenings, also one with telangiectasis. Generally, high levels of suspended solids are related to poor sh welfare as indicated by poor growth, fusion of gill lamellae (Mettam, 2005) and susceptibility to bacterial or parasite infections (Noble and Summerfelt, 1996). However, since minor abnormalities in some sh gills from both treatment and control tanks were affected, there is no evidence of potential gill damage due to presence of biooc in this experiment. The similar observation was made byVincent (2006) who compared sh raised over extended periods within BFT and RAS systems in a commercial farm. The proportion of blood haematocrit in both BFT35 and RAS35 sh was 27%. Blood cortisol concentrations also did not vary signicantly between the treatment and control tanks. However, after 1 h stress, the concentrations increased by 8 times in the treatment BFT35 and by ves times in the control RAS35 (Fig. 4). This difference might be due to an increased initial load of, and challenge from, bacteria entering the sh at the tagging site in the biooc treatment. The proximate composition and energy content of sh in different treatment and control tanks are also compared and presented in Table 5. There were no signicant differences in any sh nutritional parameters among the tanks under biooc treatments and control. 4. Conclusion and future research directions Biooc clearly contributed to the growth and production of sh in light-limited indoor systems. The nutritional quality of biooc was appropriate at least for herbivorous and omnivorous sh species including tilapias. Although there was no evidence that the prevailing biooc levels affected sh welfare, observed lower feed intake, slower growth rate and sometime chronic mortalities (Murray and Little, submitted for publication) suggest that the growth and production of sh in indoor BFT are not comparable to its counterpart indoor RAS hence unsecured economic sustainability. Uptake of biooc by the sh was insufcient to prevent its build-up and the need to remove it regularly from the system was clear. Water quality parameters were not stable. The biooc technology seems not appropriate to those species that do not graze on biooc directly. Therefore, further development of mixed systems has been advocated in which culture units are partitioned with algae, microbial oc and/or periphyton (e.g.Azim and Little, 2006; Avnimelech, 2006, 2007). Research efforts should be made

Fig. 5. Schematic diagram of a biooc-based recirculation aquaculture system.

M.E. Azim, D.C. Little / Aquaculture 283 (2008) 2935

35

to develop a hybrid technology of BFT and RAS. A simplied diagram of such technology is depicted in Fig. 5. All bioltration units of an RAS are replaced with a biooc reactor or activated suspension unit. Fish tank efuents are continuously pumped into the biooc reactor in which bacterial ocs are produced by vigorous agitation and aeration with a dome diffuser and carbon supplementation. Then the ocs are passed through a oc separator in which ocs are settled and removed periodically and processed for formulated feed. The clean water along with controlled amount of bioocs are pumped to the sh tanks again. The production and potential use of heterotrophic bacteria using sh waste concurrently improving water quality in the proposed indoor system would be a prospective technology to lower nutrient discharge and to increase nutrient retention thereby ensuring its future sustainability. A preliminary study indicated that microbial ocs generated in bioreactors using sh waste and offered as supplemental feed improved shrimp growth (Kuhn et al., 2007). However, clear understanding on the microbiological aspects particularly bacteria growth patterns, characterization of biooc, possible manipulation of microbial community is necessary for the successful design and operation of such technology. Acknowledgements The research is funded through the Marie Curie Incoming International Fellowship (IIF) of the European Commission (ASPECT: MIFT-CT2005-008965). Special thanks are due to our colleagues, Dr. James Bron for Confocal Microscopy, Mr. James Dickson for fatty acid analysis, Dr. Ben North for Radioimmunoassay, Mr. Keith Ranson for preparing experimental set up and providing experimental sh, Mr. William Struthers for the water quality analysis, Mr. Allan Porter for proximate analysis and Nufeld Fellow Miss Erin Matthews for taxonomic identication. References
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