Source: ALABAMA A&M UNIVERSITY submitted to
FOOD SAFETY OF AQUAPONIC PRODUCTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
1006260
Grant No.
(N/A)
Project No.
ALAX-011-0915
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 5, 2015
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Howell, HE, .
Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762
Performing Department
Biological & Environmental Sciences
Non Technical Summary
In this project, we will grow fish and vegetables in an aquaponics system. Fish tissue from fish grown in the system will be tested for mercury in order to determine its food safety. The mercury content from cultured fish will be compared to wild and commercial fish stocks/products. Aquaponics solution based upon fish waste will be used to grow vegetables, primarily lettuce. Aquaponics solution and vegetable products will be tested for bacteria to determine food safety. Bacteria content will be compared to other systems/products. A graduate student will be supported and trained as part of this project.
Animal Health Component
0%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7110810200025%
7121430110025%
3080810106025%
2041430106025%
Goals / Objectives
Objective 1: Determine the safety of plant food products produced in an aquaponics system.Integrate plant production with fish production.Adaptively manage nutritional needs of plants based upon fish waste nutrient output.Control pests and diseases on plants using methods compatible with fish safety and organic food production.Test plant nutrient solution for pathogenic bacteria, particularly E. coli.Test harvested plant parts for pathogenic bacteriaCompare aquaponics products to products currently on the market.Objective 2: Determine the safety of fish products produced in an aquaponics system.Establish and maintain fish production system (s).Adaptively manage fish growth based upon fish age, ambient temperature, stocking rates, etc.Control diseases in fish using methods that should not affect food safety.Test feed and water used in the system for heavy metal content, particularly mercury.Test fish tissue for heavy metal content, particularly mercury.Compare heavy metal levels in fish tissue from wild game stocks and/or products currently on the market.
Project Methods
Aquaponics SystemThe aquaponics system used in this project integrates fish production, biofiltration, and plant production. The microorganisms in the biofilter and settling tank clean the water of harmful nitrogenous compounds, and the plants uptake the soluble nutrients and reduce the amounts of harmful bacteria in a manner similar to that of a constructed wetland. Systems in temperature-controlled areas may have tilapia as the major fish component, while systems in areas where temperature cannot be controlled will have hybrid bluegill (Lepomis macrochirus x cyanellus), largemouth bass (Micropterus salmoides), channel catfish (Ictalurus punctatus), or a combination of the three fishes. The tanks for the fish and the biofilter will consist of food-grade plastic. The plant production elements will consist of aquaponics trays, which will have floating rafts of plants. The trays will be periodically filled, allowed to sit full, and then drained and refilled at intervals as required for plant health.Water will be tested for chlorine, chloramine, and ammonia using a LaMotte Smart3 colorimeter before introduction to fish. If necessary, water will be purified of chlorine using degassing columns and/or filtering through zeolite and activated charcoal. Water in the settling tanks, aquaponics trays, and discharge holding tanks will be tested for plant nutrients using a LaMotte Smart3 colorimeter. Water quality parameters will be measured in the fish tanks, settling tanks, and aquaponics trays for conductivity (mS/cm1), and temperature (oC) using a Hanna conductivity meter. Dissolved oxygen (mg/L) and pH will be measured using a Hach SensION multiparameter meter. Turbidity (FAU) will be measured using a LaMotte Smart3 colorimeter. Insect, bacterial, fungal, and other pests will be controlled through integrated pest management (IPM) with emphasis in organic, non-pesticide treatments, because of the high susceptibility of fish to chemicals. Plant matter infected by pests that are not susceptible to the aforementioned tests will be removed and burned, and involved trays, pots will be sterilized.Fish will be fed commercially prepared feed or feed developed in-house. Feed will be weighed as it is given to fish. At least a 10% subsample of fish will be weighed and measured at intervals depending on season and ambient temperature. Fish diseases will be managed with methods that do not render fish unsafe for human consumption. Fish that are wild-caught for comparison will be obtained through angling, electrofishing, or seining. Ages of wild caught fish will be estimated using scales, otoliths, or fin ray cross sections.Food Safety EvaluationSimultaneous Detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in aquaponic nutrient solutionThe objectives of the study are to use conventional methods such as with the IDEXX system to identify E. coli and fecal coliforms to check for the presence and molecular tools to confirm the identity of E. coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in the nutrient solution to determine the safety and accuracy of this aquaponic system.Initial evaluation will seek to quantify the amount of fecal indicator bacteria fecal coliforms and E. coli in water samples using Most Probable Number (MPN) Analysis. Solution will be collected in sterile polypropylene bottles immediately placed on ice and kept at 4°C until microbiological analyses were completed. The IDEXX (IDEXX laboratories, Maine) system for water quality analysis will be used for enumeration procedures of E. coli (Colilert®). The samples will be analyzed with enzymes specific for each using a Quanti-Tray and IDEXX® Quanti-Tray Sealer. The sample trays will be incubated in trays at 35°C (Colilert®) for 24 hours. Results will be compared to a Most Probable Number (MPN) chart.Sampled nutrient solutions will be extracted for DNA using the MO BIO Ultra Clean Soil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA). The DNA concentration will then be determined using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Multiplex PCR will be performed in a total volume of 50 μl containing 2 μl of template DNA and 48 μl of PCR master mix composed of 1x PCR buffer, 5.0 mM MgCl2, 80 nM of each of the detection primers, 200 μM dATP, dCTP, and dGTP, 400 μM dUTP, 0.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems Foster City, USA), and 0.5 U of AmpErase UNG (uracil-N-glycosidase; Applied Biosystems Foster City, USA) as described by Yasmin et al. (2007). Optimization of the multiplex PCR system for the simultaneous detection of E. coli O157:H7, Listeria monocytogenes and Salmonella enteritidis will be done using DNA from pure cultures of each organism. Each organism will be used as templates for DNA extraction analysis and used as positive controls for the PCR reactions. PCR amplification will be done using a Peltier Thermal Cycler PTC-200 (GMI, Inc., Ramsey, Minnesota. The expected size for E. coli O157:H7, L. monocytogenes and Salmonella are 120, 234 and 375 bp, respectively. This study will aid us in determining if the presence of indicator bacteria signifies the presence of pathogenic bacteria. Additionally, the potential detection of the target pathogen (s) will aid us in identifying the safety of this aquaponic system.Mercury Analysis with DMA-80Cultured fishes, their food, and water from the fish tanks, settling tanks, and aquaponics trays will be analyzed for mercury. Fish samples will be wrapped in aluminum foil, immediately frozen and transported to the laboratory and kept frozen at -80oC. Before analysis, fish samples will be gently thawed, sorted into similar sizes, and carefully dissected with a clean scalpel to select desirable tissues (muscle, gills, liver, skin, and kidney) for analysis. Pooled samples from each tissue will be homogenized and freeze dried before extraction of methyl Hg according to the procedure of Carbonell et al. (2009). At the end of the extraction, 10ul of the extract will be loaded in quartz boats and introduced into DMA-80 system with thermal combustion (drying 120 s at 250oC, thermal decomposition 120 s at 650oC). Fish feed samples (10-200mcg), being already dried, will not require the drying step, and will otherwise be treated similarly. Water samples will be dried for 300 s at 300oC (Rüdel, et al. 2011). The amalgamator in the DMA-80 selectively traps Hg after the system is flushed with oxygen for 60s to remove any remaining gases or decomposition products. At the end of the elapsed flush time, the amalgamator is rapidly heated (12s), releasing Hg vapor. Absorbance is measured at 253.7 nm as a function of Hg concentration. Total mercury in the fish samples will be quantified by thermal combustion of 0.05g wet weight at 750oC for 120s using DMA-80. The difference between total Hg and MeHg will be classified as inorganic Hg. A-two range calibration (0-40 and 40-500 ng of Hg) of the instrument will be performed before Hg analysis because the instrument operates on two measuring cells. After initial calibration, a blank run will be performed to free the system from Hg residual and contamination. All sample boats to be used for analysis will be run through the system to get rid of any residual Hg on the boats. Certified reference material will be analyzed at the beginning and end of each set of samples (typically 10) to verify that the instrument remains calibrated during the course of the study. A blank (i.e., an empty sample boat or boat with purified water) will also be analyzed at the end of each sample set to prevent Hg from being carried over between samples.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Our target audiences are undergraduate students, graduate students, other researchers and the general public. Changes/Problems:The disease on the fish was likely Saprolegniasis (winter fungus) because of its ocurrence congruently with fall and spring temperature changes and its response to treatment with peroxide. The water in the greenhouse routinely has ammonia levels high enough to have sublethal (growth depressive) and lethal effects upon fish. There was a general lack of coordination and cooperation between the former PI, the current PI, and the graduate student that resulted in the overfeeding of fish, the unsuccessful feed formulation, and the lack of aquaculture systems maintenance assistance provided by the graduate student. What opportunities for training and professional development has the project provided?We have trained four students in aquaponics systems maintenance, water quality testing, and microbiological testing. How have the results been disseminated to communities of interest?Alisha Bailey won a first place prize for her poster at the Alabama A&M University STEM day (April 2017). Undergraduate students have received instruction in aquaculture using the systems. What do you plan to do during the next reporting period to accomplish the goals?With a long enough grow out, mercury testing can occur on fish tissue. We will also test the fish feeds for mercury content. We need additional info on fish growth response and tissue analysis in response to a modified feed formulation. We also need additional information about the mercury content of fish waste, so we will measure that.

Impacts
What was accomplished under these goals? We have accompolished the following towards objective 1: Integrate plant production with fish production. Adaptively manage nutritional needs of plants based upon fish waste nutrient output. Control pests and diseases on plants using methods compatible with fish safety and organic food production. Test plant nutrient solution for pathogenic bacteria, particularly E. coli. Test harvested plant parts for pathogenic bacteria Compare aquaponics products to products currently on the market. We have accomplished the following goals related to objective 2: Establish and maintain fish production system (s). Adaptively manage fish growth based upon fish age, ambient temperature, stocking rates, etc. Control diseases in fish using methods that should not affect food safety. Test water used in the system for mercury.

Publications

  • Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2017 Citation: Bailey, Alisha M. (2017) WATER QUALITY, SENSORY, AND FISH PRODUCTIVITY DIFFERENCES IN A CLOSED AQUAPONICS SYSTEM (Master's Thesis)


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience for our project are individuals who are interested in designing and operating an Aquaponics system. Specifically, designed our system for the home user and small farmer who wishes to grow their own fish and vegetables either for consumption or to sell to the public. Additionally our project is designed as a model project to show end-users how to set up systems and to make them productive. Changes/Problems:To date we have had major problems with diseases of the fish in our tanks. These fish have been affected with an airborne disease called cotton fin (http://fishkeepingadvice.com/cotton-fin-fungus/). This has greatly delayed our project as we develop a mechanism to reduce infections in our systems. As a result of these infections we have lost a lot of fish in our experiments which have delayed our progress. We have been successfully treating the remaining fish for the disease. Once they have completely healed we will resume the project to meet the specified objectives. Additionally we have added new fish to our project to complete the objectives. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We have had several school groups tour our plant and fish systems. We explain the concept of Aqua upon it and the design of the system. As a result we have introduced approximately 150 primary school age children to Aquaponics. Additionally several students from Alabama A&M are involved in the project and have a working knowledge of the Aquaponics concept and design What do you plan to do during the next reporting period to accomplish the goals?During this last year the project we will continue to meet the objectives outlined in our proposal. We hope to have the final portion of the project completed by August 2017. The graduate student associated with the project will complete her thesis in August 2017 and subsequent manuscripts will be prepared between now and the end of the project. We expect that to professional manuscripts will be developed and we plan to use the results from this study to obtain additional funding to expand the project based on our findings and results.

Impacts
What was accomplished under these goals? To date we have had major problems with diseases of the fish in our tanks. These fish have been affected with an airborne disease called cotton fin (http://fishkeepingadvice.com/cotton-fin-fungus/). This has greatly delayed our project as we develop a mechanism to reduce infections in our systems. As a result of these infections we have lost a lot of fish in our experiments which have delayed our progress. We have been successfully treating the remaining fish for the disease. Once they have completely healed we will resume the project to meet the specified objectives. Additionally we have added new fish to our project to complete the objectives. Our initial project results indicate that the bacteria on the plants have been very minimal to nonexistent. We been able to maintain the fish production systems despite the disease suffered by the fish. We have further established appropriate stocking rates in each of the tanks to maximize growth and to minimize the crowding effects and nutrient overloads in the tank water. We have also determined that we can grow both Bass and bluegill to a consumable size within 5 to 6 months. This is all determined on the feed type and water temperatures in the tanks. The production of plants has been going as expected. We have not tested for heavy metal content. The heavy metal testing will occur at the end of the project because we have to sacrifice the fish to conduct those measurements. We expect however that the heavy metals in the tissues of the fish will be less than wild game stocks because there is no direct source of heavy metals from our current water source.

Publications


    Progress 05/05/15 to 09/30/15

    Outputs
    Target Audience:The target audience for our project are individuals who are interested in doing Aqua Pontic's. Our system is designed for the home user and small business user who wishes to grow their own fish and vegetables either for consumption or to sell to the public. Additionally our project is designed as a model project to show end-users how to set up systems and to make them productive. Changes/Problems:The only major problems we've had is the initial ordering specific items and setting up of the tanks. This is taken much longer than we anticipated primarily because the project started a little late and we were faced with ordering items at the end of our fiscal year. However we have overcome these obstacles and we are getting back on track to meet our original timeline What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?For the next reporting system we will be in full operations to test objectives one and two. We have had to run some new electrical lines and water lines to accommodate our system. We've had to make some modifications to our original design system based on logistics and space availability.

    Impacts
    What was accomplished under these goals? For the first fiscal year we were in the process of ordering materials and setting up the experimental area. We experienced some delay with ordering items which set the project back about four months. Additionally we hired a graduate student to assist on the project. We anticipate the second fiscal year the project will be up and running and we will be able to complete objectives one and two.

    Publications