1. Background Overfishing of natural fisheries is a global issue that is becoming more urgent as the human population increases exponentially. According to the FAO, over 70% of the world’s seafood species are fully exploited or depleted. This high demand for seafood protein is not going away. In fact, an astonishing one out of five people depend on this source of protein. To meet the growing demand for seafood, aquaculture (the farming of aquatic organisms) is on the rise and reportedly the fastest growing sector of agriculture worldwide. Traditional aquacultural practices use pond and flow-through systems which are often responsible for discharging pollutants (e.g. nutrients and solids) into the environment. Furthermore, aquacultural feeds often contain high levels of fish or seafood protein, so the demand on wild fisheries is not completely eased. Even though traditional aquaculture has these drawbacks, there is a significant movement towards more sustainable practices, especially in developed countries. For example, recirculating aquaculture systems (RAS) are being used to maximize the reuse of water; and, alternative proteins (e.g., soy bean) are replacing fish and seafood proteins in aquaculture diets. Recycle of Nutrients in Wastewater Effluent to Sustain Fish Polyculture David Kuhn, Ph.D. Student Dr. Gregory D. Boardman Department of Civil & Environmental Engineering 418 Durham Hall Virginia Tech Blacksburg, VA 24061 email: davekuhn@vt.edu Objectives Objective 1: Determine ion supplementation requirements to sustain marine shrimp culture in freshwater tilapia effluent. Objective 2: Determine if shrimp can utilize microbial flocs generated in bioreactors as a supplemental feed (e.g., alternative source of protein). Microbial flocs are generated through the biological treatment of the tilapia effluent. Objective 3: Characterize the biological treatability of fish effluent and the capacity of the treatment system to recycle nutrients in the effluent for shrimp feed. Implications Culturing marine shrimp in fish effluent could potentially reduce water use, reduce the discharge volume of aquacultural wastewaters to receiving streams, serve as a model for the treatment of fish farm effluent, help a fish farm expand into polyculture while creating job opportunities, and offer a sustainable option for the culture of shrimp. Methods/Analysis Objective 1 methods: Twelve 37 liter (L) aquaria with RAS were used to test tilapia effluent supplemented with various salts/ions versus a seawater control. Shrimp survival/growth were monitored. Four independent 42 day trials were used to collect data. Objective 2 methods: Twenty-four 5 L aquaria were used to test biomass flocs as a potential supplemental feed for shrimp culture. Biomass flocs were produced in 37 liter aerobic bioreactors as tilapia effluent was treated. Shrimp survival rates, specific growth rates (SGRs), and food conversion ratios were monitored over two separate 35 to 40 day trials. Objective 3 methods: Three 4 L lab-scale sequencing batch reactors (SBRs) are currently being used to determine the treatability of the tilapia effluent and how to best recycle nutrients into biomass (microbial floc) proteins. Kinetic modeling, substrate supplementation, and microbial floc nutritional properties are being investigated. Findings/Results Water quality during this study was within safe levels and no differences (P > 0.05) between shrimp treatments were observed for dissolved oxygen, nitrite, pH, ammonia, and temperature between treatments for objectives 1 and 2. Objective 1 results: Results from this study indicated that marine shrimp can be cultured in freshwater tilapia effluent with sufficient ion supplementation; 0.6 g/L synthetic sea salt, 50 mg/L Ca 2+ (CaO), and 30 mg/L Mg 2+ (MgSO 4 ). Discussion Fish effluent can be responsible for negative cash flows for producers, but using shrimp as a “cash crop” in the effluent is potentially an option that might enable the operation to be environmentally sustainable. Results from objective 1 are encouraging because marine shrimp can be cultured in freshwater tilapia effluent with minimum ion supplementation and without compromising survival or growth compared to shrimp reared in sea water. The requirement of Ca 2+ and Mg 2+ supplementation are not surprising, given that shrimp exoskeletons are largely composed of these cations. Culturing shrimp in fish effluent maximizes the reuse of a waste stream that would have been normally discharged to the environment. Evidence that shrimp can utilize microbial flocs generated in bioreactors used to treat the tilapia effluent (objective 2) is extremely encouraging. This alternative source of nutrition could reduce the demand for commercial shrimp feed. Consequently, less fishmeal would be required, thereby easing the pressures on wild fisheries. Results from objective 3 are promising. Even though carbon input (to increase C:N ratios) is required to generate a healthy population of biomass, nitrogenous constituents in the effluent are recycled into bacterial protein which will be offered a supplemental feed to shrimp. Carbon is relatively inexpensive compared to protein. High values for observed yield, carbon uptake rate, biomass generation rate, organic matter content, and Kjeldahl protein content are being noted and are a reflection of high efficiencies in terms of nutrient recycling. For example, microbial flocs contain > 50% protein and shrimp feeds typically contain 35-40% protein. Acknowledgements We would like to thank Dr. George Flick, Dr. Steven Craig, Dr. Ewen McLean, Dr. Lori Marsh, and Peter Van Wyk for their research advisement and support. We would also like to thank Blue Ridge Aquaculture (Martinsville, VA) for providing logistical support. This project is funded by USDA-CSREES. Objective 2 results: Results from this study demonstrated that microbial flocs generated in bioreactors, and offered as a supplemental feed, significantly (P < 0.05) improved shrimp growth and SGRs in shrimp fed a restricted ration of commercial shrimp feed. This figure demonstrates the growth of shrimp fed various diets over 35 days. Shrimp fed diet three (6% body weight of shrimp feed per day with microbial flocs significantly outperformed other diets (P < 0.05), even diet one in which shrimp were fed higher rates of shrimp feed but without flocs. Objective 3 results: This project is currently underway. The following kinetic coefficients are reported for treating the effluent with carbon supplementation (sugar): operating biomass concentration 1,380±150 (mg biomass)/(L), observed yield 0.71±0.05 (g biomass)/(g soluble COD), soluble COD uptake rate 9.1±0.8 (mg soluble carbon/L)/(min), and biomass generation rate 6.3±0.9 (mg biomass/L)/(min). Organic content of the microbial flocs generated include, organic matter 89±1% and Kjeldahl protein content 54±1% on a dry weight basis. 5 L aquaria systems Three 4 L sequencing batch reactors in a water bath maintained at 28±0.3 o C outfitted with a programmable electronic controller, power head pumps, control valves, float switches, air flow meters, air manifolds, solenoid valves, and peristaltic pumps. Doctoral student, David Kuhn, examining the health of an adult shrimp. This table presents correlation values (P-values in parenthesis) between shrimp survival/growth and ion concentrations. Correlation values range from no correlation 0 to perfectly correlated 1.0 or -1.0. Lower P-values denotes more significance, * denotes P < 0.05 and ** denotes P < 0.01.