component in maintaining fish viability, especially at high densities. We used a low head oxygenation (LHO) unit to maintain dissolved oxygen above 4.5 ppm at all times. The overflow from the protein skim- mer was added to the feed water of the LHO providing additional oxygen from the breakdown of the ozone.
Additional Controls Photoperiod
All of our experimental tanks were cov- ered by cone-shaped lids, under which were installed incandescent (natural light) greenhouse bulbs. Through comput- erized software, the light intensity and the photoperiod (hours of light and dark per day, and their gradual change-over time) were continuously controlled. An electronic system provided light dim- ming, enabling the simulation of dawn and dusk. Control of photoperiod is important to enable year-round spawning and juvenile production in hatcheries, and to enable optimal feed consumption at the growout stage.
Salinity and seawater make-up system Our main objective was to develop recircu- lating marine aquaculture systems based on the use of artificial seawater. For experi- mental purposes, we developed a flexible system able to provide salinity levels rang- ing from fresh to full seawater (0–35 ppt) on a continuous basis. Salt water was pre- pared from Baltimore city water in a water- makeup system that delivered the desired salinity to all tanks. A salt storage tank contained 18,000 kg of technical grade sodium chloride. City water was pumped through charcoal filters and then through the salt storage tank where a saturated brine solution of sodium chloride was pro- duced. This solution was then moved into a 9.5 m3 reservoir where it was manually mixed with essential chemical ingredients found in seawater to provide a 35 ppt salt- water solution. Making our own salt mix from individual ingredients reduced the cost of saltwater by 30% as compared to the use of ready-made, commercially avail- able salt mixes. A second reservoir of 9.5 m3 held charcoal-treated freshwater. Water at the desired salinity was provided auto- matically and on-demand to each of the culture tanks.
Disinfecting wastewater
Our entire facility was designed to house non-indigenous species. As such, it was built with safety measures to ensure strict biosecurity. One such measure was the dis- charge of all waste effluents to a 9.5 m3 reservoir, where the water was automati- cally chlorinated prior to its release to the municipal sanitary sewer.
Control of environmental and operational parameters
All the above-mentioned environmental and operational parameters of the system were continuously tracked through a real- time monitoring system connected to spe- cific probes located in the tanks or the sumps. The monitored information was fed to a desktop computer that responded to readings outside the allowed ranges by trig- gering an appropriate warning. Actual con- trol of these parameters was handled at each tank (e.g. oxygen delivery, pH buffer- ing, etc.).
Operational Performance: Growing a High-value Marine Fish – Gilthead
Seabream Experimental conditions
Gilthead seabream (Sparus aurata) fry weighing 0.25–0.6 g were purchased from a Greek producer (referred to as Greek strain).
They were air-shipped to Baltimore in plas- tic bags and grown to market size of 400–425 g under intensive conditions in the above-described tank system (Experiment 1). In Experiment 2, fry from our Spanish strain seabream broodstock were grown under identical conditions.
Effluent from the culture tanks was gravity- fed to the 60 m drum screen filter to remove solids, after which the water was collected in a sump basin. From the sump, the water was pumped through the tita- nium heat exchanger, into the nitrifying mixed bed bioreactor. Upon leaving the Commercially Feasible Urban Recirculating Aquaculture 165
MBB, water was gravity-fed into the LHO for carbon dioxide ‘stripping’ and supple- mentation of liquid oxygen, before return- ing to the fish tanks. A side stream fed water from the MBB to the ozone protein skimmer and then into the LHO. Additional oxygen was supplied to the fish via a pair of diffusers placed directly in the fish tanks.
This latter addition was done to ensure that the tanks would be oxygenated even if flow stopped. The average flow rate in the sys- tem was 10 m3 per hour. New water was added to the system at a rate of 7–10% of the total tank volume per day to make up for water lost to evaporation and during the self-cleaning cycle of the drum filter. pH levels were maintained at 7.5 as described above. Several water quality parameters, including temperature, O2, CO2, pH and ORP were monitored in real-time via com- puter, while other water chemistry parame- ters (NH3, NO3, NO2, alkalinity and phosphate) were measured daily by staff.
Water temperature was maintained at 26°C. Photoperiod in the tanks was regu- lated via computer and maintained at 18:6 h light/dark cycle. The fish were grown at salinities ranging from 15 to 25 ppt, depend- ing on the stage of their growth cycle. They were fed a commercially available red seabream extruded diet (EWOS Seabream Omega LE) containing 14% fat, 47% pro- tein, 2% fibre and 28% carbohydrate. Food was delivered twice every hour via two automatic feeders per tank. Feeding rate was determined by body weight (BW). The fingerlings were fed at an initial rate of 6%
BW/day. As they grew, this percentage was gradually reduced to 3.2% BW/day by the time they reached an average size of 40 g after which they were fed 1.5% BW/day.
Growth rates were tracked by determining the average weight of 40 fish every 2–4 weeks. The fish were graded by size three times over the duration of the experiment.
Results
The results of two seabream growout experiments are illustrated in Fig. 10.2A and B. Figure 10.2A provides the full
growth curve for the first experiment, in which seabream fingerlings of a Greek strain grew from 0.5 to 400 g commercial size in 268 days (under 9 months) and to 450 g in 300 days (10 months). Figure 10.2B presents the growth curve for the Spanish strain fingerlings, which grew from 0.5 to 410 g in 232 days (7.7 months).
Overall survival rate in both experiments was over 90%. The above growth rates are very fast for seabream compared to the best growth performances in net-pens, reported at 387 days (12.9 months) and 420 days (14 months) from 1 to 400 g (Lupatsch and Kissil, 1998; and personal communications with industry, respectively). Commercial standards for growing seabream from 1 to 400 g in the Mediterranean net-pen indus-
Fig. 10.2. Growth rate of seabream in the recirculating mariculture system. (A) Greek strain, 0.5–516 g. (B) Spanish strain, 0.5–410 g. The dotted lines indicate the time (days) needed to grow fish to 400 g. Note difference in x-axis (time) scales.
try are in the range of 13–17 months, depending on locations and strain used (Sahin, 1996; Kissil et al., 2000). We attribute the excellent growth performance in our system to the fact that all environ- mental parameters (temperature, salinity, photoperiod, etc.) were tailored to opti- mally meet the specific physiological requirements of seabream at its different stages of growth and that feeding was on a high frequency schedule. Additionally, the continuous long days to which the fish were exposed delayed the onset of sexual maturation and gonadal growth, resulting in more energy diverted to muscular growth (Kissil et al., 2001).
Table 10.1 summarizes some of the growth performance parameters of gilthead seabream in our system. As can be seen, the above-discussed growth rates were obtained at densities of 44–47 kg/m3, much higher than the average 20 kg/m3standard densities in commercial seabream net- pens. In more recent experiments, we have been able to grow seabream at stocking densities exceeding 60 kg/m3. Moreover, as can be seen in Table 10.1, at least for the first stages of growout to 76 g, we obtained excellent food conversion rates (FCR) of under 1 (0.9–40.8 g and 0.87–87 g). These values are much better than the 1.31–1.58 FCRs predicted for those weight ranges in the net-pen industry (Lupatsch and Kissil, 1998). We suggest that our better FCR val- ues reflect the fact that fish in our tanks, in addition to all the enhanced environmental parameters noted above, have better access to food, compared to fish in floating net- pens. Our FCR value increased to 1.89 for fish in the 83–403 g size range. A similar increase in FCR, associated with the growth of the fish, has also been predicted
by Lupatsch and Kissil (1998), who list the- oretical FCR values of 1.58–1.79 for seabream in the size range of 100–400 g.
We attribute the large FCR increase observed in our experiments to the fact that at a feeding rate of 1.5% of their body weight daily, we slightly overfed our large fish. Lupatsch and Kissil (1998) predict a 1.2–1.3% BW daily feed intake in seabream weighing 300–400 g. Nevertheless, overall the experimental FCR we observed for seabream grown in the marine recirculating systems is much better than the net-pen industry standard of 1.8 (Theodorou, 2002).
The water chemistry of the culture sys- tems during our growout experiments is summarized in Table 10.2. Average total ammonia and nitrite levels were quite low, much below concentrations that are consid- ered to be stressful to fish or to interfere with fish growth (Losordo and Westers, 1994; Timmons et al., 2002). This is a reflection of the high efficiency of our nitri- fying moving bed reactors. Surprisingly, rel- ative to our earlier freshwater studies, the tank nitrate levels never built up. This is mainly the result of the 7–10% daily water exchange (which will have to be reduced, see below), but also due to the high het- erotrophic bacterial growth in these organic rich systems. However, another factor that is probably responsible for the low nitrate level was revealed by a recent study, which characterized the microbial communities present in our biofilters (Tal et al., 2003).
This study demonstrated considerable deni- trification and anaerobic ammonia oxida- tion (anammox) activities in our nitrifying filters. Anammox is a microbial process responsible for the direct conversion of ammonia to free nitrogen.
Commercially Feasible Urban Recirculating Aquaculture 167
Table 10.1. Grow-out densities and FCR values for gilthead seabream grown in recirculating mariculture systems (Experiment 1).
Weight (g) Duration (days) Density (kg/m3) FCR
0.62–40.8 91 44 0.9
6.5–76 89 47 0.87
83–403 158 44 1.89
Increasing the Cost-efficiency of Marine Aquaculture Systems:
Improving Nitrogen Removal Through Biofiltration
As explained earlier, the major goal of our studies is to develop the technology and protocols that will enable the industry to expand the production of marine finfish in urban recirculating systems. Cost-analysis of our technology has indicated that despite the excellent farm-gate value of seabream, its production cost is still rela- tively high. Dissecting the components of the production cost has shown that salt used for producing seawater accounts for about 25% of that cost. This figure is based on daily discharge and renewal of 10% of the total tank saltwater volume.
As discussed above, our encouraging results and growth performance for seabream were obtained in systems in which we replaced 7–10% of the system’s volume daily. In an effort to reduce the production cost in the marine RAS, we initiated a research programme aimed at decreasing the daily saltwater discharge from our tanks. Since the most important limiting factor to increasing the degree of recirculation is the build up of chemical waste, specifically of nitrogenous com- pounds, we set out to develop a better understanding of the biofiltration process as a basis for enhancing its biological nitrogen removal efficiency.
The process of biological nitrogen removal in recirculating aquaculture sys- tems is carried out by microorganisms that colonize the biofilters (for review see Wheaton et al., 1994; van Rijn, 1996;
Timmons et al., 2002). Despite the impor- tance of nitrogen removal in recirculating aquaculture, there is a dearth of informa- tion about the identity of the microbial communities present in the biofilters, their biology, ecology and beneficial activities.
Generally speaking, RAS biofilters are largely ‘black boxes’, and very little is known about the marine biological filtra- tion process and the specific contribution of each microorganism to the process.
Consequently, the main objectives of our study were to: (i) use modern tools of mole- cular biology to identify the microbial com- munities associated with our marine moving bed bioreactors (MBB); (ii) study the physiology of the identified microor- ganisms and their contribution to the nitro- gen cycle; (iii) enhance the nitrogen removal activity of the MBBs by modifying and engineering its microbial consortia;
and (iv) add a denitrifying, anaerobic com- ponent to our biological filtration process.
Our initial studies resulted in the iden- tification of several unique autotrophic and heterotrophic bacteria associated with ammonia and nitrite oxidation (Tal et al., 2003). In addition to nitrification activity, the microbial consortia associated with the MBRs were shown to carry out denitrifica- tion. More significantly, we have identified two unique marine Planctomycetesspecies capable of nitrogen removal via the anaer- obic ammonia oxidation (anammox) process (Tal et al., 2003). Anammox, which has only recently been shown to occur in the marine environment (Strous et al., 1999), involves the reduction of ammonia to nitrogen gas using nitrite as an electron acceptor. Laboratory scale studies using the MBBs demonstrated that the anammox process could be induced in the system (Fig. 10.3). This complete nitrogen removal process is likely due to the participation of all nitrogen and carbon utilizers within the filter’s microbial community.
Table 10.2.Physical and chemical parameters (average where applicable) recorded during the seabream growth (Experiment 1).
Parameter Mean ±SD
Water exchange 7–10% per day
Temperature 26 ± 2°C
pH 7.47 ± 0.11
Alkalinity 202 ± 36 mg/l (CaCO3)
Total NH3 0.50 ± 0.25 mg/l
NO2 0.85 ± 0.45 mg/l
NO3 50.4 ± 10.5 mg/l
PO4 9.71 ± 2.6 mg/l
Our innovative results demonstrated that the microbial consortia present in the MBRs have the potential to support differ- ent nitrogen transformation processes that enable closing the nitrogen cycle and releasing nitrogen back to the atmosphere.
We are now in the process of studying the potential of using MBRs for complete nitro- gen removal in our marine systems by com- bining nitrification with denitrification and the anammox process. We expect that these improvements will enable us to establish standardized marine nitrogen-removing consortia for use in inoculating MBR bio- logical filters in recirculating mariculture systems.
Another avenue to achieving our goal of decreasing saltwater discharge from our system is the addition of an anaerobic den- itrification unit (ADU) to our MBR nitrify- ing module, thus creating a two-stage nitrogen removal process. In our recent studies (Tal and Schreier, 2004), we sup- plemented our system (Fig. 10.1) with an ADU that was directly attached to the main biofiltration tank (label 6 in Fig. 10.1). The denitrification unit was found to reduce 90–100% of the daily nitrate production of the nitrifying filter resulting in minimal
nitrate accumulation in the system. The two-stage biofiltration device was tested in our two 3.8 m3 tanks in which adult seabream were grown at 40–50 kg/m3 and fed daily at 1% of their body weight.
During a 4-month experimental period, we were able to obtain daily water exchanges that averaged as low as 1% of the tank vol- ume. This water exchange was significantly lower than the 7–10% used in our earlier study and is expected to reduce the costs associated with salt use in recirculating marine systems from as high as 25% to as little as 3% of the total production cost, thereby considerably enhancing the prof- itability of such systems. Presently, we are also engineering ways to recover salts lost through solids removal in order to gain additional savings in system costs.
Conclusions
This chapter describes the successful development and characterization of a small-scale pilot of environmentally com- patible urban mariculture, based on the use of water recirculating technologies. Using a high-value marine species, the gilthead Commercially Feasible Urban Recirculating Aquaculture 169
Fig. 10.3. Anammox potential of the bacterial consortia in MBRs. Ammonia removal rates of MBRs incubated with ammonia and nitrite (closed boxes) or ammonia alone (open boxes). Three separate incubations were performed under conditions that would stimulate anammox activity, i.e. anaerobic conditions with no added organic carbon source. The ammonia oxidation rate with nitrite as electron acceptor was between 0.29 and 0.33 mg NH3–N/m2/h compared to values of 0.092–0.20 mg NH3–N/m2/h during incubations without nitrite, suggesting the presence of anammox activity. For additional details see Tal et al. (2003).
seabream, we demonstrated both the feasi- bility of this approach and excellent perfor- mances in terms of growth and food conversion rates, production densities and water quality. In additional studies not pre- sented here, we closed the entire life cycle of seabream and other marine species in recirculating systems, including spawning and larval rearing. Based on our initial eco- nomic feasibility studies, we believe that, when properly scaled-up, our technology will support a profitable operation produc- ing high-quality marine fish. The first such urban, commercial operation to be located in the city of Baltimore is now in the plan- ning stages. While this technology is ready for scaling up, it is clear that much more research is required to strengthen its long- term outlook and success. Our current and future research efforts focus on diversifying the species to be farmed to include addi- tional finfish, as well as shellfish species,
and decreasing production cost associated with the loss of water and salt, through understanding and improving the effi- ciency of the biological filtration process.
Acknowledgements
We are grateful to Steven Rodgers, Eric Evans, James Frank and Jon Deeds for the technical help provided in the develop- ment and maintenance of our systems.
Stanley Serfling and Dr Moti Harel also contributed to the design of the systems.
Dan Gross contributed useful technical editing to the final text. This work was supported in part by a Maryland Industrial Partnership grant to ARP and a Binational Agricultural Research and Development Foundation award to HJS. This is contri- bution #05–112 from the Center of Marine Biotechnology.
References
Bromage, N.R. and Roberts, R.J. (1995) Broodstock Management and Egg and Larval Quality. Blackwell Scientific, Oxford.
Devlin, R.H., Yesaki, T.Y., Biagi, C.A., Donaldson, E.M., Swanson, P. and Chan, W.K. (1994) Extraordinary salmon growth. Nature371, 209–210.
FAO (2002) The State of World Fisheries and Aquaculture. FAO Press, Rome, pp. 1–125.
Goldburg, R.J., Elliott, R.L. and Naylor, R.L. (2001) Marine Aquaculture in the United States:
Environmental Impacts and Policy Options. Pew Oceans Commission, Arlington, Virginia, pp. 1–33.
Hew, C.L. and Fletcher, G.L. (1997) Transgenic fish for aquaculture. Chemistry & Industry21, 311–314.
Hew, C.L. and Fletcher, G.L. (2001) The role of aquatic biotechnology in aquaculture. Aquaculture197, 191–204.
Hew, C.L., Fletcher, G.L. and Davies, P.L. (1995) Transgenic salmon: tailoring the genome for food pro- duction.Journal of Fish Biology47, 1–19.
Kissil, G.W., Tandler, A., Elizur, A., Colorni, A. and Zohar, Y. (2000) Gilthead seabream culture. In:
Stickney, R.S. (ed.) Encyclopedia of Aquaculture. John Wiley & Sons, Chichester, pp. 408–416.
Kissil, G.W., Lupatsch, I., Elizur, A. and Zohar, Y. (2001) Long photoperiod delayed spawning and increased somatic growth in gilthead seabream (Sparus aurata).Aquaculture200, 363–379.
Losordo, T.M. and Westers, H. (1994) System carrying capacity and flow estimation. In: Timmons, M.B.
and Losordo, T.M. (eds) Aquaculture Water Reuse Systems. Engineering Design and Management.
Elsevier Press, Amsterdam.
Lupatsch, I. and Kissil, G.W. (1998) Predicting aquaculture waste from gilthead seabream (Sparus aurata) culture using a nutritional approach. Aquatic Living Resources 11, 265–268.
Naylor, R.L., Goldburg, R.J., Mooney, H., Beveridge, M., Clay, J., Folke, C., Kautsky, N., Lubchenco, J., Primavera, J. and Williams, M. (1998) Nature’s subsidies to shrimp and salmon farming. Science 282, 883–884.
Naylor, R.L., Goldburg, R.J., Primavera, J.H., Kautsky, N., Beveridge, M.C.M., Clay, J., Folke, C., Lubchenco, J., Mooney, H. and Troell, M. (2000) Effect of aquaculture on world fish supplies. Nature 405, 1017–1024.